RESUMO
A randomized, active-controlled, double-blind, first-in-human, phase 1 study was conducted in healthy Korean adults to evaluate the safety, tolerability, and immunogenicity of EuNmCV-5, a new pentavalent meningococcal vaccine targeting serogroups A, C, W, X, and Y. Sixty participants randomly received a single dose of either EuNmCV-5 or MenACWY-CRM, a quadrivalent vaccine containing serogroups A, C, W, and Y. Safety was assessed through monitoring anaphylactic reactions, adverse events for 28 days, and serious adverse events over 180 days. Immunogenicity was assessed via rabbit complement-dependent serum bactericidal antibody (rSBA) assay. EuNmCV-5 was safe, well-tolerated, and elicited a substantial antibody titer increase. The seroprotection rates exceeded 96.7%, and the seroconversion rates were over 85% for all the targeted serogroups. It showed higher seroconversion rates against serogroups A and C (p = 0.0016 and 0.0237, respectively) and elicited a substantial increase in GMT for all targeted serogroups compared to the MenACWY-CRM.ClinicalTrials.gov identifier: NCT05739292.
RESUMO
During the COVID-19 pandemic, expedient vaccine production has been slowed by the shortage of safe and effective raw materials, such as adjuvants, essential components to enhance the efficacy of vaccines. Monophosphoryl lipid A (MPLA) is a potent and safe adjuvant used in human vaccines, including the Shingles vaccine, Shingrix. 3-O-desacyl-4'-monophosphoryl lipid A (MPL), a representative MPLA adjuvant commercialized by GSK, was prepared via chemical conversion of precursors isolated from Salmonella typhimurium R595. However, the high price of these materials limits their use in premium vaccines. To combat the scarcity and high cost of safe raw materials for vaccines, we need to develop a feasible MPLA production method that is easily scaled up to meet industrial requirements. In this study, we engineered peptidoglycan and outer membrane biosynthetic pathways in Escherichia coli and developed a Escherichia coli strain, KHSC0055, that constitutively produces EcML (E. coli-produced monophosphoryl lipid A) without additives such as antibiotics or overexpression inducers. EcML production was optimized on an industrial scale via high-density fed-batch fermentation, and obtained 2.7 g of EcML (about 135,000 doses of vaccine) from a 30-L-scale fermentation. Using KHSC0055, we simplified the production process and decreased the production costs of MPLA. Then, we applied EcML purified from KHSC0055 as an adjuvant for a COVID-19 vaccine candidate (EuCorVac-19) currently in clinical trial stage III in the Philippines. By probing the efficacy and safety of EcML in humans, we established KHSC0055 as an efficient cell factory for MPLA adjuvant production.
Assuntos
Adjuvantes de Vacinas , Lipídeo A/análogos & derivados , Vacinas , Humanos , Escherichia coli/genética , Vacinas contra COVID-19 , Pandemias , Adjuvantes ImunológicosRESUMO
OBJECTIVE: EuCorVac-19 (ECV-19), an adjuvanted liposome-displayed receptor binding domain (RBD) COVID-19 vaccine, previously reported interim Phase 2 trial results showing induction of neutralizing antibodies 3 weeks after prime-boost immunization. The objective of this study was to determine the longer-term antibody response of the vaccine. METHODS: To assess immunogenicity 6 and 12 months after vaccination, participants in the Phase 2 trial (NCT04783311) were excluded if they: 1) withdrew, 2) reported COVID-19 infection or additional vaccination, or 3) exhibited increasing Spike (S) antibodies (representing possible non-reported infection). Following exclusions, of the 197 initial subjects, anti-S IgG antibodies and neutralizing antibodies were further assessed in 124 subjects at the 6-month timepoint, and 36 subjects at the 12-month timepoint. RESULTS: Median anti-S antibody half-life was 52 days (interquartile range [IQR]:42-70), in the "early" period from 3 weeks to 6 months, and 130 days (IQR:97-169) in the "late" period from 6 to 12 months. There was a negative correlation between initial antibody titer and half-life. Anti-S and neutralizing antibody responses were correlated. Neutralizing antibody responses showed longer half-lives; the early period had a median half-life of 120 days (IQR:81-207), and the late period had a median half-life of 214 days (IQR:140-550). CONCLUSION: These data establish antibody durability of ECV-19, using a framework to analyze COVID-19 vaccine-induced antibodies during periods of high infection.