TianTan Vaccinia Virus-based EBV vaccines targeting both latent and lytic antigens elicits potent immunity against lethal EBV challenge in humanized mice.

AbstractEpstein-Barr virus (EBV) infection has been related to multiple epithelial cancers and lymphomas. Current efforts in developing a prophylactic EBV vaccine have focused on inducing neutralizing antibodies. However, given the lifelong and persistent nature of EBV infection following primary infection, it is rationalized that an ideal vaccine should elicit both humoral and cellular immune responses targeting multiple stages of the EBV lifecycle. This study used a DNA vector and a TianTan vaccinia virus to express key EBV antigens, including BZLF1, EBNA1, EBNA3B, and gH/gL, to generate multi-antigen vaccines. The multi-antigen vaccine expressing all four antigens and the multi-antigen vaccine expressing BZLF1, EBNA1, and EBNA3B showed comparable protection effects and prevented 100% and 80% of humanized mice, respectively, from EBV-induced fatal B cell lymphoma by activating BZLF1, EBNA1, and EBNA3B specific T cell. The vaccine expressing lytic protein BZLF1 elicited stronger T cell responses and conferred superior protection compared to vaccines targeting single latent EBNA1 or EBNA3B. The vaccine solely expressing gH/gL exhibited no T cell protective effects in our humanized mice model. Our study implicates the potential of EBV vaccines that induce potent cellular responses targeting both latent and lytic phases of the EBV life cycle in the prevention of EBV-induced B cell lymphoma.

Role of lamin A/C on dendritic cell function in antiviral immunity.

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.

Polystyrene nanoplastics enhance poxvirus preference for migrasome-mediated transmission.

Since the emergence of a global outbreak of mpox in 2022, understanding the transmission pathways and mechanisms of Orthopoxviruses, including vaccinia virus (VACV), has become paramount. Nanoplastic pollution has become a significant global issue due to its widespread presence in the environment and potential adverse effects on human health. These emerging pollutants pose substantial risks to both living organisms and the environment, raising serious health concerns related to their proliferation. Despite this, the effects of nanoparticles on viral transmission dynamics remain unclear. This study explores how polystyrene nanoparticles (PS-NPs) influence the transmission of VACV through migrasomes. We demonstrate that PS-NPs accelerate the formation of migrasomes early in the infection process, facilitating VACV entry as soon as 15 h post-infection (hpi), compared to the usual onset at 36 hpi. Immunofluorescence and transmission electron microscopy (TEM) reveal significant co-localization of VACV with migrasomes induced by PS-NPs by 15 hpi. This interaction coincides with an increase in lipid droplet size, attributed to higher cholesterol levels influenced by PS-NPs. By 36 hpi, migrasomes exposed to both PS-NPs and VACV exhibit distinct features, such as retraction fibers and larger lipid droplets, emphasizing their critical role in cargo transport during viral infections. These results suggest that PS-NPs may act as modulators of viral transmission dynamics through migrasomes, with potential implications for antiviral strategies and environmental health.

Replication-deficient Sendai virus expressing human norovirus capsid protein elicits robust NoV-specific antibody and T-cell responses in mice.

Human norovirus (HuNoV) is a major global cause of acute gastroenteritis, with vaccine development facing several challenges. Despite years of research, there are currently no licensed vaccines available for controlling HuNoVs. Here, we describe the construction and testing of a replication-deficient Sendai virus (SeV) vector as a potential vaccine candidate against the HuNoV GII.4 genotype. SeV was chosen as the vaccine backbone due to its non-pathogenic nature in humans, its capability for long-term antigen expression in mammalian cells, and its suitability for mucosal administration. By inserting the HuNoV GII.4 capsid gene, VP1, into the SeV genome, we generated a replication-deficient SeV (SeV/dP.VP1) vector. The resultant SeV/dP.VP1 virus were observed to successfully express the inserted NoV VP1 gene upon infection. Inoculating the vaccine into wild-type mice elicited NoV-specific IgG antibodies, along with INF-γ and IL-2-producing T cells, through both intranasal (i.n.) and intramuscular (i.m.) immunization. Furthermore, a significant level of NoV-specific IgA was detected in lung homogenates after i.n. immunization, particularly using a high dose of the viral vector. Additionally, a synergistic effect was observed with heterologous prime-boost regimens using SeV/dP.VP1 and MVA.VP1 vectors, indicating the potential for more robust immune responses when the vaccine design is optimized. Our study demonstrates the potential of a SeV vaccine candidate in eliciting a broad immune response and lays the foundation for further exploration of the SeV vector platform's potential as a HuNoV vaccine. Additionally, the results emphasize the importance of vaccine dosage and administration route, highlighting the need for tailored immunization strategies.

