Broad Protection Research Articles

Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity

Background Botulinum neurotoxin type A (BoNT/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (HC) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively HC of other BoNT/A subtypes. This study aimed to generate HC domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains. Methods HC domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/HCA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for HC domains of BoNT/A2-A8 were derived from pET45b-HCA1 by site-directed mutagenesis and/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of HC domains and to evaluate the reactivity of VHHs to these domains. Results SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the HC domains of all eight BoNT/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for HC of BoNT/A1, two new VHHs were found to recognize the HC domains of all BoNT/A subtypes, of which VHH-A3 displayed EC50 values for these domains close to those of ciA-C2. Conclusion This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A.

Cross-protection against homo and heterologous influenza viruses via intranasal administration of an HA chimeric multiepitope nanoparticle vaccine

BackgroundInfluenza A viruses (IAVs) cause seasonal influenza epidemics and pose significant threats to public health. However, seasonal influenza vaccines often elicit strain-specific immune responses and confer little protection against mismatched strains. There is an urgent need to develop universal influenza vaccines against emerging and potentially re-emerging influenza virus infections. Multiepitope vaccines combining multiple conserved epitopes can induce more robust and broader immune responses and provide a potential solution.ResultsHere, we demonstrated that an HA chimeric multiepitope nanoparticle vaccine, delivered intranasally conferred broad protection against challenges with various influenza viruses in mice. The nanoparticle vaccine co-expresses the ectodomain of haemagglutinin (H), three repeated highly conserved ectodomains of matrix protein 2 (M), and the M-cell-targeting ligand Co4B (C) in a baculovirus-insect cell system. These elements (C, H and M) were presented on the surface of self-assembling ferritin (f) in tandem to generate a nanoparticle denoted as CHM-f. Intranasal vaccination with CHM-f nanoparticles elicited robust humoral and cellular immune responses, conferring complete protection against a variety of IAVs, including the A/PR8/34 H1N1 strain, the swine flu H3N2 strain, the avian flu H5N8 strain, and H9N2. When CHM-f nanoparticles adjuvanted with CpG IAMA-002, the weight loss protective effect, cellular immune responses and mucosal IgA responses were significantly augmented. Compared with controls, mice immunized with CHM-f nanoparticles with or without CpG IAMA-002 showed significant reductions in weight loss, lung viral titres and pathological changes.ConclusionsThese results suggest that CHM-f nanoparticle with or without CpG IAMA-002 is a promising candidate as a universal influenza vaccine.Graphical

Diversifying T-cell responses: safeguarding against pandemic influenza with mosaic nucleoprotein.

The World Health Organization (WHO) estimates that seasonal influenza causes 3-5 million cases of severe illness annually. The influenza virus frequently undergoes genetic changes through antigenic drift and antigenic shift, resulting in annual epidemics and occasional pandemics. Consequently, a major public health objective is to develop a universal influenza vaccine that offers broad protection against both current and pandemic influenza A strains. In this study, we designed a nucleoprotein (NP) antigen (termed mosaic NP) comprising antigenic regions found in thousands of influenza viruses, aiming to use it as a vaccine to induce broad anti-influenza T-cell responses. Our findings indicate that the mosaic NP vaccine provided significant protection against seasonal H1N1 and H3N2, as well as the pandemic H1N1 strain, demonstrating its effectiveness across various influenza subtypes. These findings suggest that the mosaic NP is a potential universal influenza vaccine antigen, capable of protecting against diverse strains of influenza viruses.

P-608. Clinical Profile of GSK's Pentavalent Meningococcal ABCWY Vaccine: An Overview