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , Vacinas contra COVID-19/efeitos adversos , Lipossomos , COVID-19/prevenção & controle , Anticorpos Neutralizantes , Vacinas de Subunidades Antigênicas , República da Coreia , Anticorpos AntiviraisRESUMO
There is a need for next-generation cholera vaccines that provide high-level and durable protection in young children in cholera-endemic areas. A cholera conjugate vaccine (CCV) is in development to address this need. This vaccine contains the O-specific polysaccharide (OSP) of Vibrio cholerae O1 conjugated via squaric acid chemistry to a recombinant fragment of the tetanus toxin heavy chain (OSP:rTTHc). This vaccine has been shown previously to be immunogenic and protective in mice and found to be safe in a recent preclinical toxicological analysis in rabbits. We took advantage of excess serum samples collected as part of the toxicological study and assessed the immunogenicity of CCV OSP:rTTHc in rabbits. We found that vaccination with CCV induced OSP-, lipopolysaccharide (LPS)-, and rTTHc-specific immune responses in rabbits, that immune responses were functional as assessed by vibriocidal activity, and that immune responses were protective against death in an established virulent challenge assay. CCV OSP:rTTHc immunogenicity in two animal model systems (mice and rabbits) is encouraging and supports further development of this vaccine for evaluation in humans.
Assuntos
Vacinas contra Cólera , Cólera , Vibrio cholerae O1 , Criança , Coelhos , Humanos , Animais , Camundongos , Pré-Escolar , Cólera/prevenção & controle , Antígenos O , Toxina Tetânica , Vacinas Conjugadas , Imunoglobulina M , Vacinação , Formação de Anticorpos , Modelos Animais de Doenças , Anticorpos Antibacterianos , Toxina da CóleraRESUMO
The typhoid conjugate vaccine (TCV) ensures a long-lasting protective immune response, requires fewer doses and is fit for children under 2 years of age. From Phase I study, EuTCV displayed considerable immunogenicity and reliable safety, thus endorsing further examination in Phase II/III trials. Therefore, a clinical Phase II/III study (NCT04830371) was conducted to evaluate its efficacy in healthy Filipino participants aged 6 months to 45 years through administration of the test vaccine (Arm A, B, and C) or comparator vaccine Typbar-TCV® (Arm D). Sera samples were collected pre-vaccination (Visit 1) and post-vaccination (Visit 4, Day 28) to assess the immunogenicity of EuTCV and Typbar-TCV®. During the study, participants were regularly monitored through scheduled visits to the clinic to report any adverse events associated with the vaccine. For vaccine safety, the proportion of solicited and unsolicited Treatment-Emergent Adverse Events was all comparable between EuTCV and Typbar-TCV® groups. A single dose of EuTCV produced seroconversion in 99.4% of treated participants, with seroconversion rates non-inferior to that of Typbar-TCV®. Batch-to-batch consistency was concluded based on the 90% Confidence Interval of the geometric mean ratio (EuTCV Arm A, B, and C) at Week 4, lying within the equivalence margin of 0.5 to 2.0 for all batches. Results from this Phase II/III clinical trial of EuTCV in healthy volunteers show comparable safety and considerable immunogenicity, compared to Typbar-TCV®, meeting the objectives of this pivotal study. ClinicalTrials.gov registration number: NCT04830371.