Dose Considerations for Vaccinia Oncolytic Virus Based on Retrospective Reanalysis of Early and Late Clinical Trials.

Over the past decade, oncolytic viruses (OVs) have been developed as a promising treatment alone or in combination in immuno-oncology but have faced challenges in late-stage clinical trials. Our retrospective reanalysis of vaccinia oncolytic virus (VOV) clinical trials indicates that lower doses-rather than the maximum tolerated dose (MTD)-are associated with better tumor response rates. Patients who responded well to lower doses generally had prolonged survival rates in the early phase clinical trial. The association between poor outcomes and an increase in OV-induced neutrophils (OV-N) but not baseline neutrophil counts suggests the need for a comprehensive characterization of OV-N. Although this reanalysis is limited by patient heterogeneity-including differences in cancer type and stage, treatment schedules, and administration routes-it remains informative given the complexities of translational studies in the tumor-bearing mouse models of vaccinia oncolytic viruses. Notably, while OV-N increases with higher viral doses, the immune state shaped by tumor progression likely amplifies this tendency. These findings highlight the importance of OV-N immune modulation as well as dose optimization for the successful clinical development of VOV.

Targeted Inhibition of p21 Promotes the Growth of Breast Cancer Cells and Impairs the Tumor-Killing Effect of the Vaccinia Virus.

PURPOSE: Vaccinia virus is widely used as an oncolytic agent for human cancer therapy, and several versions of vaccinia virus have demonstrated robust antitumor effects in breast cancer. Most vaccinia viruses are modified by thymidine kinase (TK) deletion. The function of the cyclin-dependent kinase inhibitor p21 in breast cancer remains controversial. We explored the impact of p21 gene knockdown (KD) on breast cancer cells and whether p21 KD interferes with the antitumor effect of TK-negative vaccinia virus. METHODS: p21 KD MDA-MB-231 and p21 KD MCF-7 cells were prepared, and cell proliferation and migration rates were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch healing assays. The tumor growth of xenografts originating from p21KD MDA-MB-231 cells and control cells was compared in a mouse model. The colony formation and sphere-forming abilities of p21 KD breast cancer cells were also determined using low-melting agarose and serum-free culture. The tumor-killing effect of the vaccinia virus was determined in breast cancer cells and mouse models using an MTT assay and tumor cell xenografts. RESULTS: p21 KD increased the growth and migration of MDA-MB-231 and MCF-7 cells and promoted the cell growth of MDA-MB-231 cells in mice, while decreasing the colony formation and sphere formation abilities. Expression of TK was reduced in p21 KD MDA-MB-231 cells. Oncolytic effects of both wild-type and TK-deleted vaccinia viruses were attenuated in p21KD MDA-MB-231 cells. The tumor-killing effect of TK-deleted vaccinia virus was also weakened in xenografted mice bearing p21 KD MDA-MB-231 cells. CONCLUSION: Targeted inhibition of p21 accelerates the proliferation and migration of breast cancer cells and impairs the tumor-killing effect of vaccinia virus, suggesting that p21 levels in cancer cells interfere with vaccinia virus oncolytic therapy.

Oncolytic Vaccinia Virus Encoding Aphrocallistes vastus Lectin Suppresses the Proliferation of Gastric Cancer Cells.

Our previous research has demonstrated that the oncolytic vaccinia virus encoding Aphrocallistes vastus lectin (oncoVV-AVL), an oncolytic vaccinia virus engineered to carry the AVL, exhibits potent cytotoxic effects on colorectal and hepatocellular cancer cells. Based on this foundation, we undertook a series of experiments to explore its efficacy on gastric cancer (GC) cells. Our findings revealed that oncoVV-AVL significantly increased reactive oxygen species levels and suppressed the expression of nuclear factor erythroid 2-related factor 2, thereby enhancing viral replication and disrupting the cellular redox balance, ultimately leading to the demise of cancer cells. Additionally, our investigations uncovered that oncoVV-AVL reprogrammed the metabolic microenvironment to favor viral replication, culminating in the lysis of cancer cells. Furthermore, we observed that oncoVV-AVL not only regressed tumor growth but also induced tumor tissue necrosis. These promising results suggest potential new avenues for the therapeutic management of GC.

