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AdCLD-CoV19-1, a chimeric adenovirus-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, was previously reported to elicit robust antibody responses in mice and non-human primates after a single dose. In this study, we conducted a systems serology analysis to investigate changes in humoral immune responses induced by varying doses of the AdCLD-CoV19-1 vaccine in a phase I clinical trial. Serum samples from participants receiving either a low or a high dose of the vaccine were analyzed for antibody features against prototype SARS-CoV-2 spike (S) domains (full-length S, S1, S2, and receptor binding domain), as well as Fc receptor binding and effector functions. While both low- and high-dose vaccines induced robust humoral immune responses following vaccination, the quality of antibody features differed between the dose groups. Notably, while no significant difference was observed between the groups in the induction of most S1-specific antibody features, the high-dose group exhibited higher levels of antibodies and a stronger Fc receptor binding response specific to the S2 antigen. Moreover, univariate and multivariate analyses revealed that the high-dose vaccine induced higher levels of S2-specific antibodies binding to FcγR2A and FcγR3B, closely associated with antibody-dependent neutrophil phagocytosis (ADNP). Further analysis using the Omicron BA.2 variant demonstrated that the high-dose group maintained significantly higher levels of IgG and FcγR3B binding to the S2 antigen and exhibited a significantly higher ADNP response for the S2 antigen compared with the low-dose group. These findings underscore the importance of considering diverse humoral immune responses when evaluating vaccine efficacy and provide insights for optimizing adenovirus vector-based SARS-CoV-2 vaccine doses.IMPORTANCEOptimization of vaccine dose is crucial for eliciting effective immune responses. In addition to neutralizing antibodies, non-neutralizing antibodies that mediate Fc-dependent effector functions play a key role in protection against various infectious diseases, including coronavirus disease 2019. Using a systems serology approach, we demonstrated significant dose-dependent differences in the humoral immune responses induced by the AdCLD-CoV19-1 chimeric adenovirus-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine, particularly against the SARS-CoV-2 spike 2 domain. These findings highlight the importance of assessing not only neutralizing antibody titers but also the quality and functionality of antibody responses when evaluating vaccine efficacy.

Bacterial pathogens remain poorly characterized in bats, especially in North America. We describe novel (and in some cases panmictic) hemoplasmas (10.1% positivity) and bartonellae (25.6% positivity) across three colonies of Mexican free-tailed bats (Tadarida brasiliensis), a partially migratory species that can seasonally travel hundreds of kilometers. Molecular analyses identified three novel Candidatus hemoplasma species most similar to another novel Candidatus species in Neotropical molossid bats. We also detected novel hemoplasmas in sympatric cave myotis (Myotis velifer) and pallid bats (Antrozous pallidus), with sequences in the latter 96.5% related to Candidatus Mycoplasma haematohominis. We identified nine Bartonella genogroups, including those in cave myotis with 96.1% similarity to Candidatus Bartonella mayotimonensis. We also detected Bartonella rochalimae in migratory Mexican free-tailed bats, representing the first report of this human pathogen in the Chiroptera. Monthly sampling of migratory Mexican free-tailed bats during their North American occupancy period also revealed significant seasonality in infection for both bacterial pathogens, with prevalence increasing following spring migration, peaking in the maternity season, and declining into fall migration. The substantial diversity and seasonality of hemoplasmas and bartonellae observed here suggest that additional longitudinal, genomic, and immunological studies in bats are warranted to inform One Health approaches.

Importance: Bats have been intensively sampled for viruses but remain mostly understudied for bacterial pathogens. However, bacterial pathogens can have significant impacts on both human health and bat morbidity and even mortality. Hemoplasmas and bartonellae are facultative intracellular bacteria of special interest in bats, given their high prevalence and substantial genetic diversity. Surveys have also supported plausible zoonotic transmission of these bacteria from bats to humans, including Candidatus Mycoplasma haematohominis and Candidatus Bartonella mayotimonensis. Greater characterization of these bacteria across global bat diversity (over 1,480 species) is therefore warranted to inform infection risks for both bats and humans, although little surveillance has thus far been conducted in North American bats. We here describe novel (and in some cases panmictic) hemoplasmas and bartonellae across three colonies of Mexican free-tailed bats and sympatric bat species. We find high genetic diversity and seasonality of these pathogens, including lineages closely related to human pathogens, such as Bartonella rochalimae.

