Biology subjects

Leone, P.

Publications and source records attributed to Leone, P..

8 recordsLinked to original sources

bioRxiv2024

Oral vancomycin treatment alters serum levels of indole derivatives and secondary bile acids modulating the expression of mTOR pathway genes in astrocytes during EAE

Astrocytes play important roles in the central nervous system (CNS) during health and disease. Prior studies have shown that gut commensals derived indole derivatives as well as secondary bile acids modulate astrocyte function during the late stage of EAE (recovery phase). Here we show that administering vancomycin to mice starting during the early stage of EAE improved disease recovery, an effect that is mediated by the gut microbiota. We observed that 6 taxa within the Clostridia vadin BB60 group were enriched in vancomycin treated mice compared to untreated EAE mice. Vancomycin-treated EAE mice also had elevated serum levels of the anti-inflammatory tryptophan derived metabolite, indole-3-lactic acid and decreased levels of deoxycholic acid, a pro-inflammatory secondary bile acid. RNA sequencing revealed altered expression of several genes belonging to the mammalian target of rapamycin (mTOR) pathway in astrocytes obtained during the late stage of EAE from vancomycin treated EAE mice. Furthermore, we observed a link between serum levels of indole derivatives and bile acids and expression of several genes belonging to the mTOR pathway. Interestingly, the mTOR signaling cascades have been implicated in several key biological processes including innate (e.g., astrocyte) immune responses as well as neuronal toxicity/degeneration. In addition, rapamycin, a specific inhibitor of mTOR, has been shown to inhibit the induction and progression of established EAE. Collectively, our findings suggest that the neuroprotective effect of vancomycin is at least partially mediated by indole derivatives and secondary bile acids modulating the expression of mTOR pathway genes in astrocytes. HIGHLIGHTSO_LIVancomycin attenuated established EAE through regulation of the microbiota C_LIO_LIVancomycin induced increased serum levels of indole-3-lactic acid, decreased serum levels of indoxyl-3-sulfate, p-cresol and decreased stool levels of deoxycholic acid C_LIO_LIVancomycin modulated the expression of mTOR pathway genes in astrocytes C_LIO_LILactobacillus reuteri regulated the expression of mTOR pathway genes in astrocytes C_LIO_LISerum levels of indole-3-lactic acid, indoxyl-3-sulfate, p-cresol and deoxycholic acid correlated with expression of mTOR pathway genes in astrocytes C_LI

neuroscience↗

bioRxiv2023

Induction of SARS-CoV-2 N-specific CD8+ T cell immunity in lungs by engineered extracellular vesicles associates with strongly impaired viral replication

Induction of effective immunity in lungs should be a pre-requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. In the case of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection, vaccination is expected to associate with significant inhibition of viral replication in lungs. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of SARS-CoV-2 Nucleocapsid (N) protein can protect K18-hACE2 transgenic mice from the lethal intranasal infection with the ancestral Wuhan isolate. Actually, it was widely demonstrated that these transgenic mice succumb to SARS-CoV-2 intranasal infection mainly as a consequence of the viral invasiveness of central nervous system, a pathogenetic mechanism almost absent in humans. On the other hand, K18-hACE2 transgenic mice support viral replication in lungs, an event strictly mirroring the major pathogenic signature linked to the severe disease in humans. However, nothing is known about the ability of N-specific CD8+ T cell immunity induced by engineered EVs in controlling viral replication in lungs. To fill the gap, we investigated the immunity generated in lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8+ T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in lungs were evaluated. We found that three weeks after second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8+ T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting. This inhibitory effect associated with the persistence of a N-specific CD8+ T-resident memory lymphocytes in lungs of N-immunized mice. In view of the quite conserved sequence of the N protein among SARS-CoV-2 variants, these results support the idea that a vaccine strategy focused on the induction of anti-N CD8+ T cell immunity in lungs has the potential to control the replication of emerging variants.

immunology↗

bioRxiv2022

Longitudinal monitoring of maternal care and maternal neglect

Parental care is required for offspring survival, because infants require nearly-continual oversight for extensive periods. Parents must balance caretaking with their own survival, and benefit from help provided by other experienced adults. We built a system for 24/7 long-term monitoring of wild-type or oxytocin receptor knockout (OXTR-KO) mouse mothers ( dams) over litters. Some wild-type dams had high litter survival rates, but others consistently lost pups due to neglect and hypothermia. Maternal caretaking in low-pup-survival dams improved after co-housing with an experienced dam and litter, from reorganized maternal behavior including increased nest-building. In contrast, singly-housed OXTR-KOs died in childbirth and pups died due to prolonged parturition. Co-housing with a lactating female prevented OXTR-KO maternal-infant mortality, because the other female acted as a midwife by removing and cleaning pups from the pregnant dam. Thus for single mothers that continually lose litters or die in labor, maternal-infant survival is enhanced by experienced helpers.

neuroscience↗

bioRxiv2022

Three-dimensional structure of the single domain cupredoxin AcoP

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 [A] resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network.

biophysics↗

bioRxiv2022

Strong SARS-CoV-2 N-specific CD8+ T immunity induced by engineered extracellular vesicles associates with protection from lethal infection in mice.