Abstract Background GSK’s MenABCWY vaccine was developed to provide broad protection against invasive meningococcal disease by combining the antigenic components of the licensed 4-component meningococcal serogroup B vaccine (MenB-4C) and serogroups A,C,W,Y vaccine (MenACWY-CRM). Methods An overview of the MenABCWY vaccine’s profile was derived from its clinical development program of 12 controlled studies,1-10 which enrolled over 7000 adolescents and adults in 13 countries. Results For serogroup B (MenB), non-inferiority of MenABCWY (2 doses, 6 months apart) to MenB-4C (2 doses, 2 months apart) was demonstrated in a phase 3 study9 in terms of vaccine-induced bactericidal immune responses measured by endogenous complement human serum bactericidal antibody (enc-hSBA) assay. The enc-hSBA assay uses each vaccinee’s serum as source of complement and tests a panel of 110 MenB strains representing 95% of disease-causing isolates in the US and ∼89% of global circulating strains,11 and is thereby used to assess immunological vaccine effectiveness (iVE) in clinical studies, in conditions close to real-world settings. MenABCWY iVE was 78% (95% CI: 77–79) against the panel of 110 MenB strains; serum from 84% of participants killed ≥ 70% of strains.9 For serogroups A,C,W,Y, non-inferiority of MenABCWY (2 doses, 6 months apart) to MenACWY-CRM (1 dose) was demonstrated in phase 3 studies in MenACWY-primed10 and -naïve9 participants, with robust immune responses against each serogroup.9,10 Phase 2 data for all 5 serogroups show 2-year and 4-year antibody persistence5,8 and anamnestic booster responses2,5,8 at 2 years after primary MenABCWY vaccination, and similar immune responses with MenABCWY 0-6 and 0-11 months schedules.8 In phase 3, 1 MenABCWY dose was non-inferior to 1 MenACWY-CRM dose as booster in participants primed with MenACWY ≥ 4 years previously.10 The safety and reactogenicity profile of MenABCWY was favorable overall and consistent with that of MenB-4C.1-3,8,9 Conclusion GSK’s combined MenABCWY vaccine is immunogenic, with demonstrated non-inferiority to MenB-4C and MenACWY-CRM vaccines, and a safety profile similar to that of MenB-4C. FUNDING: GSK Disclosures Daniela Toneatto, MD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Maria Lattanzi, MD, PhD, GSK: Employee|GSK: Stocks/Bonds (Private Company) Ellen Ypma, Msc, GSK: Employee Karen O’Brien, PhD, GSK: Employee Steven Rubin, PhD, GSK: Employee Laura Tomasi Cont, PhD, GSK: Employee|GSK: Stocks/Bonds (Private Company) Paola Pedotti, PhD, GSK: Employee|GSK: Stocks/Bonds (Private Company) Woo-Yun Sohn, MD, GSK: Employed by GSK|GSK: Stocks/Bonds (Private Company)

A locally administered single-cycle influenza vaccine expressing a non-fusogenic stabilized hemagglutinin stimulates strong T-cell and neutralizing antibody immunity.

Influenza is a serious public health concern, causing seasonal epidemics as well as pandemics in people. Influenza can also cause severe agricultural losses due to its circulation in farmed poultry and swine. A major challenge in the control of influenza is the diversity of circulating viruses. Developing vaccines that stimulate immunity to a wide array of influenza viruses is therefore important for protecting human and animal populations from disease and death. In this study, we describe an approach for developing influenza vaccines that trigger immune mechanisms shown to induce broad protection against a diversity of viruses, while also conserving the strong protection against specific strains observed in existing vaccines.

A Novel Vaccine for Bovine Diarrhea Complex Utilizing Recombinant Enterotoxigenic Escherichia coli and Salmonella Expressing Surface-Displayed Chimeric Antigens from Enterohemorrhagic Escherichia coli O157:H7

Background/Objectives: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a zoonotic pathogen primarily found in cattle, causes Hemolytic Uremic Syndrome (HUS) in humans, often through contaminated food. Its Type Three Secretion System (T3SS) facilitates gut colonization. In contrast, neonatal calf diarrhea (NCD) is mainly caused by pathogens like enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Bovine Coronavirus (BCoV), and Bovine Rotavirus type A (BRoVA). This study engineered a chimeric protein combining EspB and Int280γ, two T3SS components, expressed in the membranes of Salmonella Dublin and ETEC. Methods: Immune responses in vaccinated mice and guinea pigs were assessed through ELISA assays. Results: Successful membrane anchorage and stability of the chimera were confirmed. Immune evaluations showed no enhancement from combining recombinant bacteria, indicating either bacterium suffices in a single formulation. Chimeric expression yielded immunogenicity equivalent to 10 µg of recombinant protein, with similar antibody titers. IgG1/IgG2a levels and Th1, Th2, and Th17 markers indicated a mixed immune response, providing broad humoral and cellular protection. Responses to BCoV, BRoVA, ETEC, and Salmonella antigens remained strong and did not interfere with chimera-specific responses, potentially boosting NCD vaccine efficacy. Conclusions: The chimera demonstrated robust immunogenicity, supporting its potential as a viable vaccine candidate against EHEC O157:H7. This approach could enhance NCD vaccine valency by offering broader protection against calf diarrhea while reducing HUS transmission risks to humans.

