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Lyme disease is the most common tick-borne disease in North America. A vaccine for use in humans is not available. Here, we detail the development of two chimeric vaccine antigens, BAF and Chv2M. BAF elicits Abs that target proteins and protein variants produced by Borreliella species in ticks (OspB and OspA) and mammals (FtlA/B). OspB serves as the backbone structure for the BAF chimeric. Two OspA_221-240 epitope-containing domain (ECD) variants (#A1 and #A15) were inserted into a loop in OspB. The N-terminal region of the FtlA protein was joined to the C-terminus of the chimeric. The second chimeric, Chv2M, consists of L5 (loop 5) and H5 (helix 5) ECDs derived from diverse OspC proteins. Borreliella species produce OspC upon exposure to the bloodmeal and during early infection in mammals. Here, we demonstrate that BAF and Chv2M are potent immunogens that elicit Abs that bind to each component protein (FtlA, FtlB, OspB, and multiple OspA and OspC variants). Anti-BAF and anti-Chv2M Abs kill Borreliella burgdorferi strains through Ab-mediated complement-dependent and complement-independent mechanisms. Eighty percent (32/40) of mice that received a three-dose vaccine regimen were protected from infection with B. burgdorferi B31. Efficacy increased to 90% (18/20) when the amount of Chv2M was increased in the third vaccine dose. Readouts for infection were flaB PCR and seroconversion to VlsE. This study establishes proof of principle for a chimeric immunogen vaccine formulation that elicits Abs to multiple targets on the B. burgdorferi cell surface produced during tick and mammalian stages of the enzootic cycle.IMPORTANCELyme disease is a growing public health threat across parts of the Northern Hemisphere. Regions that can support sustained tick populations are expanding, and the incidence of tick-borne diseases is increasing. In light of the increasing risk of Lyme disease, effective preventive strategies are needed. Most vaccine development efforts have focused on outer surface protein A, a Borreliella burgdorferi protein produced only in ticks. Herein, we describe the development of a novel vaccine formulation consisting of two multivalent chimeric proteins that are immunogenic and elicit antibodies with bactericidal activity that target several cell surface proteins produced by the Lyme disease spirochetes in feeding ticks and mammals. In a broader sense, this study advances efforts to develop custom-designed vaccinogens comprised of epitope-containing domains from multiple proteins.

Toxoplasma gondii is an intracellular parasitic protozoan that poses a significant risk to the fetus carried by a pregnant woman or to immunocompromised individuals. T. gondii tachyzoites duplicate rapidly in host cells during acute infection through endodyogeny. This highly regulated division process is accompanied by complex gene regulation networks. TgAP2XII-9 is a cell cycle-regulated transcription factor, but its specific role in the parasite cell cycle is not fully understood. In this study, we demonstrate that TgAP2XII-9 is identified as a nuclear transcription factor and is dominantly expressed during the S/M phase of the tachyzoite cell cycle. Cleavage Under Targets and Tagmentation (CUT&Tag) results indicate that TgAP2XII-9 targets key genes for the moving junction machinery (RON2, 4, and 8) and daughter cell inner membrane complex (IMC). TgAP2XII-9 deficiency resulted in a significant downregulation of rhoptry proteins and rhoptry neck proteins, leading to a severe defect in the invasion and egress efficiency of tachyzoites. Additionally, the loss of TgAP2XII-9 correlated with a substantial downregulation of multiple IMC and apicoplast proteins, leading to disorders of daughter bud formation and apicoplast inheritance and further contributing to the inability of cell division and intracellular proliferation. Our study reveals that TgAP2XII-9 acts as a critical S/M-phase regulator that orchestrates the endodyogeny and apicoplast division in T. gondii tachyzoites. This study contributes to a broader understanding of the complexity of the parasite's cell cycle and its key regulators.

Importance: The intracellular apicoplast parasite Toxoplasma gondii poses a great threat to the public health. The acute infection of T. gondii tachyzoites relies on efficient invasion by forming a moving junction structure and also fast replication by highly regulated endodyogeny. This study shows that an ApiAP2 transcription factor, TgAP2XII-9, acts as an activator for the S/M-phase gene expression, including genes related to daughter buds and moving junction formation. Loss of TgAP2XII-9 results in significant growth defects and disorders in endodyogeny and apicoplast inheritance of the parasites. Our results provide valuable insights into the transcriptional regulation of the parasite cell cycle and invading machinery in T. gondii.

