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Faced with the global monkeypox outbreak, current vaccine development predominantly focuses on the mRNA platform despite its limitations in stability and long-term efficacy. Here, we engineered a recombinant vesicular stomatitis virus (rVSV)-vectored cocktail vaccine encoding four conserved monkeypox virus (MPXV) antigens (A35R, A29L, M1R, and B6R; >94% clade homology), leveraging the thermostable properties of the VSV platform validated for 4°C storage in Ebola vaccines. In BALB/c mice, this multi-antigen vaccine elicited a rapid humoral response with specific IgG detectable by day 7, effectively neutralized the virus, and induced a robust Th1/Th2 balanced cytokine response. Immunization conferred 100% survival against lethal vaccinia virus WR strain challenge, with undetectable viral loads in the lungs and serum, and sustained efficacy against secondary infection at 60 days. Histopathology confirmed minimal lung damage in vaccinated mice. Crucially, upon the successive challenges, mutations in key poxvirus immune evasion genes (E3L and B7R) emerged in the single-component vaccine groups but were absent in the cocktail vaccine group. This finding provides direct evidence that the cocktail strategy suppresses viral escape, underscoring a fundamental advantage over single-antigen approaches. Our findings demonstrate the rVSV-based cocktail vaccines as a potent, scalable, and thermostable candidate for global MPXV control, particularly in regions with limited settings.

Importance: The global emergence of the monkeypox virus (MPXV) underscores the urgent need for effective and accessible vaccines. We developed a recombinant vesicular stomatitis virus (rVSV)-vectored cocktail vaccine expressing four conserved MPXV antigens. This multivalent vaccine elicits rapid and potent immune responses in mice, conferring complete protection against lethal vaccinia virus challenge. A critical finding is that while successive viral challenges selected for mutations in key immune evasion proteins in single-antigen vaccine groups, these mutations were absent in the cocktail-vaccinated group. This suggests that the cocktail strategy may suppress viral genetic drift, potentially limiting escape pathways. Combined with the thermostability of the VSV platform, our vaccine presents a promising and scalable candidate for combating monkeypox.

Heteroresistance is a transient resistance phenotype characterized by the presence of small subpopulations of bacterial cells with elevated antibiotic resistance within a susceptible main population. In gram-negative pathogens, heteroresistance is frequently caused by tandem amplification of genes encoding resistance proteins with low activity toward the antibiotic, a process commonly mediated by homologous recombination between flanking repeated sequences. However, the specific roles of individual recombination proteins in this mechanism remain largely undefined. In this study, we systematically evaluated the contribution of 19 recombination-associated genes to tandem amplification-driven heteroresistance in Escherichia coli. A clinical plasmid causing tobramycin heteroresistance by tandem amplification of the aac(3)-IId gene was conjugated into recombination gene-deficient mutants and the wild-type parental strain. While heteroresistance was observed with all mutants, the frequency of resistant subpopulations was decreased in recA and recB mutants, and a shift in resistance mechanism toward increased plasmid copy number and resistance mutations was observed. Partially reduced frequencies of tandem amplifications and a shift toward other heteroresistance mechanisms were also observed with recC, recJ, ruvA, and ruvC mutants, whereas other deletions of recombination genes had no or little impact on tandem amplifications. These findings identify RecABC as a key pathway in heteroresistance via tandem amplification, but even when these genes are deleted, resistant subpopulations can still be generated by other mechanisms.IMPORTANCEHeteroresistance poses a threat to efficient antibiotic treatment, as the rare resistant subpopulations often go undetected by standard laboratory tests. In Escherichia coli, heteroresistance often arises by tandem gene amplification of a resistance gene with low activity toward the specific antibiotic. This amplification is thought to be mediated by homologous recombination between repeat sequences. However, the specific roles of individual recombination proteins in this process are unclear. Here, we systematically determined the specific roles of individual recombination proteins in this process by the individual deletion of 19 recombination-associated genes. The RecABC pathway was identified as a major contributor to amplification-driven heteroresistance, but even when this pathway was disrupted, resistant subpopulations still emerged through alternative mechanisms, revealing the remarkable adaptability of bacterial populations under antibiotic stress. These findings advance our understanding of the molecular flexibility underlying heteroresistance and highlight that strategies aimed at preventing gene amplification to reduce heteroresistance are unlikely to succeed.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remains a major public health threat, particularly in vulnerable populations. SARS-CoV-2 spike proteins interact with the human angiotensin-converting enzyme 2 (ACE2) receptor, together with accessory molecules that facilitate viral entry, through its spike receptor-binding domain (RBD). Although ACE2 is the primary receptor required for viral replication, its expression patterns do not fully correlate with viral distribution or tissue pathology. Moreover, SARS-CoV-2 has been shown to infect cells and tissues lacking detectable ACE2 expression. Viral entry via ACE2-independent pathways may also confer resistance to some monoclonal antibodies (Abs) targeting the spike RBD that block ACE2-mediated binding. These observations highlight the potential significance of ACE2-independent entry factors in SARS-CoV-2 infection, particularly in vaccinated individuals with Abs directed against ACE2-dependent viral entry. In this review, we discuss the emerging roles of ACE2-independent entry factors in SARS-CoV-2 infection and the immune responses. These factors include CD147, AXL, CD169/Siglec-1, CD209L, CD209, CLEC4G, ASGR1, LDLRAD3, TMEM30A, TMEM106B, transferrin receptor 1, GPR78, integrin α5β1, KREMEN1, LFA-1, and CD4. While ACE2 remains central to viral replication, ACE2-independent entry appears sufficient to elicit immune responses. Therefore, future investigations are warranted to elucidate the roles of ACE2-independent mechanisms in immune-mediated pathology and viral evolution, independent of immune pressure targeting ACE2-mediated entry in previously infected or vaccinated individuals.

