Peptide-ligand conjugate based immunotherapeutic approach for targeted dismissal of non-structural protein 1 of dengue virus: A novel therapeutic solution for mild and severe dengue infections.

Dengue virus infection has significantly increased, with reported cases soaring from 505,430 in 2000 to 2,809,818 in 2022, emphasizing the need for effective treatments. Among the eleven structural and non-structural proteins of DENV, Non-structural protein 1 (NS1) has emerged as a promising target due to its diverse role in modulating the immune response, inducing vascular leakage, and facilitating viral replication and assembly. Monoclonal antibodies are the sole therapeutics to target NS1, but concerns about their cross-reactivity persist. Given these concerns, our study focuses on designing a novel Peptide Ligand Conjugate (PLC) as a potential alternative immunotherapeutic agent against NS1. This PLC aims to mediate the immune elimination of soluble NS1 and NS1-presenting DENV-infected host cells by pre-existing vaccine-induced immunity. By employing the High Throughput Virtual Screening (HTVS) method, QikProp analysis, and Molecular Dynamics studies, we identified three hits from Asinex Biodesigned Ligands out of 220,177 compounds that show strong binding affinity towards the monoclonal binding site of NS1 protein. After a rigorous analysis of physicochemical characteristics, antigenicity, allergenicity, and toxicity using various servers, we selected two peptides: the minimum epitopic region of the Diphtheria and Tetanus toxins as the peptide components of the PLCs. A non-cleavable, non-reactive oxime linker connected the ligand with the peptide through oxime and amide bonds. DPT vaccine is widely used in dengue-endemic countries, and it has been reported that antibodies titer against MER of Diphtheria toxin and Tetanus toxins persist lifelong in DPT-vaccinated people. Therefore, once the rationally designed PLCs bind to NS1 through the ligands, the peptide will induce an immune response against NS1 by triggering pre-existing DPT antibodies and activating memory cells. This orchestrated immune response will destroy soluble NS1 and NS1-expressing DENV-infected cells, thereby reducing the illness of severe dengue hemorrhagic fever and the DENV infection, respectively. Given the increasing demand for new therapeutics for DENV treatment, further investigation into this novel immune-therapeutic strategy may offer a new avenue for treating mild and severe dengue infections.

In situ delivery of nanoparticles formulated with micron-sized crystals protects from murine melanoma.

INTRODUCTION: Intratumoral injections of novel therapeutics can activate tumor antigen-specific T cells for locoregional tumor control and may even induce durable systemic protection (against distant metastases) via recirculating T cells. Here we explored the possibility of a universal immunotherapy that promotes T-cell responses in situ and beyond, upon intratumoral injection of nanoparticles formulated with micron-sized crystals. METHODS: Cucumber mosaic virus-like particles containing a tetanus toxin peptide (CuMVTT) were formulated with microcrystalline tyrosine (MCT) adjuvant and injected directly in B16F10 melanoma tumors. To further enhance immunogenicity, we loaded the nanoparticles with a TLR7/8 ligand and incorporated a universal tetanus toxin T-helper cell peptide. We assessed therapeutic efficacy and induction of local and systemic immune responses, including RNA sequencing, providing broad insight into the tumor microenvironment and correlates of protection. RESULTS: MCT crystals were successfully decorated with CuMVTT nanoparticles. This 'immune-enhancer' formed immunogenic depots in injected tumors, enhanced polyfunctional CD8+ and CD4+ T cells, and inhibited B16F10 tumor growth locally and systemically. Local inflammation and immune responses were associated with upregulation of genes involved in complement activation and collagen formation. CONCLUSIONS: Our new immune-enhancer turned immunologically cold tumors into hot ones and inhibited local and distant tumor growth. This type of immunotherapy does not require the identification of (patient-individual) relevant tumor antigens. It is well tolerated, non-infectious, and affordable, and can readily be upscaled for future clinical testing and broad application in melanoma and likely other solid tumors.

Immunization with placenta-specific 1 (plac1) induces potent anti-tumor responses and prolongs survival in a mouse model of melanoma.