Fungal pathogens are underappreciated causes of significant morbidity and mortality worldwide. In previous studies, we determined that a heat-killed, Cryptococcus neoformans fbp1-deficient strain (HK-fbp1) is a potent vaccine candidate. We determined that vaccination with HK-fbp1 confers protective immunity against lethal Cryptococcosis in an interferon γ (IFNγ)-dependent manner. In this study, we set out to uncover cellular sources and relevant targets of the protective effects of IFNγ in response to the HK-fbp1 vaccine. We found that early IFNγ production peaks at day 3 and that monocytes and neutrophils are important sources of this cytokine after vaccination. Neutralization of IFNγ at day 3 results in impaired CCR2⁺ monocyte recruitment and reduced differentiation into monocyte-derived dendritic cells (Mo-DC). In turn, depletion of CCR2⁺ cells prior to immunization results in impaired activation of IFNγ-producing CD4 and CD8 T cells. Thus, monocytes are important targets of innate IFNγ and help promote further IFNγ production by lymphocytes. We employed monocyte-fate mapper and conditional STAT1 knockout mice to uncover that STAT1 activation in CD11c⁺ cells, including alveolar macrophages, Mo-DCs, and monocyte-derived macrophages (Mo-Mac) is essential for HK-fbp1 vaccine-induced protection. Altogether, our aggregate findings suggest critical roles for innate cells as orchestrators of vaccine-induced protection against Cryptococcus infection.IMPORTANCEThe number of patients susceptible to invasive fungal infections across the world continues to rise at an alarming pace yet current antifungal drugs are often inadequate. Immune-based interventions and novel antifungal vaccines hold the promise of significantly improving patient outcomes. In previous studies, we identified a Cryptococcus neoformans mutant strain (Fbp1-deficient) as a potent, heat-inactivated vaccine candidate capable of inducing homologous and heterologous antifungal protection. In this study, we used a combination of methods together with a cohort of conditional knockout mouse strains to interrogate the roles of innate cells in the orchestration of vaccine-induced antifungal protection. We uncovered novel roles for neutrophils and monocytes as coordinators of a STAT1-dependent cascade of responses that mediate vaccine-induced protection against invasive cryptococcosis. This new knowledge will help guide the future development of much-needed antifungal vaccines.

When a virus crosses from one host species to another, the consequences can be devastating. However, animal models to empirically evaluate cross-species transmission can fail to recapitulate natural transmission routes, physiologically relevant doses of pathogens, and population structures of naturally circulating viruses. Here, we present a new model of cross-species transmission where deer mice (Peromyscus maniculatus) are exposed to the natural virome of pet store mice (Mus musculus). Using RNA sequencing, we tracked viral transmission via fecal-oral routes and found the evidence of transmission of murine astroviruses, coronaviruses, and picornaviruses. Deep sequencing of murine kobuvirus revealed tight bottlenecks during transmission and purifying selection that leaves limited diversity present after transmission from Mus to Peromyscus. This work provides a structure for studying viral bottlenecks across species while keeping natural variation of viral populations intact and a high resolution look at within-host dynamics that occur during the initial stages of cross-species viral transmission.IMPORTANCEViral spillover events can have devastating public health consequences. Tracking cross-species transmission in real-time and evaluating viral evolution during the initial spillover event are useful for understanding how viruses adapt to new hosts. Using our new animal model and next generation sequencing, we develop a framework for understanding intrahost viral evolution and bottleneck events, which are very difficult to study in natural transmission settings.

The ongoing outbreak of the Goose/Guangdong/1/1996 (Gs/Gd) H5 lineage of highly pathogenic avian influenza (HPAI) viruses has caused higher mortality than all other HPAI outbreaks taken together. It is unique in its spillover and adaptation to wild waterfowl, which has facilitated its spread worldwide to many other species. However, avian influenza virus (AIV) surveillance, which historically aims to protect the poultry sector, is inadequate to document the spread and impact of Gs/Gd H5 virus in wild birds and other wildlife in most countries. A positive exception is Canada's AIV surveillance in wild birds, applied in a recent study (J. A. Giacinti, A. V. Signore, M. E. B. Jones, L. Bourque, et al., mBio 15:e03203-23, 2024, https://doi.org/10.1128/mbio.03203-23), which aims to protect wildlife, domestic animals, and human health according to the One Health approach. It is recommended to follow this approach in other countries to fill knowledge gaps in the epidemiology of Gs/Gd H5 virus in wild birds and other wildlife and to help control and, above all, prevent future HPAI outbreaks.