Human papillomavirus 16 (HPV16) is a causative agent of oropharyngeal, cervical, and anogenital cancers. The viral E2 protein is essential for viral genome replication, transcriptional regulation, episome maintenance, and activation of the host DNA damage response. Despite its central role, the full network of HPV16 E2 interactions with host proteins remains incompletely defined, particularly under differentiating conditions, which support the complete viral life cycle. In this study, we used TurboID-based proximity labeling to characterize the interactome of HPV16 E2 and its known host partner protein TOPBP1 in both undifferentiated monolayer and differentiating keratinocytes. We generated stable keratinocyte lines expressing doxycycline-inducible TurboID-tagged HPV16 E2 and confirmed that the tagged protein retained transcriptional, replicative, and DNA damage-inducing functions. Mass spectrometry analysis of streptavidin-enriched proteins identified both known and novel E2-associated host factors, including chromatin regulators, DNA repair proteins, and nucleolar components. Comparative analysis revealed substantial overlap between E2 and TOPBP1 interactomes, and in situ validation by proximity ligation assay identified nucleolin (NCL) as a differentiation-dependent factor whose interaction with E2 is stabilized by TOPBP1. Functional studies demonstrated that NCL is required for episomal genome maintenance, highlighting a cooperative E2-TOPBP1-NCL axis critical for viral genome stability during differentiation. These findings provide a comprehensive view of the E2-associated protein landscape in stratified epithelial cells and reveal a mechanistic pathway through which HPV16 co-opts host factors to support genome maintenance, productive replication, and persistence.

Importance: Human papillomaviruses (HPVs) establish persistent infections in stratified epithelia and rely on host DNA damage and repair factors to support their replication. The E2 protein is central to viral genome replication and maintenance and depends heavily on its interaction with the host factor TOPBP1 for these functions. Here, we define the E2 and TOPBP1 interactomes in differentiating keratinocytes and identify nucleolin (NCL) as a critical differentiation- and TOPBP1-dependent E2 partner required for episomal genome stability. These findings expand the understanding of how HPV16 coordinates viral replication with host chromatin and DNA repair networks, uncovering a cooperative E2-TOPBP1-NCL axis that may represent a new target for antiviral intervention.

Bacteriophages exhibit strict host specificity, primarily determined by adsorption to bacterial surface receptors. However, the molecular basis underlying this specificity remains incompletely understood. Here, we investigate the interaction between outer membrane protein C (OmpC) of Escherichia coli O157 and gp38, the receptor-binding protein located at the tip of the long tail fibers of phage PP01. We determined the crystal structure of the receptor-binding domain (RBD) of gp38_PP01 at 2.1 Å resolution. The structure reveals a lattice of poly-glycine type II helices with protruding receptor recognition loops, resembling that of gp38 from Salmonella phage S16. To identify interaction sites, we performed site-specific photo-crosslinking using p-benzoyl-L-phenylalanine (pBPA), followed by liquid chromatography-tandem mass spectrometry. Two critical contacts were identified: Gly208 in loop-D of gp38_PP01 crosslinked to Ser225 and Pro226 in extracellular loop-5 of OmpC_O157, and Tyr230 in loop-E of gp38_PP01 to the Val304-Arg308 region in loop-7 of OmpC_O157. A structural model of the gp38_PP01-OmpC_O157 complex was constructed using distance-constrained prediction and validated by targeted mutagenesis. Our findings demonstrate that PP01 phage specificity is governed by loop-E of gp38_PP01 engaging a cleft formed by loops -5 and -7 of OmpC_O157. These structural and functional insights enhance our understanding of phage-host recognition and may inform the rational design of engineered bacteriophages with altered host ranges.IMPORTANCEBacteriophages must precisely recognize and bind to specific molecules on the surface of their bacterial hosts to initiate infection, but the details of these interactions are often unclear. In this study, we examined how phage PP01 targets Escherichia coli O157. Using structural analysis of the phage tail fiber and a technique to capture contact points between the phage and a bacterial surface protein, we mapped the molecular basis of host recognition. We also developed a simple test system using a modified phage to identify which parts of the tail fiber are essential for binding. These methods can be broadly applied to other phages to better understand how they select their hosts. This work provides valuable insights and tools that could aid the design of phages with customized host specificity for therapeutic or biotechnological applications.