PURPOSE: Melanoma is a malignant and metastatic form of skin cancer, which is not diagnosed in early stages of the disease. Nowadays, immunotherapy is changing the treatment landscape for metastatic melanoma. Placenta-specific1 (PLAC1) is a cancer-testis-placenta (CTP) antigen with differential expression in melanoma tissues. Here, we evaluated the potential of plac1 to induce anti-cancer immune responses as well as to prevent cancer development in a mouse model of melanoma. METHODS: Two proteins containing full extracellular domain (ED) of mouse plac1+KDEL3 and full ED of mouse plac1+ tetanus toxin P2 and P30+ pan DR epitope (PADRE) â€‹+ â€‹KDEL3 were produced and injected in mice to evaluate their capacity to induce anti-cancer immune responses as well as their potential to prevent melanoma development. Induction of plac1-specific humoral and cellular responses as well as tumor-associated parameters were tested in a series of 36 mice. RESULTS: Sera of mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 contained antibodies able to react with surface plac1 in B16F10 â€‹cells. Both proteins induced proliferative cellular immune responses against B16F10 â€‹cells and plac1-specific cytotoxic T cells (CTL) and CD107a â€‹+ â€‹CTL responses, which was higher in mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3. Splenocytes of mice vaccinated with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 exerted a significant cytotoxicity against B16F10 â€‹cells. Vaccination with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 significantly delayed B16F10-induced tumor onset, reduced tumor growth, and increased survival. Tumors induced by B16F10 expressed plac1 in vivo. CONCLUSION: Our results pave the way for development of effective melanoma preventive vaccine in humans, although further studies are needed.

[Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain].

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P < 0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P < 0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.

Intramuscular tetanus neurotoxin reverses muscle atrophy: a randomized controlled trial in dogs with spinal cord injury.

BACKGROUND: Motor symptoms of spinal cord injury (SCI) considerably impair quality of life and are associated with a high risk of secondary diseases. So far, no pharmacological treatment is available for these symptoms. Therefore, we conducted a randomized, double-blinded, placebo-controlled study in dogs with spontaneous SCI due to disc herniation to test whether a reduction of spinal inhibitory activity by intramuscular injections of tetanus neurotoxin (TeNT) alleviates motor symptoms such as muscle atrophy or gait function. METHODS: To this end, 25 dogs were treated with injections of either TeNT or placebo into their paretic hindlimb muscles. Effects of TeNT on muscle thickness were assessed by ultrasound, while effects on gait function were measured using the modified functional scoring system in dogs. RESULTS: Four weeks after the TeNT injections, muscle thickness of the gluteus medius muscle (before median 1.56 cm [inter-quartile range {IQR} 1.34-1.71 cm] and after median 1.56 cm [IQR 1.37-1.85 cm], P-value 0.0133) as well as of the rectus femoris muscle (before median 0.76 cm [IQR 0.60-0.98 cm] and after median 0.93 cm [IQR 0.65-1.05 cm], P-value 0.0033) significantly increased in the TeNT group. However, there was no difference in gait function between the TeNT and placebo groups. The treatment was well tolerated by all dogs without any signs of generalized tetanus symptoms or any spreading of effects beyond the lumbar level of the injected hindlimbs. CONCLUSIONS: With regard to the beneficial effects on muscle thickness, intramuscular injections of TeNT represent the first pharmacological approach that focally reverses muscle atrophy in SCI. Moreover, the study data support the safety of this treatment when TeNT is used at low dose.

Tétanos generalizado: Una amenaza persistente, reporte de tres casos y revisión de la literatura / Generalized tetanus: A persistent threat, review of the literature and report of three cases

RESUMEN INTRODUCCIÓN: El tétanos es una enfermedad inmunoprevenible, ocasionada por la bacteria Clostridium tetani, desencadenando una enfermedad caracterizada por espasmos musculares, insuficiencia respiratoria y disautonomías, potencialmente mortal. MATERIALES Y MÉTODOS: Presentamos una serie de tres pacientes que consultaron al servicio de urgencias por presentar trismus, rigidez muscular generalizada y dificultad respiratoria, requiriendo manejo en la unidad de cuidados intensivos, con relajación muscular y administración intramuscular e intratecal de inmunoglobulina antitetánica, con evolución satisfactoria en todos los casos. DISCUSIÓN: Su tratamiento está divido en dos grandes secciones; la primera parte, el control de la infección y eliminación del agente causal, con lavado y desbridamiento de heridas, administración de antibióticos y neutralización de la neurotoxina. La segunda parte del tratamiento está en el soporte vital en la unidad de cuidados intensivos, con la administración de sedación, relajación muscular, control de disautonomías y manejo de complicaciones. CONCLUSIONES: El tétanos a pesar de los avances en vacunación aún es una enfermedad presente, cuyo diagnóstico y tratamiento rápido y adecuado, permite sobrevivir a los pacientes, como en los casos aquí reportados.

