Biology subjects

Pravetoni, M.

Publications and source records attributed to Pravetoni, M..

6 recordsLinked to original sources

bioRxiv2025

A cationic liposome-formulated Toll Like Receptor (TLR)7/8 agonist enhances the efficacy of a vaccine against fentanyl toxicity

The U.S. opioid epidemic is an extraordinary public health crisis that started in 1990 and significantly accelerated in the last decade. Since 2020, over 100,000 fatal drug overdoses have been reported annually, and 75% of those involved fentanyl and its analogs (F/FA). Accelerating the translation of innovative, effective, and safe treatments is needed to augment existing measures to counteract such a crisis. Active immunization against F/FA and other opioids represents a promising therapeutic and prophylactic strategy for opioid use disorder (OUD) and opioid-induced overdose toxicity. Previously we demonstrated that the anti-fentanyl vaccine comprising a fentanyl-based hapten (F) conjugated to the diphtheria cross-reactive material (CRM), admixed with the novel lipidated toll-like receptor 7/8 (TLR7/8) agonist INI-4001 adsorbed on Alhydrogel(R) (alum) induced high-affinity fentanyl-specific polyclonal antibodies that protected against fentanyl-induced pharmacological effects in mice, rats, and mini-pigs. Here, INI-4001 was formulated into liposomes with different surface charges, and their impact on F-CRM adsorption, INI-4001 adjuvanticity, and vaccine efficacy were explored. Additionally, as the role of innate immunity in mediating the efficacy of addiction vaccines is largely unknown, we tested these formulations on the activation of innate immunity in vitro. Cationic INI-4001 liposomes surpassed other liposomal and aluminum-based formulations of INI-4001 by enhancing the efficacy of fentanyl vaccines and protecting rats against bradycardia and respiratory depression by blocking the distribution of fentanyl to the brain. Fentanyl vaccines adjuvanted with either cationic INI-4001 liposomes or the aqueous INI-4001 adsorbed to alum induced significant surface expression of co-stimulatory molecules and maturation markers in a murine dendritic cell line (JAWS II), while the former was superior in enhancing the macrophages surface expression of CD40, CD86 and inducible nitric oxide synthase (iNOS), indicative of maturation and activation. These results warrant further investigation of liposome-based formulations of TLR7/8 agonists for improving the efficacy of vaccines targeting F/FA and other opioids of public health interest. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/631964v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@178c8fdorg.highwire.dtl.DTLVardef@d38c09org.highwire.dtl.DTLVardef@8ec41forg.highwire.dtl.DTLVardef@edcf82_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

CD4+ but not CD8+ T cells are required for protection against severe guinea pig cytomegalovirus infections

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and the leading cause of infectious disease related birth defects worldwide. How the immune response modulates the risk of intrauterine transmission of HCMV after maternal infection remains poorly understood. Maternal T cells likely play a critical role in preventing infection at the maternal-fetal interface and limiting spread across the placenta, but concerns exist that immune responses to infection may also cause placental dysfunction and adverse pregnancy outcomes. This study investigated the role of CD4+ and CD8+ T cells in a guinea pig model of primary cytomegalovirus infection. Monoclonal antibodies specific to guinea pig CD4 and CD8 were used to deplete T cells in non- pregnant and in pregnant guinea pigs after mid-gestation. CD4+ T cell depletion increased the severity of illness, caused significantly elevated viral loads, and increased the rate of congenital guinea pig cytomegalovirus (GPCMV) infection relative to animals treated with control antibody. CD8+ T cell depletion was comparably well tolerated and did not significantly affect the weight of infected guinea pigs or viral loads in their blood or tissue. However, significantly more viral genomes and transcripts were detected in the placenta and decidua of CD8+ T cell depleted dams post-infection. This study corroborates earlier findings made in nonhuman primates that maternal CD4+ T cells play a critical role in limiting the severity of primary CMV infection during pregnancy while also revealing that other innate and adaptive immune responses can compensate for an absent CD8+ T cell response in -CD8-treated guinea pigs. Author SummaryCongenital cytomegalovirus infection is a leading cause of adverse pregnancy outcomes and preventable disability in children. Using guinea pigs, a well-established small animal model of congenital infection and intrauterine development, this study tested how depleting T cells affects the course of primary cytomegalovirus infections. Severe illness and high rates of congenital infection were observed when helper CD4+ T cells were depleted. The depletion of killer CD8+ T cells did not affect the severity of disease or the rate of congenital infection but did increase the amount of virus that was detected in the placenta. A greater understanding how an immune response can prevent the infection of the placenta and developing offspring is needed to inform vaccine and therapeutic development. This study not only describes a new reagent that can be used to study the guinea pig immune system but also sheds new light in how adaptive immunity regulates congenital viral infection.

immunology↗

bioRxiv2024

ACE2 decoy Fc-fusions and bi-specific killer engager (BiKEs) require Fc engagement for in vivo efficacy against SARS-CoV-2