The bioRxiv and medRxiv preprint servers brought preprinting to the life sciences and played a critical role in disseminating COVID research during the pandemic. Here, I reflect on the birth of bioRxiv and medRxiv and the crucial role so many members of the community played, our experience during the pandemic, and the launch of the new non-profit organization set up to oversee the servers. The pandemic was a stress test for bioRxiv and medRxiv that demonstrated their value and robustness. Under the umbrella of openRxiv, they are now poised to become long-term infrastructure underpinning a new publishing ecosystem.

Toxoplasma gondii exploits host phagocytes to disseminate systemically and establish infection in the central nervous system (CNS). Yet, the mechanisms governing the interaction between parasitized phagocytes and the brain endothelium remain elusive. Here, we show that T. gondii infection, but not parasite lysates, robustly induces transcriptional and secretory upregulation of the chemokine C-C motif ligand 5 (CCL5/RANTES) in primary brain endothelial cells and dendritic cells (DCs). This response was triggered by the parasite effector GRA15 through NF-κB signaling, while the effector TEEGR counteracted CCL5 induction in an MYR-translocon-dependent manner. In response to recombinant CCL5, infected DCs displayed increased hypermotility, chemotaxis toward CCL5 gradients, and enhanced transmigration across polarized endothelial monolayers. Intraperitoneally infected mice rapidly upregulated CCL5 in the blood and Ccl5 expression in the cerebral microvasculature, thereby increasing the adhesion of parasitized DCs and cerebral parasite loads. Pretreatment of mice with recombinant CCL5 dramatically elevated the sequestration of infected DCs, while treatment with the selective chemokine receptor 5 (CCR5) antagonist Maraviroc reverted sequestration. Together, these findings reveal that T. gondii co-opts the host CCL5/CCR5 axis via GRA15-mediated signaling to promote leukocyte-dependent dissemination and early colonization of the CNS.

Importance: The intracellular parasite Toxoplasma gondii invades immune cells to spread through the circulatory system, eventually reaching the brains of humans and animals. It is not well understood how parasitized immune cells interact with blood vessel walls, a process that ultimately helps Toxoplasma colonize the brain tissue. We found that when Toxoplasma infects the cells lining the blood vessels (endothelium), these produce C-C motif chemokine ligand 5 (CCL5), a potent signaling and attractant molecule. CCL5 production was triggered by a parasite-derived secreted protein, GRA15. CCL5 activated and attracted infected immune cells. In mice, the levels of CCL5 increased quickly in the brain microvasculature after infection, helping the infected immune cells adhere to brain vessels. When the effect of CCL5 was pharmacologically blocked, fewer infected cells sequestered in the brain vessels, lowering the parasite loads. These findings reveal a mechanism through which Toxoplasma manipulates host cells to produce factors that facilitate its colonization of the brain.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. Inbred mouse models of TB do not reflect the pathological states and heterogeneity seen in human TB disease. Thus, we recently established a model of TB in diversity outbred (DO) mice, which displayed heterogeneity in inflammatory and protective responses following aerosol Mtb infection. In the current study, we show that DO mice vaccinated with M. bovis Bacille Calmette Guerin (BCG) are significantly protected upon Mtb HN878 infection, and protection is associated with the induction of transcriptional pathways involved in transforming growth factor B (TGF-β) and Toll-like receptor (TLR)-10 signaling. Targeting lung innate pathways in BCG-vaccinated DO mice using adjuvants also further improved protection upon Mtb infection by inducing genes associated with cellular responses to external stimuli, B-cell responses, as well as IL-17-producing CD4⁺ T-cell responses. Depletion of CD4⁺ T cells resulted in loss of vaccine-induced protection in DO BCG-vaccinated and adjuvant-treated Mtb-infected mice. Together, our new results show that innate targeting of the lung by activating TLR pathways could induce protective pathways in T cells that significantly improve upon the protection induced by BCG vaccination. Additionally, the DO mouse model of vaccination and Mtb infection can provide novel insights into immune pathways that are important for improving vaccine-induced protection against TB.

Importance: Bacille Calmette Guerin (BCG) vaccination in genetically diverse outbred (DO) mice provides significant protection against Mycobacterium tuberculosis (Mtb) challenge. This protection induced pathways associated with transforming growth factor B (TGF-β) receptor complex, genes associated with lung repair, and Toll-like receptor (TLR)-10 pathway. The enhanced protection observed in BCG-vaccinated mice correlated with improved formation of B-cell follicles and IL-17-producing CD4⁺ T-cell responses. CD4⁺ T-cell responses mediated the enhanced protection in the lungs of DO mice vaccinated with BCG + adjuvant, as depletion of CD4⁺ T-cell responses reversed the enhanced protection. The DO mouse model of tuberculosis vaccination is a highly relevant model to probe mechanisms of vaccine-induced protection and provide novel insights into lung pathways that mediate protection. The study also found that genes associated with lung repair, including TGF-β receptor complex pathways, were induced in BCG-vaccinated Mtb-infected DO mouse lungs. The study suggests that the activation of lung innate pathways in BCG vaccination through the use of mucosal Amph CpG delivery, CD40L activation, and IL-10 neutralization could significantly enhance protection upon Mtb challenge.

Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.

Importance: Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.