Assuntos
Vacina Antivariólica , Febre Tifoide , Vacinas Tíficas-Paratíficas , Criança , Humanos , Lactente , Febre Tifoide/prevenção & controle , Vacinas Conjugadas , Vacinação , Imunogenicidade da VacinaRESUMO
BACKGROUND: Numerous vaccine strategies are being advanced to control SARS-CoV-2, the cause of the COVID-19 pandemic. EuCorVac-19 (ECV19) is a recombinant protein nanoparticle vaccine that displays the SARS-CoV-2 receptor-binding domain (RBD) on immunogenic nanoliposomes. METHODS: Initial study of a phase 2 randomized, observer-blind, placebo-controlled trial to assess the immunogenicity, safety, and tolerance of ECV19 was carried out between July and October 2021. Two hundred twenty-nine participants were enrolled at 5 hospital sites in South Korea. Healthy adults aged 19-75 without prior known exposure to COVID-19 were vaccinated intramuscularly on day 0 and day 21. Of the participants who received two vaccine doses according to protocol, 100 received high-dose ECV19 (20 µg RBD), 96 received low-dose ECV19 (10 µg RBD), and 27 received placebo. Local and systemic adverse events were monitored. Serum was assessed on days 0, 21, and 42 for immunogenicity analysis by ELISA and neutralizing antibody response by focus reduction neutralization test (FRNT). RESULTS: Low-grade injection site tenderness and pain were observed in most participants. Solicited systemic adverse events were less frequent, and mostly involved low-grade fatigue/malaise, myalgia, and headache. No clinical laboratory abnormalities were observed. Adverse events did not increase with the second injection and no serious adverse events were solicited by ECV19. On day 42, Spike IgG geometric mean ELISA titers were 0.8, 211, and 590 Spike binding antibody units (BAU/mL) for placebo, low-dose and high-dose ECV19, respectively (p < 0.001 between groups). Neutralizing antibodies levels of the low-dose and high-dose ECV19 groups had FRNT50 geometric mean values of 129 and 316, respectively. Boosting responses and dose responses were observed. Antibodies against the RBD correlated with antibodies against the Spike and with virus neutralization. CONCLUSIONS: ECV19 was generally well-tolerated and induced antibodies in a dose-dependent manner that neutralized SARS-CoV-2. The unique liposome display approach of ECV19, which lacks any immunogenic protein components besides the antigen itself, coupled with the lack of increased adverse events during boosting suggest the vaccine platform may be amenable to multiple boosting regimes in the future. Taken together, these findings motivate further investigation of ECV19 in larger scale clinical testing that is underway. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov as # NCT04783311.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Adulto , Humanos , Anticorpos Neutralizantes , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Pandemias , Proteínas Recombinantes/genética , SARS-CoV-2 , Adulto Jovem , Pessoa de Meia-Idade , IdosoRESUMO
The capability of emulating neural functionalities efficiently in hardware is crucial for building neuromorphic computing systems. While various types of neuro-mimetic devices have been investigated, it remains challenging to provide a compact device that can emulate spiking neurons. In this work, we propose a non-volatile spin-based device for efficiently emulating a leaky integrate-and-fire neuron. By incorporating an exchange-coupled composite free layer in spin-orbit torque magnetic tunnel junctions, multi-domain magnetization switching dynamics is exploited to realize gradual accumulation of membrane potential for a leaky integrate-and-fire neuron with compact footprints. The proposed device offers significantly improved scalability compared with previously proposed spin-based neuro-mimetic implementations while exhibiting high energy efficiency and good controllability. Moreover, the proposed neuron device exhibits a varying leak constant and a varying membrane resistance that are both dependent on the magnitude of the membrane potential. Interestingly, we demonstrate that such device-inspired dynamic behaviors can be incorporated to construct more robust spiking neural network models, and find improved resiliency against various types of noise injection scenarios. The proposed spintronic neuro-mimetic devices may potentially open up exciting opportunities for the development of efficient and robust neuro-inspired computational hardware.
Assuntos
Modelos Neurológicos , Neurônios , Potenciais da Membrana , Redes Neurais de Computação , Neurônios/fisiologia , RuídoRESUMO
There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.