The number of Vibrio-related infections in humans, e.g., by Vibrio vulnificus, has increased along the coasts of the Baltic Sea. Due to climate change, vibriosis risk is expected to increase. It is, therefore, pertinent to design a strategy for mitigation of the vibriosis threat in the Baltic Sea area, but a prerequisite is to identify the environmental conditions promoting the occurrence of pathogenic Vibrio spp., like V. vulnificus. To address this, we sampled three coastal Baltic sites in Finland, Germany, and Denmark with salinities between 6 and 21 from May to October 2022. The absolute and relative abundances of Vibrio spp. and V. vulnificus in water were compared to environmental conditions, including the presence of the eelgrass Zostera marina, which has been suggested to reduce pathogenic Vibrio species abundance. In the water column, V. vulnificus only occurred at the German station between July and August at salinity 8.1-11.2. Temperature and phosphate (PO₄^3-) were identified as the most influencing factors for Vibrio spp. and V. vulnificus. The accumulation of Vibrio spp. in the sediment and the co-occurrence with sediment bacteria in the water column indicate that sediment resuspension contributed to V. vulnificus abundance. Interestingly, V. vulnificus co-occurred with specific cyanobacteria taxa, as well as specific bacteria associated with cyanobacteria. Although we found no reduction in Vibrio spp. or V. vulnificus associated with eelgrass beds, our study underscores the importance of extended heatwaves and sediment resuspension, which may elevate the availability of PO₄^3-, for Vibrio species levels at intermediate salinities in the Baltic Sea.

Importance: Elevated sea surface temperatures are increasing the prevalence of pathogenic Vibrio at higher latitudes. The recent increase in Vibrio-related wound infections and deaths along the Baltic coasts is, therefore, of serious health concern. We used culture-independent data generated from three Baltic coastal sites in Denmark, Germany, and Finland from May to October (2022), with a special focus on Vibrio vulnificus, and combined it with environmental data. Our temporal model shows that temperature, combined with sediment resuspension, drives the prevalence of V. vulnificus at intermediate salinities in the coastal Baltic Sea.

RNA viruses have evolved sophisticated strategies to exploit the limited encoded information within their typically compact genomes. One of them, named transcriptional slippage (TS), is characterized by the appearance of indels in nascent viral RNAs, leading to changes in the open reading frame (ORF). Although members of unrelated viral families express key proteins via TS, the available information about this phenomenon is still limited. In potyvirids (members of the Potyviridae family), TS has been defined by the insertion of an additional A at A_n motifs (n ≥ 6) in newly synthesized transcripts at a low frequency, modulated by nucleotides flanking the A-rich motif. Here, by using diverse experimental approaches and a collection of plant/virus combinations, we discover cases not following this definition. In summary, we observe (i) a high rate of single-nucleotide deletions at slippage motifs, (ii) overlapping ORFs acceded by slippage at an U₈ stretch, and (iii) changes in slippage rates induced by factors not related to cognate viruses. Moreover, a survey of whole-genome sequences from potyvirids shows a widespread occurrence of species-specific A_n/U_n (n ≥ 6) motifs. Even though many of them, but not all, lead to the production of truncated proteins rather than access to overlapping ORFs, these results suggest that slippage motifs appear more frequently than expected and play relevant roles during virus evolution. Considering the potential of this phenomenon to expand the viral proteome by acceding to overlapping ORFs and/or producing truncated proteins, a re-evaluation of TS significance during infections of RNA viruses is required.IMPORTANCETranscriptional slippage (TS) is used by RNA viruses as another strategy to maximize the coding information in their genomes. This phenomenon is based on a peculiar feature of viral replicases: they may produce indels in a small fraction of newly synthesized viral RNAs when transcribing certain motifs and then produce alternative proteins due to a change of the reading frame or truncated products by premature termination. Here, using plant-infecting RNA viruses as models, we discover cases expanding on previously established features of plant virus TS, prompting us to reconsider and redefine this expression strategy. An interesting conclusion from our study is that TS might be more relevant during RNA virus evolution and infection processes than previously assumed.

Microbiota-mediated impacts on host physiology and behavior have been widely reported in honey bees (Apis mellifera). However, most of these studies are conducted in artificial lab settings and fail to take into account, or make incorrect assumptions about, the complex physical and social structures inherent to natural hive conditions. A new study by Liberti et al. (J. Liberti, E. T. Frank, T. Kay, L. Kesner, et al., mBio 15:e01034-24, 2024, https://doi.org/10.1128/mbio.01034-24) identifies one such overlooked aspect-the behavioral maturation from nurses to foragers-that can be a serious confounding factor in bee microbiota experiments. Using cuticular hydrocarbon profiling to discern between the two maturation states, they find that multiple physiological and behavioral differences between age-matched lab bees could potentially be explained by their maturation state instead of the intended treatment conditions, such as microbial inoculation. This study serves as a stark wake-up call on the necessity of careful replication and cross-disciplinary knowledge transfer (e.g., between animal specialists and microbiologists) in order to truly understand complex host-microbe systems.