ABSTRACT INTRODUCTION: Tetanus is an immuno-preventable disease, produced by the bacterium Clostridium tetani, that causes a disease characterized by muscle spasms, respiratory insufficiency and life-threatening dysautonomia. MATERIALS AND METHODS: We present a series of three patients who consulted for trismus, muscle stiffness and respiratory failure, which required intensive care management, muscle relaxation, intramuscular and intrathecal administration of tetanus immu-noglobulin, with satisfactory outcomes in all the cases. DISCUSSION: Its treatment is divided into two main sections; the first part, the control of infection and elimination of the causative agent, with washing and debridement of wounds, administration of antibiotics and neutralization of the neurotoxin. The second part is life support in the intensive care unit, with the administration of sedation, muscular relaxation and control of dysautonomia and the management of complications. CONCLUSIONS: Despite the advances in vaccination, tetanus is still a present disease, whose diagnosis and rapid and adequate treatment allows patients to survive, as in the cases reported here.

Characterising the Phenotypic Diversity of Antigen-Specific Memory B Cells Before and After Vaccination.

The diversity of B cell subsets and their contribution to vaccine-induced immunity in humans are not well elucidated but hold important implications for rational vaccine design. Prior studies demonstrate that B cell subsets distinguished by immunoglobulin (Ig) isotype expression exhibit divergent activation-induced fates. Here, the antigen-specific B cell response to tetanus toxoid (TTd) booster vaccination was examined in healthy adults, using a dual-TTd tetramer staining flow cytometry protocol. Unsupervised analyses of the data revealed that prior to vaccination, IgM-expressing CD27+ B cells accounted for the majority of TTd-binding B cells. 7 days following vaccination, there was an acute expansion of TTd-binding plasmablasts (PB) predominantly expressing IgG, and a minority expressing IgA or IgM. Frequencies of all PB subsets returned to baseline at days 14 and 21. TTd-binding IgG+ and IgA+ memory B cells (MBC) exhibited a steady and delayed maximal expansion compared to PB, peaking in frequencies at day 14. In contrast, the number of TTd-binding IgM+IgD+CD27+ B cells and IgM-only CD27+ B cells remain unchanged following vaccination. To examine TTd-binding capacity of IgG+ MBC and IgM+IgD+CD27+ B cells, surface TTd-tetramer was normalised to expression of the B cell receptor-associated CD79b subunit. CD79b-normalised TTd binding increased in IgG+ MBC, but remained unchanged in IgM+IgD+CD27+ B cells, and correlated with the functional affinity index of plasma TTd-specific IgG antibodies, following vaccination. Finally, frequencies of activated (PD-1+ICOS+) circulating follicular helper T cells (cTFH), particularly of the CXCR3-CCR6- cTFH2 cell phenotype, at their peak expansion, strongly predicted antigen-binding capacity of IgG+ MBC. These data highlight the phenotypic and functional diversity of the B cell memory compartment, in their temporal kinetics, antigen-binding capacities and association with cTFH cells, and are important parameters for consideration in assessing vaccine-induced immune responses.

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion.

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.(AU)

Neurotrophic Properties of C-Terminal Domain of the Heavy Chain of Tetanus Toxin on Motor Neuron Disease.

The carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts a neuroprotective effect in neurodegenerative diseases via the activation of signaling pathways related to neurotrophins, and also through inhibiting apoptotic cell death. Here, we demonstrate that Hc-TeTx preserves motoneurons from chronic excitotoxicity in an in vitro model of amyotrophic lateral sclerosis. Furthermore, we found that PI3-K/Akt pathway, but not p21ras/MAPK pathway, is involved in their beneficial effects under chronic excitotoxicity. Moreover, we corroborate the capacity of the Hc-TeTx to be transported retrogradely into the spinal motor neurons and also its capacity to bind to the motoneuron-like cell line NSC-34. These findings suggest a possible therapeutic tool to improve motoneuron preservation in neurodegenerative diseases such as amyotrophic lateral sclerosis.

Combination Vaccination With Tetanus Toxoid and Enhanced Tumor-Cell Based Vaccine Against Cervical Cancer in a Mouse Model.