The Salmonella Paratyphi A O-Antigen Glycoconjugate Vaccine Is Able to Induce Antibodies with Bactericidal Activity Against a Panel of Clinical Isolates

Background: Typhoid and paratyphoid fevers represent a global health burden, especially in Southern Asia, exacerbated by the increase in antimicrobial resistance. While vaccines against Salmonella Typhi have been successfully introduced, a vaccine against S. Paratyphi A is not available, yet. Efforts to develop an effective vaccine targeting both Salmonella serovars are currently ongoing. GVGH is developing a bivalent vaccine constituted by the Vi-CRM197 typhoid conjugate vaccine (TCV), and the Salmonella Paratyphi A O-antigen (O:2), also conjugated to the CRM197 carrier protein (O:2-CRM197). In this work we have characterized a panel of S. Paratyphi A clinical isolates from endemic regions, differing in terms of their O:2 structural features. Methods: Rabbits were immunized with the S. Paratyphi A component of the vaccine candidate and the resulting sera were tested for their ability to bind and kill the isolates using flow cytometry and luminescence-based serum bactericidal assay (L-SBA). Results: The O:2-CRM197 glycoconjugate induced a functional immune response in rabbits, effectively binding and killing a diverse panel of clinical isolates. The sera demonstrated bactericidal activity independent of the O:2 structural variations, including differences in O-acetylation and glucosylation levels. Additionally, the study found that the O:2-CRM197 conjugate’s adsorption to Alhydrogel did not significantly impact its immunogenicity or bactericidal efficacy. Conclusions: The O:2-CRM197 component of the bivalent vaccine candidate shows promise in providing broad protection against S. Paratyphi A isolates, regardless of their O-antigen structural variations. The ongoing clinical studies on human sera are expected to confirm these results.

A bivalent COVID-19 mRNA vaccine elicited broad immune responses and protection against Omicron subvariants infection

Continuously emerging SARS-CoV-2 Omicron subvariants pose a threat thwarting the effectiveness of approved COVID-19 vaccines. Especially, the protection breadth and degree of these vaccines against antigenically distant Omicron subvariants is unclear. Here, we report the immunogenicity and efficacy of a bivalent mRNA vaccine, PTX-COVID19-M1.2 (M1.2), which encodes native spike proteins from Wuhan-Hu-1 (D614G) and Omicron BA.2.12.1, in mouse and hamster models. Both primary series and booster vaccination using M1.2 elicited potent and broad nAbs against Wuhan-Hu-1 (D614G) and some Omicron subvariants. Strong spike-specific T cell responses against Wuhan-Hu-1 and Omicron subvariants, including JN.1, were also induced. Vaccination with M1.2 protected animals from Wuhan-Hu-1 and multiple Omicron subvariants challenges. Interestingly, protection against XBB.1.5 lung infection did not correlate with nAb levels. These results indicate that M1.2 generated a broadly protective immune response against antigenically distant Omicron subvariants, and spike-specific T cells probably contributed to the breadth of the protection.

Monoclonal Antibody Production Against Filoviruses from Immunized Mice.

Currently, targeted therapeutics against pathogenic filoviruses are very limited, with only two monoclonal antibody (mAb)-based products targeting Ebola virus having received approval from health authorities. More therapeutics are needed to fight against other fatal filoviruses. Here, we describe a protocol using hybridoma technology to generate mAbs that can target not only a single filovirus species but also multiple species for broad protection.

