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.

Therapeutic potential of interleukin-21 in cancer.

Interleukin-21 (IL-21) is an immunostimulatory cytokine which belongs to the common gamma-chain family of cytokines. It plays an import role in the development, differentiation, proliferation, and activation of immune cells, in particular T and natural killer (NK) cells. Since its discovery in 2000, IL-21 has been shown to regulate both adaptive and immune responses associates with key role in antiviral and antitumor responses. Recent advances indicate IL-21 as a promising target for cancer treatment and encouraging results were obtained in preclinical studies which investigated the potency of IL-21 alone or in combination with other therapies, including monoclonal antibodies, checkpoint inhibitory molecules, oncolytic virotherapy, and adoptive cell transfer. Furthermore, IL-21 showed antitumor effects in the treatment of patients with advanced cancer, with minimal side effects in several clinical trials. In the present review, we will outline the recent progress in IL-21 research, highlighting the potential of IL-21 based therapy as single agent or in combination with other drugs to enhance cancer treatment efficiency.

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.

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.

Differing Susceptibilities to Certain Microbicidal Chemistries among Three Representative Enveloped Viruses.

Three lipid-enveloped viruses (bovine viral diarrhea virus [BVDV], vaccinia virus, and severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) were evaluated in side-by-side liquid inactivation efficacy studies of low pH (3.0 to 3.1) treatment and of the non-formulated microbicidal actives sodium hypochlorite (100 ppm), ethanol (70%), quaternary ammonium compound BTC® 835 (100 ppm), and peracetic acid (100 ppm). Low pH was evaluated at 10 and 60 min contact times, and the microbicides were evaluated at 1 min contact time at room temperature per the ASTM E1052 standard. In each case, 5% animal serum was included in the viral inoculum as a challenge soil load. The three viruses displayed similar susceptibility to sodium hypochlorite and ethanol, with complete inactivation resulting. Significant differences in susceptibility to BTC® 835 and peracetic acid were identified, with the ordering of the three viruses for susceptibility to BTC® 835 being SARS-CoV-2 > vaccinia virus = BVDV, and the ordering for peracetic acid being vaccinia virus > SARS-CoV-2 > BVDV. The ordering for susceptibility to low pH treatment (60 min contact time) was vaccinia virus > SARS-CoV-2 > BVDV. Not all enveloped viruses display equivalent susceptibilities to inactivation approaches. For the chemistries evaluated here, BVDV appears to represent a worst-case enveloped virus.

HDAC5 enhances IRF3 activation and is targeted for degradation by protein C6 from orthopoxviruses including Monkeypox virus and Variola virus.

Histone deacetylases (HDACs) regulate gene expression and innate immunity. Previously, we showed that HDAC5 is degraded during Vaccinia virus (VACV) infection and is a restriction factor for VACV and herpes simplex virus type 1. Here, we report that HDAC5 promotes interferon regulatory factor 3 (IRF3) activation downstream of Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 or Sendai virus-mediated stimulation without requiring HDAC activity. Loss of HDAC5-mediated IRF3 activation is restored by re-introduction of HDAC5 but not HDAC1 or HDAC4. The antiviral activity of HDAC5 is antagonized by VACV protein C6 and orthologs from the orthopoxviruses cowpox, rabbitpox, camelpox, monkeypox, and variola. Infection by many of these viruses induces proteasomal degradation of HDAC5, and expression of C6 alone can induce HDAC5 degradation. Mechanistically, C6 binds to the dimerization domain of HDAC5 and prevents homodimerization and heterodimerization with HDAC4. Overall, this study describes HDAC5 as a positive regulator of IRF3 activation and provides mechanistic insight into how the poxviral protein C6 binds to HDAC5 to antagonize its function.