Assuntos
Vacinas contra Cólera , Cólera , Vibrio cholerae O1 , Administração Oral , Anticorpos Antibacterianos , Pré-Escolar , Cólera/prevenção & controle , Humanos , Imunoglobulina A , Imunoglobulina G , Imunoglobulina M , Células B de Memória , Vacinas ConjugadasRESUMO
Convolutional neural networks (CNNs) are a state-of-the-art technique for speech emotion recognition. However, CNNs have mostly been applied to noise-free emotional speech data, and limited evidence is available for their applicability in emotional speech denoising. In this study, a cascaded denoising CNN (DnCNN)-CNN architecture is proposed to classify emotions from Korean and German speech in noisy conditions. The proposed architecture consists of two stages. In the first stage, the DnCNN exploits the concept of residual learning to perform denoising; in the second stage, the CNN performs the classification. The classification results for real datasets show that the DnCNN-CNN outperforms the baseline CNN in overall accuracy for both languages. For Korean speech, the DnCNN-CNN achieves an accuracy of 95.8%, whereas the accuracy of the CNN is marginally lower (93.6%). For German speech, the DnCNN-CNN has an overall accuracy of 59.3-76.6%, whereas the CNN has an overall accuracy of 39.4-58.1%. These results demonstrate the feasibility of applying the DnCNN with residual learning to speech denoising and the effectiveness of the CNN-based approach in speech emotion recognition. Our findings provide new insights into speech emotion recognition in adverse conditions and have implications for language-universal speech emotion recognition.
Assuntos
Redes Neurais de Computação , Fala , Emoções , Idioma , PercepçãoRESUMO
An injectable typhoid conjugate vaccine (TCV) provides longer-lasting protection, requires fewer doses, and is suitable for children aged >2 years. In addition, TCV is preferred at most ages owing to its improved immunological properties as it overcomes the limitation of Vi polysaccharide vaccines. Here, we assessed the safety, tolerability, and immunogenicity of a TCV, Vi-CRM197, termed EuTCV, in an open-label clinical phase I study in healthy Filipino adults. This study was conducted in 75 healthy adults aged 18-45 years who were randomized in a 1:1:1 ratio based on the vaccines administered: EuTCV (Test), Typbar-TCV® (WHO prequalified vaccine) and Typhim Vi® (Vi polysaccharide vaccine). The study vaccines were administered at a dose of 25 µg of Vi-CRM197 conjugate by intramuscular injection as a single dose to each of the 25 participants/group, and their immunogenicity and overall safety were assessed for 42 days post-vaccination. All study participants (n = 25/group) completed the trial without dropouts. There were no deaths, SAEs, or events leading to premature withdrawal from the study. Anti-Vi IgG antibody titer (geometric mean titer) of EuTCV group on day 42 was 65.325 [95% CI (36.860, 115.771)], which was significantly higher than that of the WHO prequalified TCV [24.795, 95% CI (16.164, 38.033) p = 0.0055] and the Vi polysaccharide vaccine [7.998, 95% CI (3.800, 16.835) p < 0.0001]. Moreover, the seroconversion rate of EuTCV and Typbar-TCV® was 100%, but that of Typhim Vi® was only 84%. The IgG1-3 subclass titers and serum bactericidal assay results in the EuTCV group showed higher and better bactericidal capacity than the other groups. EuTCV was well tolerated and exhibited an acceptable safety profile in the study population. The Vi-CRM197 conjugate dose of 25 µg may be considered effective in terms of efficacy and safety. ClinicalTrials.gov registration number: NCT03956524.