Identification of mpox M1R and B6R monoclonal and bispecific antibodies that efficiently neutralize authentic mpox virus.

In 2022, the monkeypox virus (mpox virus, MPXV) exhibited global dissemination across six continents, representing a notable challenge owing to the scarcity of targeted antiviral interventions. Passive immunotherapy, such as the use of monoclonal antibodies (mAbs) and bispecific antibodies (bsAbs), has emerged as a promising option for antiviral regimens. Here, we generated several mAbs against M1R and B6R of MPXV, and subsequently characterized the antiviral activity of these antibodies both in vitro and in vivo. Two neutralizing mAbs, M1H11 and M3B2, targeting M1R, and one B6R-specific mAb, B7C9, were identified. They exhibited varying antiviral efficacy against vaccinia virus (VACV) in vitro and in vivo. A cocktail comprising M1H11 and M3B2 demonstrated a superior protective effect in vivo. A bsAb, Bis-M1M3, was engineered by conjugating the fragment crystallizable (Fc) region of the human-mouse chimeric engineered M1H11 with the single-chain fragment variable (scFv) of M3B2. In mice challenged with MPXV, Bis-M1M3 showed a notable protective effects. Analysis of neutralization mechanism showed that these mAbs and Bis-M1M3 exerted virus-neutralizing effects before the virus infects cells. In vivo pharmacokinetic experiments showed that Bis-M1M3 has a long half-life in rhesus macaques. This study provides crucial insights for further research on broad-spectrum antiviral drugs against MPXV and other orthopoxviruses.

Novel recombinant vaccinia virus-vectored vaccine affords complete protection against homologous Borrelia burgdorferi infection in mice.

The rising prevalence of Lyme disease (LD) in North America and Europe has emerged as a pressing public health concern. Despite the availability of veterinary LD vaccines, no vaccine is currently available for human use. Outer surface protein C (OspC) found on the outer membrane of the causative agent, Borrelia burgdorferi, has been identified as a promising target for LD vaccine development due to its sustained expression during mammalian infection. However, the efficacy and immunological mechanisms of LD vaccines solely targeting OspC are not well characterized. In this study, we developed an attenuated Vaccinia virus (VV) vectored vaccine encoding type A OspC (VV-OspC-A). Two doses of the VV-OspC-A vaccine conferred complete protection against homologous B. burgdorferi challenge in mice. Furthermore, the candidate vaccine also prevented the development of carditis and lymph node hyperplasia associated with LD. When investigating the humoral immune response to vaccination, VV-OspC-A was found to induce a robust antibody response predominated by the IgG2a subtype, indicating a Th1-bias. Using a novel quantitative flow cytometry assay, we also determined that elicited antibodies were capable of inducing antibody-dependent cellular phagocytosis in vitro. Finally, we demonstrated that VV-OspC-A vaccination generated a strong antigen-specific CD4+ T-cell response characterized by the secretion of numerous cytokines upon stimulation of splenocytes with OspC peptides. This study suggests a promising avenue for LD vaccine development utilizing viral vectors targeting OspC and provides insights into the immunological mechanisms that confer protection against B. burgdorferi infection.

Discordant performance of mpox serological assays.

BACKGROUND: Since July 23, 2022, global mpox cases reached 92,546, with over 31,000 in the United States. Asymptomatic carriage is a critical mechanism influencing the global dissemination of mpox. Seroprevalence studies are crucial for determining the epidemic's true burden, but uncertainties persist in serologic assay performance and how smallpox vaccination may influence assay interpretation. OBJECTIVES: Our study aimed to assess the performance of several diagnostic assays among mpox-positive, vaccinated, and pre-outbreak negative control samples. This investigation sought to enhance our understanding and management of future mpox outbreaks. STUDY DESIGN: Serum samples from 10 mpox-positive, five vaccinated uninfected, and 137 pre-outbreak controls were obtained for serological testing. The mpox-positive samples were obtained around 100 days post symptom onset, and vaccinated patients were sampled approximately 90 days post-vaccination. Multiple diagnostic assays were employed, including four commercial ELISAs (Abbexa, RayBioTech, FineTest, ProteoGenix) and a multiplex assay (MesoScale Diagnostics (MSD)) measuring five mpox and five smallpox antigens. RESULTS: Three commercial ELISA kits had low specificity (<50 %). The Proteogenix ELISA targeting the E8L antigen had a 94 % sensitivity and 87 % specificity. The E8L antigen on the MSD assay exhibited the greatest distinction between exposure groups, with 98 % sensitivity and 93 % specificity. CONCLUSIONS: None of the assays could distinguish between mpox-positive and vaccinated samples. The MSD assay targeting the MPXV E8L antigen demonstrated the greatest differentiation between mpox-positive and pre-outbreak negative samples. Our findings underscore the imperative to identify sensitive and specific assays to monitor population-level mpox exposure and infection.

