Vaccinia Research Articles

Genomic characterization of circulating human respiratory syncytial viruses A and B in Kuwait using whole-genome sequencing.

The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait.

Canine Amniotic Fluid at Birth Holds Information about Neonatal Antibody Titres against Core Vaccine Viruses

There is a growing interest in the composition of amniotic fluid (AF) in both humans and animals. In addition to its nutritional and protective functions for the foetus, current knowledge demonstrates that AF also serves advanced diagnostic, prognostic, and therapeutic roles. Newborn dogs have an underdeveloped immune system, making them highly susceptible to dangerous pathogens such as canine parvovirus (CPV-2), canine infectious hepatitis virus (CAdV-1), and canine distemper virus (CDV), thus exposing them to a high risk of mortality in the first weeks of life. Immunoglobulins G (IgGs) represent the only antibody isotype capable of crossing the placenta in a small amount and have been detected also in canine AF. The primary aim of this study was to investigate the reliability of AF collected at birth as a marker of passive immunity in canine species. For this purpose, total and specific IgGs against CPV-2, CAdV-1, and CDV were investigated and quantified in both maternal plasma and AF collected at the time of caesarean section. The vaccination status of the bitches was also taken into consideration. Since the immune system can be influenced by gestational age, with preterm infants having immature innate and adaptive immunity, IgG concentrations were correlated with amniotic lecithin, sphingomyelin, cortisol, surfactant protein A, and pentraxin 3 levels. In a previous study from our group on foetal maturity these molecules were measured in the same samples. Finally, correlations between their amniotic content and neonatal outcomes were investigated. This study demonstrates that AF analysis at birth can provide valuable insights into neonatal immunity in puppies, offering a non-invasive method to detect potential early health risks, for improved puppy care and management.

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus.

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.

Levels of antibodies against the monkeypox virus compared by HIV status and historical smallpox vaccinations: a serological study

ABSTRACT Men who have sex with men and people living with HIV are disproportionately affected in the 2022 multi-country monkeypox epidemic. The smallpox vaccine can induce cross-reactive antibodies against the monkeypox virus (MPXV) and reduce the risk of infection. Data on antibodies against MPXV induced by historic smallpox vaccination in people with HIV are scarce. In this observational study, plasma samples were collected from people living with and without HIV in Shenzhen, China. We measured antibodies binding to two representative proteins of vaccinia virus (VACV; A27L and A33R) and homologous proteins of MPXV (A29L and A35R) using an enzyme-linked immunosorbent assay. We compared the levels of these antibodies between people living with and without HIV. Stratified analyses were performed based on the year of birth of 1981 when the smallpox vaccination was stopped in China. Plasma samples from 677 people living with HIV and 746 people without HIV were tested. A consistent pattern was identified among the four antibodies, regardless of HIV status. VACV antigen-reactive and MPXV antigen-reactive antibodies induced by historic smallpox vaccination were detectable in the people born before 1981, and antibody levels reached a nadir during or after 1981. The levels of smallpox vaccine-induced antibodies were comparable between people living with HIV and those without HIV. Our findings suggest that the antibody levels against MPXV decreased in both people living with and without HIV due to the cessation of smallpox vaccination.

Anti-monkeypox virus-neutralizing activities of human immunoglobulin manufactured between 1999 and 2021 and derived from donors in the United States and Japan.

In May 2022, the United Kingdom reported the first case of chained transmission of the monkeypox (mpox) virus without any known epidemiological links to west or central Africa. The monthly number of mpox patients currently has passed a peak and is declining globally, and infected patients include both non-vaccinated and vaccinated individuals. Herein, the virus-neutralizing (VN) activity against vaccinia viruses, which are considered to cross-react with the mpox virus, in the intravenous immunoglobulin (IVIG) lots derived from donors, including vaccinated Japanese populations, was evaluated to clarify the status of the Japanese blood donor population. VN titres against vaccinia and human mpox viruses in IVIG lots derived from donors in Japan and the United States manufactured between 1999 and 2021 and 1995 and 2001, respectively, were evaluated by neutralization testing. VN titres of IVIG derived from donors in Japan and the United States against vaccinia and mpox viruses showed a slowly decreasing trend between 1999 and 2021. VN titres are expected to decrease in the future since the percentage of vaccinated donors in the donor population seems to have decreased. Therefore, continuous monitoring of VN titres is required.