Enhanced Antibody Response to the Conformational Non-RBD Region via DNA Prime-Protein Boost Elicits Broad Cross-Neutralization Against SARS-CoV-2 Variants

Preventing immune escape of SARS-CoV-2 variants is crucial in vaccine development to ensure broad protection against the virus. Conformational epitopes beyond the RBD region are vital components of the spike protein but have received limited attention in the development of broadly protective SARS-CoV-2 vaccines. In this study, we used a DNA prime-protein boost regimen to evaluate the broad cross-neutralization potential of immune response targeting conformational non-RBD region against SARS-CoV-2 viruses in mice. Mice with enhanced antibody responses targeting conformational non-RBD region show better performance in cross-neutralization against the Wuhan-01, Delta, and Omicron subvariants. Via analyzing the distribution of conformational epitopes, and quantifying epitope-specific binding antibodies, we verified a positive correlation between the proportion of binding antibodies against the N-terminal domain (NTD) supersite (a conformational non-RBD epitope) and SARS-CoV-2 neutralization potency. The current work highlights the importance of high ratio of conformational non-RBD-specific binding antibodies in mediating viral cross-neutralization and provides new insight into overcoming the immune escape of SARS-CoV-2 variants.

Durability of Adaptive Immunity in Immunocompetent and Immunocompromised Patients Across Different Respiratory Viruses: RSV, Influenza, and SARS-CoV-2.

Research on respiratory virus immunity duration post-vaccination reveals variable outcomes. This study performed a literature review to assess the efficacy and longevity of immune protection post-vaccination against SARS-CoV-2, influenza, and respiratory syncytial virus (RSV), with a focus on immunocompromised populations. Specific objectives included examining humoral and cellular immune responses and exploring the impact of booster doses and hybrid immunity on extending protection. A literature review was conducted focusing on studies published from January 2014 to November 2024. The search targeted adaptive immunity post-vaccination, natural immunity, and hybrid immunity for SARS-CoV-2, influenza, and RSV. Selection criteria emphasized human populations, adaptive immunity outcomes, and immunocompromised individuals. The PICO framework guided the analysis, culminating in a detailed review of 30 studies. SARS-CoV-2 vaccines exhibited robust initial antibody responses, which waned significantly within six months, necessitating frequent boosters. Influenza and RSV vaccines similarly showed declines in immunity, though some influenza vaccines demonstrated moderate durability. Hybrid immunity, arising from combined natural infection and vaccination, provided more resilient and lasting protection than vaccination alone, especially against emerging variants. Immunocompromised individuals consistently exhibited reduced durability in adaptive immune responses across all studied viruses. Challenges include rapid viral mutations, limiting the broad protection of current vaccines. Immune durability varies significantly across virus types and patient populations. Frequent boosters and hybrid immunity are critical to optimizing protection, particularly for vulnerable groups. The findings underscore the need for adaptable vaccination strategies and advancements in vaccine design to counter rapidly mutating respiratory pathogens effectively.

Use of equine H3N8 hemagglutinin as a broadly protective influenza vaccine immunogen

Development of an efficacious universal influenza vaccines remains a long-sought goal. Current vaccines have shortfalls such as mid/low efficacy and needing yearly strain revisions to account for viral drift/shift. Horses undergo bi-annual vaccines for the H3N8 equine influenza virus, and surveillance of sera from vaccinees demonstrated very broad reactivity and neutralization to many influenza strains. Subsequently, vaccinating mice using the equine A/Kentucky/1/1991 strain or recombinant hemagglutinin (HA) induced similar broadly reactive and neutralizing antibodies to seasonal and high pathogenicity avian influenza strains. Challenge of vaccinated mice protected from lethal virus challenges across H1N1 and H3N2 strains. This protection correlated with neutralizing antibodies to the HA head, esterase, and stem regions. Vaccinated ferrets were also protected after challenge with H1N1 influenza A/07/2009 virus using whole viral or HA. These data suggest that equine H3N8 induces broad protection against multiple influenzas using a unique antigen that diverges from other universal vaccine approaches.

Allergen-Specific Immunotherapy and Trained Immunity.