A Pseudovirus Nanoparticle Displaying the Vaccinia Virus L1 Protein Elicited High Neutralizing Antibody Titers and Provided Complete Protection to Mice against Mortality Caused by a Vaccinia Virus Challenge.

The recent worldwide incidence of mpox infection and concerns about future emerging variants of mpox viruses highlight the need for the development of a new generation of mpox vaccines. To achieve this goal, we utilized our norovirus S nanoparticle vaccine platform to produce and evaluate two pseudovirus nanoparticles (PVNPs), S-L1 and S-J1. These PVNPs displayed the L1 neutralizing antigen target of the vaccinia virus and a yet-untested J1 antigen of the mpox virus, respectively, with the aim of creating an effective nanoparticle-based mpox vaccine. Each self-assembled PVNP consists of an inner shell resembling the interior layer of the norovirus capsid and multiple L1 or J1 antigens on the surface. The PVNPs improved the antibody responses toward the displayed L1 or J1 antigens in mice, resulting in significantly greater L1/J1-specific IgG and IgA titers than those elicited by the corresponding free L1 or J1 antigens. After immunization with the S-L1 PVNPs, the mouse sera exhibited high neutralizing antibody titers against the vaccinia virus, and the S-L1 PVNPs provided mice with 100% protection against mortality caused by vaccinia virus challenge. In contrast, the S-J1 PVNPs induced low neutralizing antibody titers and conferred mice weak protective immunity. These data confirm that the L1 protein is an excellent vaccine target and that the readily available S-L1 PVNPs are a promising mpox vaccine candidate worthy of further development.

Viral Vector-Based Chlamydia trachomatis Vaccines Encoding CTH522 Induce Distinct Immune Responses in C57BL/6J and HLA Transgenic Mice.

Chlamydia trachomatis remains a major global health problem with increasing infection rates, requiring innovative vaccine solutions. Modified Vaccinia Virus Ankara (MVA) is a well-established, safe and highly immunogenic vaccine vector, making it a promising candidate for C. trachomatis vaccine development. In this study, we evaluated two novel MVA-based recombinant vaccines expressing spCTH522 and CTH522:B7 antigens. Our results show that while both vaccines induced CD4+ T-cell responses in C57BL/6J mice, they failed to generate antigen-specific systemic CD8+ T cells. Only the membrane-anchored CTH522 elicited strong IgG2b and IgG2c antibody responses. In an HLA transgenic mouse model, both recombinant MVAs induced Th1-directed CD4+ T cell and multifunctional CD8+ T cells, while only the CTH522:B7 vaccine generated antibody responses, underscoring the importance of antigen localization. Collectively, our data indicate that distinct antigen formulations can induce different immune responses depending on the mouse strain used. This research contributes to the development of effective vaccines by highlighting the importance of careful antigen design and the selection of appropriate animal models to study specific vaccine-induced immune responses. Future studies should investigate whether these immune responses provide protection in humans and should explore different routes of immunization, including mucosal and systemic immunization.

An Attenuated and Highly Immunogenic Variant of the Vaccinia Virus.