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut- based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.

microbiology↗

bioRxiv2021

CD8+ T cell immunogenicity induced by endogenous EVs engineered by antigens fused to a truncated Nefmut EV-anchoring protein

Intramuscular injection of DNA vectors expressing the extracellular vesicle (EV)-anchoring protein Nefmut fused at its C-terminus to viral and tumor antigens elicits a potent, effective, and anti-tolerogenic CD8+ T cell immunity against the heterologous antigen. The immune response is induced through the production of EVs incorporating Nefmut-derivatives released by muscle cells. In the perspective to a possible translation into the clinic of the Nefmut-based vaccine platform, we aimed at increasing its safety profile by identifying the minimal part of Nefmut retaining the EV-anchoring protein property. We found that a C-terminal deletion of 29-amino acids did not affect the ability of Nefmut to associate with EVs. Furthermore, the EV-anchoring function was preserved when antigens from both HPV16 (i.e., E6 and E7) and SARS-CoV-2 (i.e., S1 and S2) were fused to its C-terminus. By analyzing the immune responses induced after intramuscular injection of DNA vectors expressing fusion products based on the four viral antigens, we found that the Nefmut C-terminal deletion did not impact on the levels of antigen -specific CD8+ T lymphocytes as evaluated by IFN-{gamma} EliSpot analysis and intracellular cytokine staining. In addition, immune responses at distal sites remained unaffected, as indicated by the similar percentages of SARS-CoV-2 S1- and S2-specific CD8+ T cells detected in spleens and lung airways of mice injected with DNA vectors expressing the viral antigens fused with either Nefmut or NefmutPL. We concluded that the C-terminal Nefmut truncation does not affect stability, EV-anchoring, and CD8+ T cell immunogenicity of the fused antigen. Hence, NefmutPL represents a safer alternative to full-length Nefmut for the design of CD8+ T cell vaccines for humans.

bioengineering↗

bioRxiv2021

Long-term antitumor CD8+ T cell immunity induced by endogenously engineered extracellular vesicles

We developed a novel approach to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is an innovative vaccination approach employing a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) that has lost the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by cells expressing the Nefmut_based DNA vector. We previously provided evidence that the inoculation in mice of a DNA vector expressing the Nefmut/HPV16-E7 fusion protein induced an E7-specific CTL immune response as detected 2 weeks after the second immunization. In an effort to optimize the anti-HPV16 CD8+ T cell immune response, we found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that we observed in mice injected with the single vectors. When TC-1 cells, i.e., a tumor cell line co-expressing E6 and E7, were implanted before immunization, all mice survived until day 44, whereas no mice injected with either void or Nefmut_expressing vectors survived until day 32 after tumor implantation. A substantial part of mice (7 out of 12) cleared the tumor. When cured mice were re-challenged with a second sub cute implantation of TC-1 cells, and followed for additional 135 days, whereas none of them developed tumors. Both E6- and E7-specific CD8+ T immunity was still detectable at the end of the observation time. Hence, the immunity elicited by engineered EVs, besides curing already developed tumors, is strong enough to guarantee the resistance to additional tumor attack. This results is of relevance for therapy against both metastatic and relapsing tumors.

cancer biology↗

bioRxiv2020

A new concept on anti-SARS-CoV-2 vaccines: strong CD8+ T-cell immune response in both spleen and lung induced in mice by endogenously engineered extracellular vesicles

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is spreading rapidly in the absence of validated tools to control the growing epidemic besides social distancing and masks. Many efforts are ongoing for the development of vaccines against SARS-CoV-2 since there is an imminent need to develop effective interventions for controlling and preventing SARS-CoV-2 spread. Essentially all vaccines in most advanced phases are based on the induction of antibody response against either whole or part of spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). We exploited this technology with the aim to identify a clinical candidate defined as DNA vectors expressing SARS-CoV-2 antigens inducing a robust CD8+ T-cell response. This is a new vaccination approach employing a DNA expression vector encoding a biologically inactive HIV-1 Nef protein (Nefmut) showing an unusually high efficiency of incorporation into EVs even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells are internalized by antigen-presenting cells leading to cross-presentation of the associated antigens thereby priming of antigen-specific CD8+ T-cells. To apply this technology to a design of anti-SARS-CoV-2 vaccine, we recovered DNA vectors expressing the products of fusion between Nefmut and four viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. All fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity was generated. Most important, high levels of virus-specific CD8+ T cells were found in bronchoalveolar lavages of immunized mice. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lung. EVs engineered with SARS-CoV-2 antigens proved immunogenic also in the human system through cross-priming assays carried out with ex vivo human cells. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed as anti-SARS-CoV-2 vaccine candidates.

immunology↗