The high prevalence of allergic diseases reached over the last years is attributed to the complex interplay of genetic factors, lifestyle changes, and environmental exposome. Allergen-specific immunotherapy (AIT) is the single therapeutic strategy for allergic diseases with the potential capacity to modify the course of the disease. Our knowledge of the mechanisms involved in allergy and successful AIT has significantly improved. Recent findings indicate that long-term allergen tolerance upon AIT discontinuation not only relies on the generation of proper adaptive immune responses by the generation of allergen-specific regulatory T and B cells enabling the induction of different isotypes of blocking antibodies but also relies on the restoration of proper innate immune responses. Trained immunity (TRIM) is the process by which innate immune cells acquire memory by mechanisms depending on metabolic and epigenetic reprogramming, thus conferring the host with increased broad protection against infection. This concept was initially explored for infectious diseases, as well as for vaccination against infections, but compelling experimental evidence suggests that TRIM might also play a role in allergy and AIT. Hyperinflammatory innate immune responses in early life, likely due to TRIM maladaptations, lead to aberrant type 2 inflammation-enhancing allergy. However, exposure to farming environments and specific microbes prevents recurrent infections and allergy development, likely due to mechanisms partially depending on TRIM. TRIM-based vaccines and next-generation AIT vaccines inducing metabolic and epigenetic reprogramming in innate immune cells and their precursors have shown protective antiallergic effects. A better understanding of the factors involved in early-life TRIM mechanisms in the context of allergy and the identification and characterization of novel tolerance inducers might well enable the design of alternative TRIM-based allergen vaccines for allergic diseases.

Cross-reactivity in Adaptive Immunity: An Overview

Cross-reactivity in adaptive immunity refers to the ability of immune cells to recognize and respond to similar but distinct antigens. This phenomenon, while aiding in the recognition and defense against diverse pathogens, can also lead to unintended immune responses, including autoimmune reactions. The review explores key mechanisms underlying cross-reactivity, including molecular mimicry, T cell receptor degeneracy, and antibody cross-recognition. It discusses the dual role of cross-reactivity in infectious diseases, where it contributes to both protective immunity and potential disease enhancement, as seen in infections like dengue and Zika. Cross-reactivity is also implicated in autoimmunity, where immune responses to microbial antigens can mistakenly target self-tissues, potentially triggering conditions such as type 1 diabetes and multiple sclerosis. Additionally, the review addresses the implications of cross-reactivity in vaccine design, emphasizing the need to balance broad immune protection with safety, as well as challenges in immunotherapy where cross-reactivity may lead to off-target effects. Understanding cross-reactivity is essential to leveraging adaptive immunity effectively while minimizing potential adverse effects, with applications spanning infection control, vaccine development, and therapeutic interventions in immune-mediated diseases. Keywords: Adaptive immunity, Cross-reactivity, Molecular mimicry, Autoimmunity, Vaccine development, Immunotherapy

Structural characterization of human monoclonal antibodies targeting uncommon antigenic sites on spike glycoprotein of SARS-CoV.

The function of the spike protein N terminal domain (NTD) in coronavirus (CoV) infections is poorly understood. However, some rare antibodies that target the SARS-CoV-2 NTD potently neutralize the virus. This finding suggests the NTD may contribute in part to protective immunity. Pan-sarbecovirus antibodies are desirable for broad protection, but the NTD region of SARS-CoV and SARS-CoV-2 exhibit a high level of sequence divergence, and therefore, cross-reactive NTD-specific antibodies are unexpected, and there is no structure of a SARS-CoV NTD-specific antibody in complex with NTD. Here we report a monoclonal antibody COV1-65 encoded by the IGHV1-69 gene that recognizes the NTD of SARS-CoV S protein. A prophylaxis study showed the MAb COV1-65 prevented disease when administered before SARS-CoV challenge of BALB/c mice, an effect that requires intact Fc effector functions for optimal protection in vivo. The footprint on the S protein of COV1-65 is near to functional components of the S2 fusion machinery, and the selection of COV1-65 escape mutant viruses identified critical residues Y886H and Q974H, which likely affect the epitope through allosteric effects. Structural features of the mAb COV1-65-SARS-CoV antigen interaction suggest critical antigenic determinants that should be considered in the rational design of sarbecovirus vaccine candidates.

Inhibition of Cardiac p38 Highlights the Role of the Phosphoproteome in Heart Failure Progression.