The vaccinia virus (VACV) has been used for prophylactic immunization against smallpox for many decades. However, the VACV-based vaccine had been highly reactogenic. Therefore, after the eradication of smallpox, the World Health Organization in 1980 recommended that vaccination against this infection be discontinued. As a result, there has been a rise in the occurrence of orthopoxvirus infections in humans in recent years, with the most severe being the 2022 monkeypox epidemic that reached all continents. Thus, it is crucial to address the pressing matter of developing safe and highly immunogenic vaccines for new generations to combat orthopoxvirus infections. In a previous study, we created a LAD strain by modifying the LIVP (L) VACV strain, which is used as a first-generation smallpox vaccine in Russia. This modification involved introducing mutations in the A34R gene to enhance extracellular virion production and deleting the A35R gene to counteract the antibody response to the viral infection. In this study, a strain LADA was created with an additional deletion in the DNA of the LAD strain ati gene. This ati gene directs the production of a major non-virion immunogen. The findings indicate that the LADA VACV variant exhibits lower levels of reactogenicity in BALB/c mice during intranasal infection, as compared to the original L strain. Following intradermal immunization with a 105 PFU dose, both the LAD and LADA strains were found to induce a significantly enhanced cellular immune response in mice when compared to the L strain. At the same time, the highest level of virus-specific IFN-γ producing cells for the LAD variant was detected on the 7th day post-immunization (dpi), whereas for LADA, it was observed on 14 dpi. The LAD and LADA strains induced significantly elevated levels of VACV-specific IgG compared to the original L strain, particularly between 28 and 56 dpi. The vaccinated mice were intranasally infected with the cowpox virus at a dose of 460 LD50 to assess the protective immunity at 62 dpi. The LADA virus conferred complete protection to mice, with the LAD strain providing 70% protection and the parent strain L offering protection to only 60% of the animals.

Equivalence of freeze-dried and liquid-frozen formulations of MVA-BN as smallpox and mpox vaccine.

Modified Vaccinia Ankara Bavarian Nordic (MVA-BN) as a smallpox and mpox vaccine has been approved in its liquid-frozen (LF) formulation in the US, Canada, and EU. A freeze-dried (FD) formulation may offer additional benefits, such as a longer shelf life and reduced dependence on cold chain storage and transport. In a phase 2 clinical trial, 651 vaccinia-naïve participants were vaccinated with two doses of MVA-BN LF or FD, 4 weeks apart. The objectives were to compare MVA-BN FD with LF in terms of vaccine-induced immune responses, safety, and reactogenicity. Non-inferiority of the immune response was assessed by the 95% CI of the geometric mean ratios. Both formulations induced robust vaccinia-specific humoral and cellular immune responses. At peak humoral responses (Week 6), geometric means of total antibody titers were 1096 (95% CI 1013, 1186) from the FD group and 877 (95% CI 804, 956) from the LF group, achieving the primary endpoint of non-inferiority of MVA-BN FD compared to MVA-BN LF. At peak cellular responses (Week 2), geometric means of T cell spot forming units were 449 (95% CI 341, 590) from the FD group and 316 (95% CI 234, 427) from the LF group. Both formulations of MVA-BN were well tolerated, with similar unsolicited AEs and solicited systemic reactions in both groups but slightly more local reactions in the FD group. No vaccine-related serious adverse events (SAEs) or vaccine-related AE of special interest were reported. The FD formulation of MVA-BN was shown to be equivalent to MVA-BN LF.

A clinically used anti-human papilloma virus agent (3-hydroxyphthalic anhydride-modified bovine ß-lactoglobulin) has a potential for topical application to prevent sexual transmission of monkeypox virus.

A global outbreak of monkeypox (mpox) caused by the mpox virus (MPXV) has posed a serious threat to public health worldwide, thus calling for the urgent development of antivirals and vaccines to curb its further spread. In this study, we screened 41 anhydride-modified proteins and found that 3-hydroxyphthalic anhydride-modified ß-lactoglobulin (3HP-ß-LG), a clinically used anti-HPV agent, was highly effective in inhibiting infection of vaccinia virus Tiantan strain (VACV-VTT) and MPXV. Mechanistic studies demonstrated that 3HP-ß-LG bound to the virus, not the host cell, by targeting the early stage of virus entry, possibly through the interaction between the amino acids with negatively charges in 3HP-ß-LG and the key amino acids with positive charges in the target region of A29L, a key surface protein of MPXV. A synergistic effect was observed when 3HP-ß-LG was combined with tecovirimat, a small-molecule antiviral drug approved by the United States Food and Drug Administration and the European Medicine Agency for the treatment of smallpox and mpox. Because of its clinically proven safety and stability, 3HP-ß-LG shows promise for further development as a prophylactic agent to prevent the sexual transmission of MPXV.

Exploring computational approaches to design mRNA Vaccine against vaccinia and Mpox viruses.