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

BackgroundMonkeypox (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.MethodsGEO 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.ResultsInfection 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.ResultsMPXV 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.

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.

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

Vaccinia viruses (VACV) are versatile therapeutic agents and different features of various VACV strains allow for a broad range of therapeutic applications. Modified Vaccinia virus 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 Vaccinia virus 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, 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.

Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING

Micronuclei induced by radiation, replication stress, or chromosome segregation errors do not activate cGAS-STING

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.

The Role of Tecovirimat in the Management of Monkeypox

Monkeypox is a self-limited disease with symptoms that disappear within 14 to 21 days. Clinical management of monkeypox includes general supportive care and the use of antivirals that have activity against the monkeypox virus. This literature study aims to describe the role of tecovirimat in the management of monkeypox. Tecovirimat is an antiviral drug approved by the U.S. Food and Drug Administration (FDA) as part of the treatment management of smallpox in adults and children. Evidence of the efficacy of tecovirimat as mpox therapy was obtained from animal studies, where tecovirimat reduced mortality rates, reduced duration of illness and viral shedding. Tecovirimat inhibits the vp37 viral protein encoded by the F13 gene of the variola virus. This protein is highly conserved in orthopoxviruses, allowing tecovirimat to have in vitro activity against several orthopoxviruses, including vaccinia, variola, cowpox, and monkeypox viruses. In conclusion, tecovirimat is used in monkeypox sufferers with severe clinical conditions (sepsis, encephalitis, extensive lesions, bleeding manifestations), patients at risk of experiencing severe clinical conditions (immunocompromised, pregnant or breastfeeding, history of psoriasis, varicella zoster infection) and patients with one or more complications.

Relative Vaccine Effectiveness of Cell- vs Egg-Based Quadrivalent Influenza Vaccine Against Test-Confirmed Influenza Over 3 Seasons Between 2017 and 2020 in the United States.

Influenza vaccine viruses grown in eggs may acquire egg-adaptive mutations that may reduce antigenic similarity between vaccine and circulating influenza viruses and decrease vaccine effectiveness. We compared cell- and egg-based quadrivalent influenza vaccines (QIVc and QIVe, respectively) for preventing test-confirmed influenza over 3 US influenza seasons (2017-2020). Using a retrospective test-negative design, we estimated the relative vaccine effectiveness (rVE) of QIVc vs QIVe among individuals aged 4 to 64 years who had an acute respiratory or febrile illness and were tested for influenza in routine outpatient care. Exposure, outcome, and covariate data were obtained from electronic health records linked to pharmacy and medical claims. Season-specific rVE was estimated by comparing the odds of testing positive for influenza among QIVc vs QIVe recipients. Models were adjusted for age, sex, geographic region, influenza test date, and additional unbalanced covariates. A doubly robust approach was used combining inverse probability of treatment weights with multivariable regression. The study included 31 824, 33 388, and 34 398 patients in the 2017-2018, 2018-2019, and 2019-2020 seasons, respectively; ∼10% received QIVc and ∼90% received QIVe. QIVc demonstrated superior effectiveness vs QIVe in prevention of test-confirmed influenza: rVEs were 14.8% (95% CI, 7.0%-22.0%) in 2017-2018, 12.5% (95% CI, 4.7%-19.6%) in 2018-2019, and 10.0% (95% CI, 2.7%-16.7%) in 2019-2020. This study demonstrated consistently superior effectiveness of QIVc vs QIVe in preventing test-confirmed influenza over 3 seasons characterized by different circulating viruses and degrees of egg adaptation.