Expression of GPX4 by oncolytic vaccinia virus can significantly enhance CD8+T cell function and its impact against pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is currently difficult to treat, even when therapies are combined with immune checkpoint blockade (ICB). A novel strategy for immunotherapy would be to maximize the therapeutic potential of oncolytic viruses (OVs), which have been proven to engage the regulation of tumor microenvironment (TME) and cause-specific T-cell responses. To boost tumor sensitivity to ICB therapy, this study aimed to investigate how glutathione peroxide 4 (GPX4)-loaded OVs affect CD8+ T cells and repair the immunosuppressive environment. Here, we successfully constructed a novel recombinant oncolytic vaccinia virus (OVV) encoding the mouse GPX4 gene. We found the OVV-GPX4 effectively replicated in tumor cells and prompted the expression of GPX4 in T cells. Our research indicated that OVV-GPX4 could reshape the TME, rectify the depletion of CD8+T cells, and enhance the antitumor effects of ICB therapy.

The association between BCG scars and self-reported chronic diseases: A cross-sectional observational study within an RCT of Danish health care workers.

INTRODUCTION: The live-attenuated vaccines Bacillus Calmette-Guérin (BCG) and Vaccinia have been associated with beneficial non-specific effects. We assessed the prevalence of BCG and Vaccinia vaccine scars in a cohort of Danish health care workers and investigated the association between the presence of vaccine scars and self-reported chronic diseases. METHODS: Cross-sectional study utilizing baseline data collected during 2020-2021 at enrollment in a BCG trial aiming to assess the effect of BCG vaccination on absenteeism and infectious disease morbidity during the SARS-COV-2 pandemic. In Denmark, Vaccinia was discontinued in 1977, and BCG was phased out in the early 1980s. We used logistic regression analysis (adjusted for sex, birth year, and smoking status) to estimate the association between scar status and chronic diseases, providing adjusted Odds Ratios (aORs) with 95 % Confidence Intervals, for participants born before 1977, and born from 1965 to 1976. RESULTS: The cohort consisted of 1218 participants (206 males; 1012 females) with a median age of 47 years (Q1-Q3: 36-56). Among participants born 1965-1976 (n = 403), who experienced the phase-outs, having BCG and/or Vaccinia scar(s) vs. having no vaccine scars yielded an aOR of 0.51 (0.29-0.90) of self-reported chronic disease; an effect primarily driven by BCG. In the same birth cohort, having vaccine scar(s) was most strongly associated with a lower prevalence of chronic respiratory and allergic diseases; the aORs being 0.39 (0.16-0.97) and 0.39 (0.16-0.91), respectively. CONCLUSION: Having a BCG scar was associated with a lower prevalence of self-reported chronic disease.

Vaccinia Virus Defective Particles Lacking the F17 Protein Do Not Inhibit Protein Synthesis: F17, a Double-Edged Sword for Protein Synthesis?

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

Transient inhibition of neutrophil functions enhances the antitumor effect of intravenously delivered oncolytic vaccinia virus.

Oncolytic viruses (OVs) possess the unique ability to selectively replicate within tumor cells, leading to their destruction, while also reversing the immunosuppression within the tumor microenvironment and triggering an antitumor immune response. As a result, OVs have emerged as one of the most promising approaches in cancer therapy. However, the effective delivery of intravenously administered OVs faces significant challenges imposed by various immune cells within the peripheral blood, hindering their access to tumor sites. Notably, neutrophils, the predominant white blood cell population comprising approximately 50%-70% of circulating white cells in humans, show phagocytic properties. Our investigation revealed that the majority of oncolytic vaccinia viruses (VV) are engulfed and degraded by neutrophils in the bloodstream. The depletion of neutrophils using the anti-LY6G Ab (1-A8) resulted in an increased accumulation of circulating oncolytic VV in the peripheral blood and enhanced deposition at the tumor site, consequently amplifying the antitumor effect. Neutrophils heavily rely on PI3K signaling to sustain their phagocytic process. Additionally, our study determined that the inhibition of the PI3Kinase delta isoform by idelalisib (CAL-101) suppressed the uptake of oncolytic VV by neutrophils. This inhibition led to a greater presence of oncolytic VV in both the peripheral blood and at the tumor site, resulting in improved efficacy against the tumor. In conclusion, our study showed that inhibiting neutrophil functions can significantly enhance the antitumor efficacy of intravenous oncolytic VV.