Cervical cancer is the fourth most common cancer in women with an estimated 570,000 new cases in 2018 which constitute about 6. 6% of all cancers in women according to WHO report 2018. Approximately 90% of the 270,000 deaths from cervical cancer in 2015 occurred in low- and middle-income countries. In cervical cancers, which is caused by human papillomavirus (HPV) infection, the expression of HPV 16 E6 and E7 proteins are essential for tumor cell transformation and maintenance of malignancy. Prophylactic vaccines against cervical cancer caused by human papillomavirus have not proven successful. Although virus-like particle-based (VLPs) vaccines have been developed with prophylactic activities to prevent most HPV infections, the therapeutic effect of VLP vaccines has yet to be demonstrated for those who were already infected. A recent study showed that pre-conditioning mice with a potent antigen such as tetanus toxoid significantly improves lymph node homing and efficacy of dendritic cells. Tetanus toxoid has also been used in combination with DNA vaccines designed from tumor based antigens. In the present study, we pre-conditioned mice with tetanus toxoid followed by vaccination with a Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) overexpressing tumor-cell based vaccine (GVAX). We observed that pre-conditioning with tetanus toxoid followed by vaccination with GVAX regressed tumor growth and enhanced the overall survival of the mice. Pre-conditioning with tetanus toxoid enhanced the immune response which was observed by enlarged spleen size, higher proliferation rate of lymphocytes, a higher level of IFN-γ, TNF-α, and IL-4 antigen-specific secretions by the splenocytes. Pre-conditioning with tetanus toxoid increased memory T cell migration into the tumor site and spleen. The antigen-specific cytotoxic T cell lysis percentage was also found to be higher in the group of mice vaccinated with the combination of tetanus toxoid and GVAX. Hence, pre-conditioning with tetanus toxoid prior to vaccination with a tumor-cell based vaccine overexpressing GM-CSF might be an effective strategy for targeting E7-specific HPV-associated cervical malignancy.

Neuroprotective Fragment C of Tetanus Toxin Modulates IL-6 in an ALS Mouse Model.

Neuroinflammation plays a significant role in amyotrophic lateral sclerosis (ALS) pathology, leading to the development of therapies targeting inflammation in recent years. Our group has studied the tetanus toxin C-terminal fragment (TTC) as a therapeutic molecule, showing neuroprotective properties in the SOD1G93A mouse model. However, it is unknown whether TTC could have some effect on inflammation. The objective of this study was to assess the effect of TTC on the regulation of inflammatory mediators to elucidate its potential role in modulating inflammation occurring in ALS. After TTC treatment in SOD1G93A mice, levels of eotaxin-1, interleukin (IL)-2, IL-6 and macrophage inflammatory protein (MIP)-1 alpha (α) and galectin-1 were analyzed by immunoassays in plasma samples, whilst protein expression of caspase-1, IL-1ß, IL-6 and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) was measured in the spinal cord, extensor digitorum longus (EDL) muscle and soleus (SOL) muscle. The results showed reduced levels of IL-6 in spinal cord, EDL and SOL in treated SOD1G93A mice. In addition, TTC showed a different role in the modulation of NLRP3 and caspase-1 depending on the tissue analyzed. In conclusion, our results suggest that TTC could have a potential anti-inflammatory effect by reducing IL-6 levels in tissues drastically affected by the disease. However, further research is needed to study more in depth the anti-inflammatory effect of TTC in ALS.

In silico analysis of transmembrane protein 31 (TMEM31) antigen to design novel multiepitope peptide and DNA cancer vaccines against melanoma.

Multiepitope cancer vaccines are announcing themselves as the future of melanoma treatment. Herein, high immunogenic regions of transmembrane protein 31 (TMEM31) antigen were selected according to cytotoxic T lymphocytes' (CTL) epitopes and major histocompatibility complex (MHC) binding affinity through in silico analyses. The 32-62, 77-105, and 125-165 residues of the TMEM31 were selected as the immunodominant fragments. They were linked together by RVRR and HEYGAEALERAG motifs to improve epitopes separation and presentation. In addition, to activate helper T lymphocytes (HTL), Pan HLA DR-binding epitope (PADRE) peptide sequence and tetanus toxin fragment C (TTFrC) were incorporated into the final construct. Also, the Beta-defensin conserved domain was utilized in the final construct as a novel adjuvant for Toll-like receptor 4/myeloid differentiation factor (TLR4-MD) activation. The CTL epitopes, cleavage sites, post-translational modifications, TAP transport efficiency, and B cells epitopes were predicted for the peptide vaccine. The final construct contained multiple CTL and B cell epitopes. In addition, it showed 93.55% and 99.13% population coverage in the world for HLA I and HLA II, respectively. According to these preliminary results, the multiepitope cancer vaccine can be an appropriate choice for further experimental investigations.

Antidepressant effects of C-Terminal domain of the heavy chain of tetanus toxin in a rat model of depression.