Scenarios of future mpox outbreaks among men who have sex with men: a modelling study based on cross-sectional seroprevalence data from the Netherlands, 2022.

BackgroundFollowing the 2022-2023 mpox outbreak, crucial knowledge gaps exist regarding orthopoxvirus-specific immunity in risk groups and its impact on future outbreaks.AimWe combined cross-sectional seroprevalence studies in two cities in the Netherlands with mathematical modelling to evaluate scenarios of future mpox outbreaks among men who have sex with men (MSM).MethodsSerum samples were obtained from 1,065 MSM attending Centres for Sexual Health (CSH) in Rotterdam or Amsterdam following the peak of the Dutch mpox outbreak and the introduction of vaccination. For MSM visiting the Rotterdam CSH, sera were linked to epidemiological and vaccination data. An in-house developed ELISA was used to detect vaccinia virus (VACV)-specific IgG. These observations were combined with published data on serial interval and vaccine effectiveness to inform a stochastic transmission model that estimates the risk of future mpox outbreaks.ResultsThe seroprevalence of VACV-specific antibodies was 45.4% and 47.1% in Rotterdam and Amsterdam, respectively. Transmission modelling showed that the impact of risk group vaccination on the original outbreak was likely small. However, assuming different scenarios, the number of mpox cases in a future outbreak would be markedly reduced because of vaccination. Simultaneously, the current level of immunity alone may not prevent future outbreaks. Maintaining a short time-to-diagnosis is a key component of any strategy to prevent new outbreaks.ConclusionOur findings indicate a reduced likelihood of large future mpox outbreaks among MSM in the Netherlands under current conditions, but emphasise the importance of maintaining population immunity, diagnostic capacities and disease awareness.

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Antibodies Induced by Smallpox Vaccination after at Least 45 Years Cross-React with and In Vitro Neutralize Mpox Virus: A Role for Polyclonal B Cell Activation?

To evaluate whether antibodies specific for the vaccinia virus (VV) are still detectable after at least 45 years from immunization. To confirm that VV-specific antibodies are endowed with the capacity to neutralize Mpox virus (MPXV) in vitro. To test a possible role of polyclonal non-specific activation in the maintenance of immunologic memory. Sera were collected from the following groups: smallpox-vaccinated individuals with or without latent tuberculosis infection (LTBI), unvaccinated donors, and convalescent individuals after MPXV infection. Supernatant of VV- or MPXV-infected Vero cells were inactivated and used as antigens in ELISA or in Western blot (WB) analyses. An MPXV plaque reduction neutralization test (PRNT) was optimized and performed on study samples. VV- and PPD-specific memory T cells were measured by flow cytometry. None of the smallpox unvaccinated donors tested positive in ELISA or WB analysis and their sera were unable to neutralize MPXV in vitro. Sera from all the individuals convalescing from an MPXV infection tested positive for anti-VV or MPXV IgG with high titers and showed MPXV in vitro neutralization capacity. Sera from most of the vaccinated individuals showed IgG anti-VV and anti-MPXV at high titers. WB analyses showed that positive sera from vaccinated or convalescent individuals recognized both VV and MPXV antigens. Higher VV-specific IgG titer and specific T cells were observed in LTBI individuals. ELISA and WB performed using supernatant of VV- or MPXV-infected cells are suitable to identify individuals vaccinated against smallpox at more than 45 years from immunization and individuals convalescing from a recent MPXV infection. ELISA and WB results show a good correlation with PRNT. Data confirm that a smallpox vaccination induces a long-lasting memory in terms of specific IgG and that antibodies raised against VV may neutralize MPXV in vitro. Finally, higher titers of VV-specific antibodies and higher frequency of VV-specific memory T cells in LTBI individuals suggest a role of polyclonal non-specific activation in the maintenance of immunologic memory.

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

Interferon 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, down-regulated IFN-induced expression of inhibitory ligands, up-regulated 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.

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.