Filamin B restricts vaccinia virus spread and is targeted by vaccinia virus protein C4.

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.

Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses.

We previously identified the bisbenzimide Hoechst 33342 (H42) as a potent multi-stage inhibitor of the prototypic poxvirus, the vaccinia virus (VACV), and several parapoxviruses. A recent report showed that novel bisbenzimide compounds similar in structure to H42 could prevent human cytomegalovirus replication. Here, we assessed whether these compounds could also serve as poxvirus inhibitors. Using virological assays, we show that these bisbenzimide compounds inhibit VACV spread, plaque formation, and the production of infectious progeny VACV with relatively low cell toxicity. Further analysis of the VACV lifecycle indicated that the effective bisbenzimide compounds had little impact on VACV early gene expression but inhibited VACV late gene expression and truncated the formation of VACV replication sites. Additionally, we found that bisbenzimide compounds, including H42, can inhibit both monkeypox and a VACV mutant resistant to the widely used anti-poxvirus drug TPOXX (Tecovirimat). Therefore, the tested bisbenzimide compounds were inhibitors of both prototypic and pandemic potential poxviruses and could be developed for use in situations where anti-poxvirus drug resistance may occur. Additionally, these data suggest that bisbenzimide compounds may serve as broad-activity antiviral compounds, targeting diverse DNA viruses such as poxviruses and betaherpesviruses.IMPORTANCEThe 2022 mpox (monkeypox) outbreak served as a stark reminder that due to the cessation of smallpox vaccination over 40 years ago, most of the human population remains susceptible to poxvirus infection. With only two antivirals approved for the treatment of smallpox infection in humans, the need for additional anti-poxvirus compounds is evident. Having shown that the bisbenzimide H33342 is a potent inhibitor of poxvirus gene expression and DNA replication, here we extend these findings to include a set of novel bisbenzimide compounds that show anti-viral activity against mpox and a drug-resistant prototype poxvirus mutant. These results suggest that further development of bisbenzimides for the treatment of pandemic potential poxviruses is warranted.

Live attenuated smallpox vaccine candidate (KVAC103) efficiently induces protective immune responses in mice.

Smallpox, caused by the variola virus belonging to the genus Orthopoxvirus, is an acute contagious disease that killed 300 million people in the 20th century. Since it was declared to be eradicated and the national immunization program against it was stopped, the variola virus has become a prospective bio-weapon. It is necessary to develop a safe vaccine that protects people from terrorism using this biological weapon and that can be administered to immunocompromised people. Our previous study reported on the development of an attenuated smallpox vaccine (KVAC103). This study evaluated cellular and humoral immune responses to various doses, frequencies, and routes of administration of the KVAC103 strain, compared to CJ-50300 vaccine, and its protective ability against the wild-type vaccinia virus Western Reserve (VACV-WR) strain was evaluated. The binding and neutralizing-antibody titers increased in a concentration-dependent manner in the second inoculation, which increased the neutralizing-antibody titer compared to those after the single injection. In contrast, the T-cell immune response (interferon-gamma positive cells) increased after the second inoculation compared to that of CJ-50300 after the first inoculation. Neutralizing-antibody titers and antigen-specific IgG levels were comparable in all groups administered KVAC103 intramuscularly, subcutaneously, and intradermally. In a protective immunity test using the VACV-WR strain, all mice vaccinated with CJ-50300 or KVAC103 showed 100% survival. KVAC103 could be a potent smallpox vaccine that efficiently induces humoral and cellular immune responses to protect mice against the VACV-WR strain.

Residual Immunity from Smallpox Vaccination and Possible Protection from Mpox, China.

Among persons born in China before 1980 and tested for vaccinia virus Tiantan strain (VVT), 28.7% (137/478) had neutralizing antibodies, 71.4% (25/35) had memory B-cell responses, and 65.7% (23/35) had memory T-cell responses to VVT. Because of cross-immunity between the viruses, these findings can help guide mpox vaccination strategies in China.

Palisade structure in intact vaccinia virions.

Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

Long-lasting humoral and cellular memory immunity to vaccinia virus Tiantan provides pre-existing immunity against mpox virus in Chinese population.

Investigating immune memory to vaccinia virus and pre-existing immunity to mpox virus (MPXV) among the population is crucial for the global response to this ongoing mpox epidemic. Blood was sampled from vaccinees inoculated with vaccinia virus Tiantan (VTT) strain born before 1981 and unvaccinated control subjects born since 1982. After at least 40 years of the inoculation, 60% or 5% VTT vaccinees possess neutralizing antibodies (NAbs) to VTT or MPXV, with at least 50% having T cell memory to VTT protein antigens. Notably, 46.7% vaccinees show pre-existing T cell responses to MPXV. Broad pre-existing CD8+ T cell reactivities to MPXV are detected not only against conserved epitopes but also against variant epitopes between VTT and MPXV. Persistent NAbs and T cell memory to VTT among vaccinees, along with pre-existing T cells to MPXV among both vaccinees and the unvaccinated population, indicate a particular immune barrier to mpox.

Design and selection of anti-PD-L1 single-domain antibody and tumor necrosis factor superfamily ligands for an optimal vectorization in an oncolytic virus.

Arming oncolytic viruses with transgenes encoding immunomodulators improves their therapeutic efficacy by enhancing and/or sustaining the innate and adaptive anti-tumoral immune responses. We report here the isolation, selection, and vectorization of a blocking anti-human PDL1 single-domain antibody (sdAb) isolated from PDL1-immunized alpacas. Several formats of this sdAb were vectorized into the vaccinia virus (VV) and evaluated for their programmed cell death protein 1 (PD1)/PD1 ligand (PDL1) blocking activity in the culture medium of tumor cells infected in vitro. In those conditions, VV-encoded homodimeric sdAb generated superior PDL1 blocking activity compared to a benchmark virus encoding full-length avelumab. The sdAb was further used to design simple, secreted, and small tumor necrosis factor superfamily (TNFSF) fusions with the ability to engage their cognate receptors (TNFRSF) only in the presence of PDL1-positive cells. Finally, PDL1-independent alternatives of TNFRSF agonists were also constructed by fusing different variants of surfactant protein-D (SP-D) oligomerization domains with TNFSF ectodomains. An optimal SP-D-CD40L fusion with an SP-D collagen domain reduced by 80% was identified by screening with a transfection/infection method where poxvirus transfer plasmids and vaccinia virus were successively introduced into the same cell. However, once vectorized in VV, this construct had a much lower CD40 agonist activity compared to the SP-D-CD40L construct, which is completely devoid of the collagen domain that was finally selected. This latest result highlights the importance of working with recombinant viruses early in the payload selection process. Altogether, these results bring several complementary solutions to arm oncolytic vectors with powerful immunomodulators to improve their immune-based anti-tumoral activity.

Enhancing the efficacy of vaccinia-based oncolytic virotherapy by inhibiting CXCR2-mediated MDSC trafficking.

Oncolytic virotherapy is an innovative approach for cancer treatment. However, recruitment of myeloid derived suppressor cells (MDSC) into the tumor microenvironment (TME) after oncolysis-mediated local inflammation leads to tumor resistance to the therapy. Using the murine malignant mesothelioma model, we demonstrated that the in-situ vaccinia virotherapy recruited primarily polymorphonuclear MDSC (PMN-MDSC) into the TME where they exhibited strong suppression of cytotoxic T lymphocytes (CTL) in a reactive oxygen species (ROS)-dependent way. Single-cell RNA sequencing analysis confirmed the suppressive profile of PMN-MDSC at the transcriptomic level and identified CXCR2 as a therapeutic target expressed on PMN-MDSC. Abrogating PMN-MDSC trafficking by CXCR2-specific small molecule inhibitor during the vaccinia virotherapy exhibited enhanced antitumor efficacy in three syngeneic cancer models, through increasing CD8+/MDSC ratios in the TME, activating CTL and skewing suppressive TME into an antitumor environment. Our results warrant clinical development of CXCR2 inhibitor in combination with oncolytic virotherapy.

Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.