The re-emerging human adenovirus (HAdV) types 3, 7, 14, and 55 of species B have caused severe or even fatal acute respiratory disease. Therefore, the development of multivalent vaccines against HAdV types 3, 7, 14, and 55 remains an important goal. In our previous study, we identified a cross-neutralizing epitope that induced broadly reactive monoclonal neutralizing antibodies against the knob proteins of HAdV types 7, 11, 14, and 55. To study the immunogenicity of HAdV species B knob proteins, we evaluated humoral immune responses to the knob proteins in vivo and in vitro. We found that the knob proteins elicited robust binding and neutralizing antibody responses after three immunizations of mice. In addition, mouse antisera raised against the knob proteins exhibited cross-neutralizing activity against original species B members. Furthermore, immunization with a mix of HAdV-3, 7, and 55 knob proteins protected Chinese tree shrews against an experimental HAdV challenge. Our results provide insight into the immunogenicity of HAdV species B knob proteins.IMPORTANCEHuman adenovirus (HAdV) species B are common pathogens causing severe pneumonia in children, and there is currently no vaccine available. Because there are many HAdV species B types, developing broad-spectrum vaccines against HAdV species B is an important research goal. Our study revealed that immunization with recombinant HAdV species B knob proteins effectively elicited cross-neutralizing antibody responses against original species B members with protective immunity. This study provides a novel insight into the immunogenicity of HAdV species B knob proteins.

The toxigenic Klebsiella oxytoca strains secrete tilymicin and tilivalline enterotoxins, which cause antibiotic-associated hemorrhagic colitis. Both enterotoxins are non-ribosomal peptides synthesized by enzymes encoded in two divergent operons clustered in a pathogenicity island. The transcriptional regulator Lrp (leucine-responsive regulatory protein) controls the expression of several bacterial genes involved in virulence. In this work, we have uncovered novel findings that have significant implications. We determined the transcriptional expression of aroX and npsA, the first genes of each tilimycin (TM)/tilivalline (TV) biosynthetic operon in K. oxytoca MIT 09-7231 wild-type and its derivatives Δlrp mutant and complemented strains. Our results suggest that Lrp directly activates the transcription of both aroX and npsA genes by binding to the intergenic regulatory region in a leucine-dependent manner. Furthermore, the lack of Lrp significantly diminished the cytotoxicity of K. oxytoca on HeLa cells due to reduced production of TM and TV. Altogether, our data present a new perspective on the role of Lrp as a regulator in cytotoxin-producing K. oxytoca strains and how it controls the expression of genes involved in the biosynthesis of their main virulence factors.IMPORTANCETilimycin (TM) and tilivalline (TV) are enterotoxins that are a hallmark for the cytotoxin-producing Klebsiella oxytoca strains, which cause antibiotic-associated hemorrhagic colitis. The biosynthesis of TM and TV is driven by enzymes encoded by the aroX- and NRPS-operons. In this study, we discovered that the transcriptional regulator Lrp plays a crucial role in activating the expression of the aroX- and NRPS-operons, thereby initiating TM and TV biosynthesis. Our results underscore a molecular mechanism by which TM and TV production by toxigenic K. oxytoca strains is regulated and shed further light on developing strategies to prevent the intestinal illness caused by this enteric pathogen.

Antimicrobial peptides (AMPs) have long been considered as potential agents against non-growing, dormant cells due to their membrane-targeted action, which is largely independent of the cell's growth state. However, the relationship between the action of AMPs and the physiological state of their target cells has been unclear, with recent reports offering conflicting views on the efficacy of AMPs against bacteria in a stationary phase. In this study, we employ single-cell approaches combined with population-level experiments to examine the action of human LL37 peptides against Escherichia coli cells in different growth phases. Time-lapse, single-cell data from our experiments reveal that LL37 peptides act faster on large, dividing cells than on small, newborn cells. We extend this investigation to non-growing E. coli cells in a stationary phase, where we observe that the action of LL37 peptides is slower on non-growing cells compared to exponentially growing cells. This slower action rate is, however, not mirrored in the minimum bactericidal concentration (MBC) measurements. Notably, we find that the MBC for non-growing cells is lower than for exponentially growing cells, indicating that, given sufficient time, LL37 peptides exhibit strong potency against non-growing cells. We propose that the enhanced potency of LL37 peptides against non-growing cells, despite their slower action, can be attributed to continuous absorption of AMPs on the cell membrane over time.

Importance: Antibiotic treatments can fail because of the regrowth of a bacterial subpopulation that resumes proliferation once the treatment ceases. This resurgence is primarily driven by non-growing, dormant bacterial cells that withstand the action of antibiotics without developing resistance. In this study, we explore the potency of the human antimicrobial peptide LL37 against non-growing Escherichia coli cells. Our findings reveal that despite a slower initial action, LL37 peptides, given sufficient time, demonstrate strong efficacy against non-growing cells. These insights suggest a potential role of antimicrobial peptides in combating persistent bacterial infections by targeting the non-growing cells.