Expression, purification, and crystal structure of mpox virus A41 protein

Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries outside of Africa. The rapidly increasing number of confirmed mpox cases poses a threat to the international community. In-depth studies of key viral factors are urgently needed, which will inform the design of multiple antiviral agents. Mpox virus A41L gene encodes a secreted protein, A41, that is nonessential for viral replication, but could affect the host response to infection via interacting with chemokines. Here, mpox virus A41 protein was expressed in Sf9 cells, and purified by affinity chromatography followed by gel filtration. Surface plasmon resonance spectroscopy showed that purified A41 binds a certain human chemokine CXCL8 with the equilibrium dissociation constant (KD) being 1.22 × 10−6 M. The crystal structure of mpox virus A41 protein was solved at 1.92 Å. Structural analysis and comparison revealed that mpox virus A41 protein adopts a characteristic β-sheet topology, showing minor differences with that of vaccinia virus. These preliminary structural and functional studies of A41 protein from mpox virus will help us better understand its role in chemokine subversion, and contributing to the knowledge to viral chemokine binding proteins.

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.

Abstract LB354: A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice

Abstract Oncolytic viruses (OVs) are potent cancer therapeutics that can selectively kill tumor cells and promote anti-tumor immunity. mSJ-650 (Wyeth, K2L-, TK-, murine GM-CSF, human CD55) is the Wyeth strain of vaccinia virus with human complement regulatory protein CD55 incorporated on the intracellular mature virion membrane to evade complement-mediated attack during circulation. The virus also expresses mGM-CSF in replace of thymidine kinase to increase tumor selectivity and enhance immunogenicity. We previously reported that a single dose of mSJ-650 effectively evades complement and shows prominent anti-tumor efficacy in A549 and HCT116 xenograft model. Here, we further investigated the capability of mSJ-650 in promoting anti-tumor response after multiple systemic administration in immunocompetent breast cancer mouse model. In an orthotopic EMT6 breast cancer model, mSJ-650 or control mSJ-612 devoid of CD55 was intravenously injected at low dose (1x106 pfu) and high dose (3x106 pfu), twice weekly. mSJ-650 treatment dose-dependently suppressed tumor growth, showing superior antitumor efficacy compared to treatment of control mSJ-612. Moreover, multiple systemic administration of mSJ-650 was well-tolerated with no apparent signs of toxicity in serum tested via AST and ALT assay. To further elucidate whether mSJ-650 can evade neutralization by antibody and maintain its antitumor potency in vivo, we designed two experimental models. Firstly, endogenous vaccinia virus specific neutralizing antibody was generated by intravenous injection of mSJ-650 prior to tumor cell inoculation in tumor-bearing mouse, followed by systemic administration of mSJ-650 twice weekly. Second, serum containing vaccinia virus specific neutralizing antibody was intravenously injected 24 hours prior to mSJ-650 administration in tumor-bearing mouse. In both experimental models, mSJ-650 treated group significantly inhibited tumor growth despite the presence of neutralizing antibody, while control mSJ-612 treated group failed to suppress tumor growth. These results imply that mSJ-650 is capable of evading neutralization and maintaining its antitumor efficacy. Moreover, we performed multi-color flow cytometry analysis of tumor-infiltrating leukocytes to evaluate immunogenicity of mSJ-650. As a result, we observed a significant increase in recruitment of activated CD4+ and CD8+ T cells by 2-fold and high expression level of cytotoxic molecules (perforin, TNF-α, IFN-γ) in tumor-infiltrating T cells. In summary, multiple systemic administration of mSJ-650 markedly evaded neutralization and suppressed tumor growth by promoting anti-tumor response in immunocompetent breast cancer mouse model, thus implying that mSJ-650 is a promising candidate for cancer immunotherapy. Citation Format: Yeonsoo Yang, Solchan Won, Namhee Lee, Eunhye Kim, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. A novel oncolytic vaccinia virus with intravenous compatibility evaded complement- and neutralizing antibody-mediated inhibition and provided superior anti-tumor activity to intra-tumoral administration in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB354.