BACKGROUND: Messenger RNA (mRNA) vaccines emerged as a powerful tool in the fight against infections. Unlike traditional vaccines, this unique type of vaccine elicits robust and persistent innate and humoral immune response with a unique host cell-mediated pathogen gene expression and antigen presentation. METHODS: This offers a novel approach to combat poxviridae infections. From the genome of vaccinia and Mpox viruses, three key genes (E8L, E7R, and H3L) responsible for virus attachment and virulence were selected and employed for designing the candidate mRNA vaccine against vaccinia and Mpox viral infection. Various bioinformatics tools were employed to generate (B cell, CTL, and HTL) epitopes, of which 28 antigenic and immunogenic epitopes were selected and are linked to form the mRNA vaccine construct. Additional components, including a 5' cap, 5' UTR, adjuvant, 3' UTR, and poly(A) tail, were incorporated to enhance stability and effectiveness. Safety measures such as testing for human homology and in silico immune simulations were implemented to avoid autoimmunity and to mimics the immune response of human host to the designed mRNA vaccine, respectively. The mRNA vaccine's binding affinity was evaluated by docking it with TLR-2, TLR-3, TLR-4, and TLR-9 receptors which are subsequently followed by molecular dynamics simulations for the highest binding one to predict the stability of the binding complex. RESULTS: With a 73% population coverage, the mRNA vaccine looks promising, boasting a molecular weight of 198 kDa and a molecular formula of C8901H13609N2431O2611S48 and it is said to be antigenic, nontoxic and nonallergic, making it safe and effective in preventing infections with Mpox and vaccinia viruses, in comparison with other insilico-designed vaccine for vaccinia and Mpox viruses. CONCLUSIONS: However, further validation through in vivo and in vitro techniques is underway to fully assess its potential.

Microchip construction for migration assays: investigating the impact of physical confinement on cell morphology and motility during vaccinia virus infection.

Vaccinia virus (VACV)-induced cell migration is thought to be closely related to the rapid transmission of viral infection in the body. The limited studies are mainly based on scratch assay using traditional cell culture techniques, which inevitably ignores the influences of extracellular microenvironment. Physical confinement, inherently presenting in vivo, has proven to be a critical extern cue in modulating migration behaviors of multiple cells, while its impacts on VACV-induced cell motility remain unclear. Herein, we developed a migration assay microchip featuring confined microchannel array to investigate the effect of physical confinement on infected cell morphology and motility during VACV infection. Results showed that different from the random cell migration observed in traditional scratch assay on planar substrate, VACV-infected cells exhibited accelerated directionally persistent migration under confinement microenvironment. Moreover, single-directed elongated dominant lamella appeared to contrast distinctly with multiple protrusions stretched in random directions under unconfined condition. Additionally, the Golgi complex tended to relocate behind the nucleus confined within the microchannel axis compared to the classical reorientation pattern. These differences in characteristic subcellular architecture and organelle reorientation of migrating cells revealed cell biological mechanisms underlying altered migration behavior. Collectively, our study demonstrates that physical confinement acting as a guidance cue has profound impacts on VACV-induced migration behaviors, which provides new insight into cell migration behavior and viral rapid spread during VACV infection.

An mpox quadrivalent mRNA vaccine protects mice from lethal vaccinia virus challenge.

The outbreak of 2022 monkeypox virus (MPXV) infection in nonendemic regions is a global public health concern. A highly effective and safe MPXV vaccine that is available to the general public is urgently needed to control the mpox pandemic. Here, we developed a multivalent mRNA vaccine candidate, MPXV-1103, which expresses the full-length B6, A35, A29 and M1 proteins with three flexible linkers (G4S1)3 in a single sequence. Compared with the monovalent MPXV mRNA vaccine candidates or the quadrivalent mRNA vaccine from mixtures of the four monovalent MPXV mRNA vaccines, MPXV-1103 elicits a robust humoral response and an MPXV-specific T-cell response and protects mice from lethal vaccinia virus (VACV) challenge, with no live virus detected in the nasal or lungs even at dosages as low as 1 µg. Furthermore, analysis of complete blood counts and photomicrographs of tissue from the main organs of mice vaccinated with MPXV-1103 at doses of 5 µg and 20 µg revealed that two doses of MPXV-1103 did not cause any observable pathological changes in the mice. Collectively, our results suggest that MPXV-1103, with features of high efficacy, safety and a simplified manufacturing process, is a promising vaccine candidate for defending against MPXV infection.

Vaccines against mpox: MVA-BN and LC16m8.