Vigilin is a large and evolutionary conserved RNA-binding protein (RBP), which can interact with RNA through its KH domain. Vigilin is, therefore, a multifunctional protein reported to be associated with RNA transport and metabolism, sterol metabolism, chromosome segregation, carcinogenesis, and heterochromatin-mediated gene silencing. The receptor for activated C kinase 1 (RACK1) is another highly conserved protein involved in many cellular pathways. Functional studies in human cells indicated that RACK1 interacts with Vigilin to promote dengue virus (DENV) replication. Both proteins are associated with the endoplasmic reticulum. Here, we investigated the significance of Vigilin and RACK1 homologs in Aedes aegypti mosquitoes concerning DENV replication and Wolbachia infection. We identified the homologs of the two genes in Ae. aegypti (AeVigilin and AeRACK1), which were upregulated in DENV-infected Aag2 cells and mosquitoes, and silencing them in Aag2 cells resulted in reduced DENV replication. Co-immunoprecipitation showed that AeRACK1 and AeVigilin interact in mosquito cells. Interestingly, we also found upregulation of both genes in a Wolbachia-infected cell line (Aag2.wAlbB). Furthermore, silencing AeVigilin and AeRACK1 in Aag2.wAlbB cells reduced DENV replication but increased Wolbachia density. However, we did not find a significant effect on DENV replication after silencing both genes in Ae. aegypti mosquitoes. Overall, our results support the involvement and significance of AeVigilin and AeRACK1 in DENV replication in Ae. aegypti.IMPORTANCEDengue virus (DENV), transmitted mainly by Aedes aegypti mosquitoes, poses significant health risks. Identifying factors involved in the virus replication in mosquitoes and human hosts is essential for devising control measures. In this study, we show that Vigilin and the receptor for activated C kinase 1 (RACK1), two proteins shown to play a role in the replication of DENV in human cells, are induced in mosquitoes and cell lines following DENV replication. Both proteins reside in the cytoplasm, where they interact similarly to human cells. Silencing the genes in mosquito cells significantly reduced virus replication. Furthermore, we found that both genes are induced in mosquito cells transinfected with Wolbachia, a bacterium that blocks DENV replication. The results help better understand the role of the common factors supporting DENV replication in mosquitoes and human cells.

Miguel Chiurillo works in the field of protein kinases, studying their role in cell signaling and cell cycle progression in Trypanosoma cruzi. In this mSphere of Influence article, he reflects on how the research articles "Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival" by Baker et al. and "Screening the Toxoplasma kinome with high throughput tagging identifies a regulator of invasion and egress" by Smith et al. made an impact on his understanding of the complexity of the Trypanosoma cruzi kinome and the challenges to unravel it.

It is unclear whether human progression to active tuberculosis disease (TB) risk signatures are viable endpoint criteria for evaluations of treatments in development. TB is the deadliest infectious disease globally and more efficacious vaccines are needed to reduce this mortality. However, the immune correlates of protection for either preventing infection with Mycobacterium tuberculosis or preventing TB disease have yet to be completely defined, making the advancement of candidate vaccines through the pipeline slow, costly, and fraught with risk. Human-derived correlate of risk (COR) gene signatures, which identify an individual's risk of progressing to active TB disease, provide an opportunity for evaluating new therapies for TB with clear and defined endpoints. Though prospective clinical trials with longitudinal sampling are prohibitively expensive, the characterization of COR gene signatures is practical with preclinical models. Using a 3Rs (replacement, reduction, and refinement) approach we reanalyzed heterogeneous publicly available transcriptional data sets to determine whether a specific set of COR signatures are viable endpoints in the preclinical pipeline. We selected RISK6, Sweeney3, and BATF2 human-derived blood-based RNA biosignatures because they require relatively few genes and have been carefully evaluated across several clinical cohorts. These data suggest that in certain experimental designs and in several tissue types, human COR signatures correlate with disease progression as measured by the bacterial burden in the preclinical TB model pipeline. We observed the best performance when the model most closely reflected human infection or disease conditions. Human-derived COR signatures offer an opportunity for high-throughput preclinical endpoint criteria of vaccine and drug therapy evaluations.

Importance: Understanding the strengths or limitations of back-translating human-derived correlate of risk (COR) RNA signatures into the preclinical pipeline may help streamline down-selection of therapeutic vaccine and drug candidates and better align preclinical models with proposed clinical trial efficacy endpoints.