Heart failure (HF) is a complex condition characterized by the inability of the heart to pump sufficient oxygen to the organs to meet their metabolic needs. Among the altered signal transduction pathways associated with HF pathogenesis, the p38 mitogen-activated protein kinase (p38 MAPK) pathway-activated in response to stress- has attracted considerable attention for its potential role in HF progression and cardiac hypertrophy. However, the exact mechanisms by which p38 MAPK influences HF remain unclear. Addressing knowledge gaps may provide insight on why p38 inhibition has yielded inconsistent outcomes in clinical trials. Here we investigate the effects of p38 MAPK inhibition via SB203580 on cardiac remodeling in a guinea pig model of HF and sudden cardiac death. Using a well-established HF model with ascending aortic constriction and daily isoproterenol (ACi) administration, we assessed proteomic changes across three groups: sham-operated controls, untreated ACi, and ACi treated with SB203580 (ACiSB). Cardiac function was evaluated by M-mode echocardiography, while proteome and phosphoproteome profiles were analyzed using multiplexed tandem mass tag labeling and LC-MS/MS. Our findings demonstrate that chronic SB203580 treatment offers protection against progressive decline in cardiac function in HF. The proteomic data indicate that SB203580-treatment exerts broad protection of the cardiac phosphoproteome, beyond inhibiting maladaptive p38-dependent phosphorylation, extending to PKA and AMPK networks among others, ultimately protecting the phosphorylation status of critical myofibrillar and Ca2+-handling proteins. Though SB203580 had a more restricted impact on widespread protein changes in HF, its biosignature was consistent with preserved mitochondrial energetics as well as reduced oxidative and inflammatory stress.

Arboviral Diseases: A Rising Global Health Burden

Arboviruses, transmitted to humans through arthropod vectors like mosquitoes and ticks, pose a significant global health threat. The emergence and re-emergence of arboviral diseases, including Dengue, Zika, Chikungunya, and West Nile Virus, continue to challenge healthcare systems worldwide. Vaccination is a cornerstone of arbovirus control, and this review explores the current landscape and future perspectives of new vaccines against arboviruses. The diversity of arboviruses, including multiple serotypes and genotypes, presents a major hurdle for vaccine development. Novel vaccine platforms, such as mRNA vaccines and viral vectors, hold promise for rapid response and broad protection. Advances in structural biology provide insights into conserved viral epitopes, offering potential targets for universal arbovirus vaccines. Moreover, integrated approaches combining vaccination with vector control and surveillance strategies are crucial for breaking the transmission cycle. A one health approach that considers human, animal, and environmental health can enhance arbovirus surveillance and control. Behavioural interventions and community engagement play a pivotal role in improving vaccine acceptance and coverage. Understanding human behaviour and perceptions is essential for successful vaccination campaigns. Global collaboration and resource allocation are paramount in advancing arbovirus vaccine research and development. Partnerships between governments, academia, and industry are needed to address this multifaceted challenge. In conclusion, while arboviruses continue to threaten public health, innovative approaches, interdisciplinary efforts, and international cooperation offer hope for the development of effective vaccines against these elusive pathogens. A concerted global effort is essential to mitigate the impact of arboviral diseases and protect vulnerable populations.

Discovery of New 3-(Benzo[b]Thiophen-2-yl)Pyrrolidine-2,5-Dione Derivatives as Potent Antiseizure and Antinociceptive Agents-In Vitro and In Vivo Evaluation.