INTRODUCTION: Global outbreaks involving mpox clade IIb began in mid-2022. Today, clade IIb and clade I outbreaks continue. Reliable mpox vaccines can prevent serious mpox disease and death. AREAS COVERED: Globally, two vaccines hold mpox indications, regardless of mpox viral clade: MVA-BN (Bavarian Nordic) and LC16m8 (KM Biologics). This review summarizes the human and pivotal animal data establishing safety and efficacy for MVA-BN and LC16m8, including real-world evidence gathered during mpox outbreaks from 2022 through 2024. EXPERT OPINION: Some regulatory decisions for MVA-BN and LC16m8 followed pathways based on surrogate outcomes, including lethal-challenge studies in nonhuman primates, among other atypical aspects. Nonetheless, MVA-BN and LC16m8 hold unencumbered registration in multiple countries. Effectiveness of MVA-BN as primary preventive vaccination (PPV) in humans against clade IIb mpox is clear from real-world studies; effectiveness of LC16m8 against clade IIb is likely from surrogate endpoints. Effectiveness of MVA-BN and LC16m8 as PPV against more-lethal clade I is likely, based on animal-challenge studies with multiple orthopoxvirus species and other studies. Both vaccines have solid safety records. MVA-BN's replication incompetence favors adoption, whereas LC16m8 has more pediatric data. Additional real-world evidence, in additional geographic settings and special populations (e.g. pregnancy, immune suppression, atopic dermatitis), is needed.

Situation Mpox outbreaks spread globally in 2022, hospitalizing many people. Many recent mpox cases in Africa occur in children. Two vaccines, known as MVA-BN and LC16m8, can help prevent mpox.MVA-BN MVA-BN protects animals from lethal doses of mpox and similar viruses. During outbreaks, MVA-BN lowered the chance of mpox disease by 62% to 85%. In people already exposed to mpox, MVA-BN reduced disease risk by 20%. MVA-BN may help reduce how serious mpox cases are, even if this vaccine does not block infection fully. MVA-BN cannot grow inside the body, making it very safe, even in children. Side effects include pain, redness, swelling, and itching. Some people feel muscle pain, headache, fatigue, nausea, or chills after vaccination. Several million people have received MVA-BN so far, including thousands of people living with HIV.LC16m8 LC16m8 protects animals from lethal doses of mpox and similar viruses. There are not much data about LC16m8 used during mpox outbreaks. LC16m8 contains a weakened virus. Side effects include fever, fatigue, redness, swollen lymph nodes, and itching. Vaccine virus can spread to other parts of the body. Over 90,000 people have received LC16m8 so far. No significant safety signals were found after these doses, including 50,000 children. People who are immunosuppressed, have certain skin diseases, or are pregnant should not be given LC16m8.Mpox vaccine recommendations Health officials recommend mpox vaccine for people at risk, including children.

Vaccinia virus F1L blocks the ribotoxic stress response to subvert ZAKα-dependent NLRP1 inflammasome activation.

Inflammasomes are essential for host defense, recognizing foreign or stress signals to trigger immune responses, including maturation of IL-1 family cytokines and pyroptosis. Here, NLRP1 is emerging as an important sensor of viral infection in barrier tissues. NLRP1 is activated by various stimuli, including viral double-stranded (ds) RNA, ribotoxic stress, and inhibition of dipeptidyl peptidases 8 and 9 (DPP8/9). However, certain viruses, most notably the vaccinia virus, have evolved strategies to subvert inflammasome activation or effector functions. Using the modified vaccinia virus Ankara (MVA) as a model, we investigated how the vaccinia virus inhibits inflammasome activation. We confirmed that the early gene F1L plays a critical role in inhibiting NLRP1 inflammasome activation. Interestingly, it blocks dsRNA and ribotoxic stress-dependent NLRP1 activation without affecting its DPP9-inhibition-mediated activation. Complementation and loss-of-function experiments demonstrated the sufficiency and necessity of F1L in blocking NLRP1 activation. Furthermore, we found that F1L-deficient, but not wild-type MVA, induced ZAKα activation. Indeed, an F1L-deficient virus was found to disrupt protein translation more prominently than an unmodified virus, suggesting that F1L acts in part upstream of ZAKα. These findings underscore the inhibitory role of F1L on NLRP1 inflammasome activation and provide insight into viral evasion of host defenses and the intricate mechanisms of inflammasome activation.