Assuntos
Febre Tifoide , Vacinas Tíficas-Paratíficas , Adolescente , Adulto , Anticorpos Antibacterianos , Proteínas de Bactérias , Criança , Pré-Escolar , Humanos , Imunogenicidade da Vacina , Pessoa de Meia-Idade , Febre Tifoide/prevenção & controle , Vacinas Tíficas-Paratíficas/efeitos adversos , Vacinas Conjugadas/efeitos adversos , Adulto JovemRESUMO
Glycoconjugate vaccines are vaccines in which a bacterial polysaccharide antigen is conjugated to a carrier protein to enhance immunogenicity by promoting T cell-dependent immune response. However, the free (unreacted) polysaccharides remaining after the conjugation process can inhibit the immunogenicity of a conjugate vaccine. Thus, we aimed to reduce the unbound free polysaccharides in the polysaccharide-protein conjugation process for the development of a new 15-valent pneumococcal conjugate vaccine (PCV15) by varying some factors that may affect the conjugation results such as polysaccharide/protein ratio, polysaccharide size, and concentration of a coupling agent in a conjugation reaction mixture. Concentrations of a coupling agent, carbodiimide (EDAC), and a carrier protein (CRM197) used in PCV15 production, during the conjugation process, had little effect on the content of free polysaccharides. However, the size of the polysaccharide was identified as the critical factor to control the free polysaccharide content, with an inverse relationship observed between the molecular weight of the polysaccharide and the residual free polysaccharide content after conjugation. Based on these results, a new PCV15 with low free polysaccharide contamination was produced and tested for immunogenicity using a rabbit model to show that it induces similar level of immune responses in rabbits compared to a comparator vaccine Prevnar13®.
Assuntos
Glicoconjugados/química , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Vacinas Conjugadas/química , Glicoconjugados/imunologia , Glicoconjugados/uso terapêutico , Humanos , Imunidade/efeitos dos fármacos , Imunidade/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/química , Vacinas Pneumocócicas/uso terapêutico , Polissacarídeos/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêuticoRESUMO
Vibrio cholerae, cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2-O-methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.
Assuntos
Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Lipopolissacarídeos/imunologia , Vibrio cholerae O1/imunologia , Animais , Vacinas Bacterianas/imunologia , Bacteriófagos/genética , Epitopos , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Camundongos , Antígenos O/imunologia , Análise de Sequência , Sorogrupo , Vibrio cholerae O1/genéticaRESUMO
Emerging influenza viruses pose an extreme global risk to human health, resulting in an urgent need for effective vaccination against influenza infection. Adjuvants are vital components that can improve vaccine efficacy, yet only a few adjuvants have been licensed in human vaccines. Here, we investigate the adjuvant effects of Escherichia coli-produced monophosphoryl lipid A (MPL), named EcML, in enhancing the immunogenicity and efficacy of an influenza vaccine. Similar to MPL, EcML activated dendritic cells and enhanced the antigen processing of cells in vitro. Using ovalbumin (OVA) as a model antigen, EcML increased OVA-specific antibody production, cytotoxic T lymphocyte activity. The safety of EcML was demonstrated as being similar to that of MPL by showing not significant in vitro cell cytotoxicity but transient systemic inflammatory responses within 24 h in OVA immunized mice. Importantly, mice vaccinated with pandemic H1N1 (pH1N1) vaccine antigen, combined with EcML, were fully protected from pH1N1 virus infection by enhanced influenza-specific antibody titers, hemagglutination inhibition titers, and IFN-γ- secreting cells. Taken together, our results strongly suggest that EcML might be a promising vaccine adjuvant for preventing influenza virus infection.
RESUMO
Streptococcus pneumoniae is the causative agent of many diseases, most notably pneumonia. Most of the currently used vaccines to protect against this pathogen employ pneumococcal capsular polysaccharides (CPSs) as antigens, but purifying CPS of sufficient quality has been challenging. A purification process for CPS comprising conventional methods such as ultrafiltration, CTAB precipitation, and chromatography was previously established; however, this method resulted in high cell wall polysaccharide (CWPS) contamination, especially for serotype 5. Thus, a better purification method that yields CPS of a higher quality is needed for vaccine development. In this study, we significantly reduced CWPS contamination in serotype 5 CPS by improving the ultrafiltration and CTAB precipitation steps. Moreover, by applying an acid precipitation process to further remove other impurities, serotype 5 CPS was obtained with a lower impurity such as decreased nucleic acid contamination. This improved method was also successfully applied to 14 other serotypes (1, 3, 4, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F). To assess the immunogenicity of the CPS from the 15 serotypes, two sets of 15-valent pneumococcal conjugate vaccines were prepared using the previous purification method and the improved method developed here; these vaccines were administered to a rabbit model. Enzyme-linked immunosorbent assay and opsonophagocytic assay demonstrated higher immunogenicity of the conjugate vaccine prepared using CPS produced by the improved purification process.