SARS-CoV-2 virus has continued to evolve over time necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a key line of defense for unvaccinated or immunocompromised individuals. However, these mAbs are now ineffective against current SARS-CoV-2 variants. Here, we tested three aspects of SARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for in vivo clearance of SARS-CoV-2. Secondly, we tested bi-specific killer engagers (BiKEs) that simultaneously engage SARS-CoV-2 and a specific Fc receptor. Benefits of these engagers include the ease of manufacturing, stability, more cell-specific targeting, and high affinity binding to Fc receptors. Using both mAbs and BiKEs, we found that both neutralization and Fc receptor engagement were necessary for effective SARS-CoV-2 clearance. Thirdly, due to ACE2 being necessary for viral entry, ACE2 will maintain binding to SARS-CoV-2 despite viral evolution. Therefore, we used an ACE2 decoy Fc-fusion or BiKE, instead of an anti-SARS-CoV-2 antibody sequence, as a potential therapeutic that would withstand viral evolution. We found that the ACE2 decoy approach also required Fc receptor engagement and, unlike traditional neutralizing antibodies against specific variants, enabled the clearance of two distinct SARS-CoV-2 variants. These data show the importance of Fc engagement for mAbs, the utility of BiKEs as therapies for infectious disease, and the in vivo effectiveness of the ACE2 decoy approach. With further studies, we predict combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels. AbbreviationsACE2, scFv, mAb, BiKE, COVID-19, Fc, CD16, CD32b, CD64, d.p.i Key pointsO_LIWith equal dosing, both neutralization and Fc engagement are necessary for the optimal efficacy of in vivo antibodies and bi-specific killer engagers (BiKEs) against SARS-CoV-2. C_LIO_LIBiKEs can clear SARS-CoV-2 virus and protect against severe infection in the hACE2-K18 mouse model. C_LIO_LIACE2 decoys as part of Fc-fusions or BiKEs provide in vivo clearance of two disparate SARS-CoV-2 variants. C_LI

immunology↗

bioRxiv2024

Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C)

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease. SummaryMIS-C is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Limited studies tested cellular function ex vivo to understand the aberrant immune response in MIS-C. We found dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/589585v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16d9e0dorg.highwire.dtl.DTLVardef@1a37693org.highwire.dtl.DTLVardef@bdb0cforg.highwire.dtl.DTLVardef@1a32604_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

immunology↗

bioRxiv2022

An Epitope-Focused Trypanosome-Derived Vaccine Platform Elicits High-Affinity Antibodies and Immunity Against Fentanyl Effects

Poorly antigenic small molecules pose challenges for the production of clinically efficacious antibodies. To address this, we have developed an immunization platform derived from the antigenic surface coat of the African trypanosome. Through sortase-based conjugation of antigens to the trypanosome surface coat protein variant surface glycoprotein (VSG), we created the VAST (VSG-immunogen Array by Sortase Tagging). We used VAST to elicit protective immunity to the effects fentanyl. Immunizing mice with fentanyl-VAST generated serological memory and protection from fentanyl challenge. Employing single-cell RNAseq, we then developed a streamlined method that synergizes with the VAST to identify immunization-elicited memory B cells and the antibodies they encode. All antibodies selected by this method displayed picomolar affinities for fentanyl. Passive immunization protected animals from fentanyl, while crystallography revealed that the mAbs bind fentanyl in an unusually deep pocket suited to small molecules, demonstrating the ability of the VAST to elicit high-quality therapeutic antibodies.

immunology↗

bioRxiv2019

Interruption of Continuous Opioid Exposure Exacerbates Drug-Evoked Adaptations in Brain Function and Behavior

Drug-evoked adaptations in the mesolimbic dopamine system are postulated to drive opioid abuse and addiction. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological and behavioral impact. We exposed male and female mice to morphine for one week, with administration patterns that were either intermittent (daily injections) or continuous (osmotic minipump infusion). We then interrupted continuous morphine exposure with either naloxone-precipitated or spontaneous withdrawal. Continuous morphine exposure caused tolerance to the psychomotor-activating effects of morphine, whereas both intermittent and interrupted morphine exposure caused long-lasting psychomotor sensitization. Given links between locomotor sensitization and mesolimbic dopamine signaling, we used fiber photometry and a genetically encoded dopamine sensor to conduct longitudinal measurements of dopamine dynamics in the nucleus accumbens. Locomotor sensitization caused by interrupted morphine exposure was accompanied by enhanced dopamine signaling in the nucleus accumbens. To further assess downstream consequences on striatal gene expression, we used next-generation RNA sequencing to perform genome-wide transcriptional profiling in the nucleus accumbens and dorsal striatum. The interruption of continuous morphine exposure exacerbated drug-evoked transcriptional changes in both nucleus accumbens and dorsal striatum, dramatically increasing differential gene expression and engaging unique signaling pathways. Our study indicates that opioid-evoked adaptations in brain function and behavior are critically dependent on the pattern of drug administration, and exacerbated by interruption of continuous exposure. Maintaining continuity of chronic opioid administration may therefore represent a strategy to minimize iatrogenic effects on brain reward circuits.

neuroscience↗