Extracellular vesicles (EV), key players in cell-to-cell communication, may contribute to disease propagation in several neurodegenerative diseases, including Alzheimer's disease (AD), by favoring the dissemination of neurotoxic proteins within the brain. Interestingly, growing evidence supports the role of herpes simplex virus type 1 (HSV-1) infection in the pathogenesis of AD. Here, we investigated whether HSV-1 infection could promote the spread of phosphorylated tau (ptau) among neurons via EV. We analyzed the ptau species that were secreted via EV following HSV-1 infection in neuroblastoma cells and primary neurons, focusing particularly on T205, T181, and T217, the phosphorylation sites mainly associated with AD. Moreover, by overexpressing human tau tagged with GFP (htau^GFP), we found that recipient tau knockout (KO) neurons uptook EV that are loaded with HSV-1-induced phtau^GFP. Finally, we exploited an in vivo model of acute infection and assessed that cerebral HSV-1 infection promotes the release of ptau via EV in the brain of infected mice. Overall, our data suggest that, following HSV-1 infection, EV play a role in tau spreading within the brain, thus contributing to neurodegeneration.IMPORTANCEHerpes simplex virus type 1 (HSV-1) infection that reaches the brain has been repeatedly linked with the appearance of the pathognomonic markers of Alzheimer's disease (AD), including accumulation of amyloid beta and hyperphosphorylated tau proteins, and cognitive deficits. AD is a multifactorial neurodegenerative disease representing the most common form of dementia in the elderly, and no cure is currently available, thus prompting additional investigation on potential risk factors and pathological mechanisms. Here, we demonstrate that the virus exploits the extracellular vesicles (EV) to disseminate phosphorylated tau (ptau) among brain cells. Importantly, we provide evidence that the HSV-1-induced EV-bearing ptau can be undertaken by recipient neurons, thus likely contributing to misfolding and aggregation of native tau, as reported for other AD models. Hence, our data highlight a novel mechanism exploited by HSV-1 to propagate tau-related damage in the brain.

We previously demonstrated that the lungs of deceased COVID-19 patients were filled with a clear hydrogel consisting of hyaluronan (HA). In this translational study, we investigated the role of HA at all stages of COVID-19 disease to map the consequences of elevated HA on morbidity and identify the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced HA production. A reduced alveolar surface area was observed in the lungs of deceased COVID-19 patients compared to healthy controls, as visualized by a 3D rendering of lung morphology using light-sheet fluorescence microscopy. We confirmed the presence of HA in lung biopsies and found large quantities of proinflammatory fragmented HA. The association of systemic HA in blood plasma and disease severity was assessed in patients with mild (WHO Clinical Progression Scale, WHO-CPS, 1-5) and severe COVID-19 (WHO-CPS, 6-9) during the acute and convalescent phases and related to lung function. We found that systemic levels of HA were high during acute COVID-19 disease, remained elevated during convalescence, and were associated with a reduced diffusion capacity. In vitro 3D-lung models, differentiated from primary human bronchial epithelial cells, were used to study the effects of SARS-CoV-2 infection on HA metabolism, and transcriptomic analyses revealed a dysregulation of HA synthases and hyaluronidases, both contributing to increased HA in apical secretions. Furthermore, corticosteroid treatment reduced the inflammation and downregulated HA synthases. Our findings demonstrate that HA plays a role in COVID-19 morbidity and that sustained elevated HA concentrations may contribute to long-term respiratory impairment.IMPORTANCEThis study provides insights into the role of hyaluronan (HA) in the severity and long-term impact of COVID-19 on lung function. Through extensive morphological examination of lung tissues and a multicenter study, we identified that HA levels are significantly elevated in COVID-19 patients, correlating with a reduced lung diffusion capacity during convalescence. Using a 3D-lung model, we further uncovered how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 infection causes a dysregulated HA metabolism, leading to increased HA production. Our findings provide valuable insights into the pathogenesis of SARS-CoV-2 and suggest that targeting HA metabolism could offer new therapeutic avenues for managing COVID-19, particularly to prevent long-term lung impairment. Additionally, HA holds potential as a biomarker for predicting disease severity, which could guide personalized treatment strategies.