Background/Objectives: To address the unmet clinical needs in the treatment of epilepsy and pain, the continued development of more effective and safer anticonvulsants and analgesics is still necessary. Therefore, herein we report synthesis and antiseizure/antinociceptive evaluation of a focused series of 3-(benzo[b]thiophen-2-yl)pyrrolidine-2,5-dione derivatives. Methods: The anticonvulsant properties were investigated in acute models of seizures, namely the maximal electroshock (MES), the 6 Hz (32 mA), and subcutaneous pentylenetetrazole (scPTZ) seizure models, whereas analgesic activity was tested in the model of a tonic pain/formalin test and oxaliplatin-induced neuropathic pain (in CD-1-mice, i.p.). In addition, a number of in vitro assays were performed, aiming at the evaluation of the drug-like properties of the compounds disclosed herein. Results: We identified 33 as a lead compound with the most promising antiseizure properties, i.e., ED50 (MES) = 27.4 mg/kg and ED50 (6 Hz, 32 mA) = 30.8 mg/kg. Furthermore, 33 at a dose of 100 mg/kg significantly prolonged the latency time to the first seizure episode in the scPTZ model and at high doses did not impaire coordination of mice in the rotarod test (TD50 > 200 mg/kg). Apart from broad antiseizure protection, 33 demonstrated a significant analgesic effect in the formalin test (45 mg/kg, i.p.), and effectively alleviated allodynia in the oxaliplatin-induced neuropathic pain model (30 and 45 mg/kg). The binding assays suggest that the most plausible mechanism of action relies on interaction with the neuronal voltage-sensitive sodium channel (site 2). Furthermore, the drug-like potential of 33 supports favorable in vitro results, i.e., no hepatocytotoxicity and neurocytotoxicity at a high concentration of 100 μM, as well as a lack of mutagenicity at a concentration as high as 500 μM. Conclusions: Compound 33 identified in the current studies is proposed to be an interesting candidate for further preclinical development as therapy for epilepsy and neuropathic pain.

A naturally occurring mitochondrial genome variant confers broad protection from infection in Drosophila.

The role of mitochondria in immunity is increasingly recognized, but it is unclear how variation in mitochondrial DNA (mtDNA) contributes to variable infection outcomes. To quantify the effect of mtDNA variation on humoral and cell-mediated innate immune responses, we utilized a panel of fruit fly Drosophila melanogaster cytoplasmic hybrids (cybrids), where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We observed substantial heterogeneity in infection outcomes within the cybrid panel upon bacterial, viral and parasitoid infections, driven by the mitotype. One of the mitotypes, mtKSA2, protected against bacterial, parasitoid, and to a lesser extent, viral infections. Enhanced survival was not a result of improved bacterial clearance, suggesting mtKSA2 confers increased disease tolerance. Transcriptome sequencing showed that the mtKSA2 mitotype had an upregulation of genes related to mitochondrial respiration and phagocytosis in uninfected flies. Upon infection, mtKSA2 flies exhibited infection type and duration specific transcriptomic changes. Furthermore, uninfected mtKSA2 larvae showed immune activation of hemocytes (immune cells), increased hemocyte numbers and ROS production, and enhanced encapsulation response against parasitoid wasp eggs and larvae. Our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes can provide broad protection against infections.

Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with regulated length of lipopolysaccharide in mice

ABSTRACT Non-typhoidal Salmonella enterica (NTS) is a major global foodborne pathogen that poses a major public health concern worldwide, and no vaccines are available for protecting against infection of multiple Salmonella serotypes, therefore, the development of Salmonella vaccines to provide broad protection is valuable. In this work, we aimed to regulate lipopolysaccharide (LPS) synthesis of live Salmonella in vivo for exposing conserved protein antigens on the outer membrane while maintaining smooth LPS patterns in vitro to keep their original ability to invade host cells for inducing cross-protection against infection of multiple Salmonella serotypes. We generated a series of mutants defective in genes to affect the length of LPS. These mutants exhibit in vivo regulated-delayed attenuation and altered length of LPS, and all these mutants were derived from SW067 (ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9 Δfur9) containing ∆pagP81::Plpp lpxE mutation to reduce their endotoxic activity. Animal experiments demonstrated that all regulated delayed attenuated mutants exhibited reduced ability to colonize the organs of the mice, and SW114 (waaI), SW116 (waaJ), SW118 (waaL), and SW120 (wbaP) induced a significant production of IgG and IgA against OMPs isolated from S. Typhimurium, S. Enteritidis, and S. Choleraesuis. SW114 (waaI), SW116 (waaJ), and SW118 (waaL) were capable of conferring significant protection against infection of wild-type S. Enteritidis and S. Choleraesuis, with SW118 (waaL) triggering significant CD4+ T-cell responses as well as the B220low IgG+ BM cell. In conclusion, regulated delayed attenuated Salmonella vaccines with the whole core oligosaccharides of LPS showed a goo.d ability to expose conserved outer antigens and to trigger strong cross-immune responses against both homologous and heterologous Salmonella infections. These results give new insight into the development of the Salmonella vaccine against multiple serotypes of Salmonella.