The C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) may be of therapeutic potential in motor impairments associated with Parkinson disease (PD). Since depression is a common co-morbid condition with PD, we undertook this study to determine whether Hc-TeTx might also show antidepressant-like properties and whether central brain-derived neurotrophic factor (BDNF) and/or tumor necrosis factor (TNF)-alpha are also affected by it. Adult male Wistar-Kyoto rats, a putative animal model of depression, were treated with various doses of Hc-TeTx (0, 20, 40 and 60 µg/kg, IM) and their performance in the open field locomotor activity (OFLA) as well as in the forced swim test (FST) was evaluated at 24 h, one week and two weeks after the single injection. A separate group of rats were injected with 60 µg/kg Hc-TeTx and sacrificed 24 h later for neurochemical evaluations. Hc-TeTx resulted in a dose-dependent decrease in immobility score after 24 h, whereas OFLA was not affected. Concomitant with the 24 h behavioral effects, the levels of hippocampal and frontal cortical BDNF were significantly increased, whereas the levels of TNF-alpha in both these areas were significantly decreased. The decrease in immobility scores following higher doses of Hc-TeTx were still evident after one week, but not 2 weeks of rest. These results indicate long lasting antidepressant effects of a single Hc-TeTx dose and suggest potential utility of Hc-TeTx in PD-depression co-morbidity.

VaccHemInf project: protocol for a prospective cohort study of efficacy, safety and characterisation of immune functional response to vaccinations in haematopoietic stem cell transplant recipients.

INTRODUCTION: Immune reconstitution after haematopoietic stem cell transplantation (HSCT) is a complex and dynamic process, varying from a state of nearly complete immunosuppression to an expected full immune recovery. Specific vaccination guidelines recommend reimmunisation after HSCT but data regarding vaccine efficacy in this unique population are scarce. New immune functional assays could enable prediction of vaccine response in the setting of HSCT. METHODS AND ANALYSIS: A prospective, longitudinal single-centre cohort study of autologous and allogeneic HSCT recipients was designed in order to determine the vaccine response to five vaccine targets (pneumococcus, hepatitis B virus, Haemophilus Influenzae type b, tetanus and diphtheria) and to correlate it to immune function parameters. A workflow was set up to study serological response to vaccines and to describe the functional immune status of 100 HSCT recipients (50 autologous and 50 allogeneic) before and 3, 12 and 24 months after primary immunisation. At each time point, 'basic' immune status recording (serology, immunophenotyping of lymphocyte subsets by flow cytometry) will be assessed. The immune response will furthermore be evaluated before and 3 months after primary vaccination by two ex vivo immune functional assays assessing: (1) tumour necrosis factor alpha, interferon gamma production and host messenger RNA expression on whole-blood stimulation by lipopolysaccharide or Staphylococcus aureus enterotoxin B and (2) T-lymphocyte proliferation in response to a standard mitogen (phytohaemagglutinin) or to selected recall antigens. Reference intervals will be determined from a cohort of 30 healthy volunteers. This translational study will provide data describing vaccine response, immune functionality of HSCT recipients over time and will allow mapping HSCT recipients with regard to their immune function. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the institutional review board (no 69HCL17_0769). Results will be communicated at scientific meetings and submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03659773; Pre-results.

scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor.

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in airways and intestine. It has been associated with goblet cell metaplasia, as expression of TMEM16A is strongly up-regulated in cystic fibrosis and asthma during mucus hypersecretion. However, the possible role of TMEM16A for mucus production or mucus secretion remains obscure, and whether TMEM16A controls the function of intestinal goblet cells is entirely unknown. Basal mucus secretion in lungs occurs through low levels of ATP in the airway surface liquid. Here, we report for the first time that TMEM16A is essential for basal secretion of mucus in airways and intestine. Airway-ciliated and intestinal epithelial-specific knockout of TMEM16A ( TMEM16Aflox/floxFoxJ1, TMEM16Aflox/floxVil1) leads to accumulation of mucus in airway club (Clara) cells and intestinal goblet cells, respectively. Acute ATP-induced mucus secretion by airway club cells is inhibited when TMEM16A is knocked out in ciliated cells, possibly as a result of compromised release of prosecretory cytokines. Knockdown or inhibition of TMEM16A in human Calu3 airway epithelial cells indicates compromised IL-8 release. In intestinal goblet cells lacking expression of TMEM16A, mucus accumulates as a result of compromised ATP-induced secretion. In contrast, cholinergic mucus secretion by compound exocytosis is independent of TMEM16A. The data demonstrate a previously unrecognized role of TMEM16A for membrane exocytosis and describe a novel, ATP-driven pathway for intestinal mucus secretion. We conclude that ATP-dependent mucus secretion in both airways and intestine requires TMEM16A. The present results may form the basis for a novel, therapeutic approach for the treatment of mucus hypersecretion in inflammatory airway and intestinal disease.-Benedetto, R., Cabrita, I., Schreiber, R., Kunzelmann, K. TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Different frequencies of memory B-cells induced by tetanus, botulinum, and heat-labile toxin binding domains.

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.