RESUMO
Spiking Neural Networks (SNNs) have recently emerged as a prominent neural computing paradigm. However, the typical shallow SNN architectures have limited capacity for expressing complex representations while training deep SNNs using input spikes has not been successful so far. Diverse methods have been proposed to get around this issue such as converting off-the-shelf trained deep Artificial Neural Networks (ANNs) to SNNs. However, the ANN-SNN conversion scheme fails to capture the temporal dynamics of a spiking system. On the other hand, it is still a difficult problem to directly train deep SNNs using input spike events due to the discontinuous, non-differentiable nature of the spike generation function. To overcome this problem, we propose an approximate derivative method that accounts for the leaky behavior of LIF neurons. This method enables training deep convolutional SNNs directly (with input spike events) using spike-based backpropagation. Our experiments show the effectiveness of the proposed spike-based learning on deep networks (VGG and Residual architectures) by achieving the best classification accuracies in MNIST, SVHN, and CIFAR-10 datasets compared to other SNNs trained with a spike-based learning. Moreover, we analyze sparse event-based computations to demonstrate the efficacy of the proposed SNN training method for inference operation in the spiking domain.
RESUMO
Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.
Assuntos
Adjuvantes Imunológicos , Escherichia coli , Lipídeo A/análogos & derivados , Engenharia Metabólica , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/isolamento & purificação , Adjuvantes Imunológicos/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Imunoglobulina G/biossíntese , Lipídeo A/biossíntese , Lipídeo A/genética , Lipídeo A/isolamento & purificação , Lipídeo A/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A new 15-valent pneumococcal conjugate vaccine (PCV15) against serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F has been developed using aluminum phosphate as an adjuvant. Using the rabbit model, immunogenicity of each serotype was evaluated by measuring antigen specific antibodies and functional antibody titers and comparing them to a control vaccine, Prevnar13®. Among the shared serotypes in both PCV15 and Prevnar13®, Type 3 and 23F in PCV15 exhibited a lower opsonic index than Prevnar13®. Conversely, the other types showed greater or nearly the same immunogenic effects. Type 11A and 22F are two additional serotypes included in PCV15, and only 22F showed a reasonable opsonic index compared with other types. Type 11A exhibited a basal level fold-increase in OPA; thus, we further optimized 11A as well as 3 and 23F by controlling the polysaccharide-to-protein conjugation ratio as a variable. Antibody levels and functional antibody activities were evaluated by ELISA and OPA, and improved levels of immunogenic activities were observed for all three serotypes. In this study, we propose a new PCV15 candidate, in which the common 13 serotypes and a licensed control vaccine have equivalent efficacy while two additional serotypes showed adequate immunogenicity in the rabbit model.
Assuntos
Anticorpos Antibacterianos/imunologia , Imunogenicidade da Vacina , Vacinas Pneumocócicas , Streptococcus pneumoniae/imunologia , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Vacinas Pneumocócicas/imunologia , Vacinas Pneumocócicas/farmacologia , Coelhos , Vacinas ConjugadasRESUMO
Cross-reactive material 197 (CRM197) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM197 has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect CRM197 and CRM197 conjugate vaccines, we generated a human anti-CRM197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM197 polyclonal antibody. The affinity (KD) of 3F9 for CRM197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM197. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was ï¼1 ng/ml CRM197. In addition, the 3F9 antibody bound to the CRM197-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM197 and CRM197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM197 and to develop a sandwich ELISA for CRM197 conjugate vaccines.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos , Anticorpos Monoclonais/isolamento & purificação , Formação de Anticorpos , Reações Antígeno-Anticorpo , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Mapeamento de Epitopos , Humanos , Imunoglobulina G/imunologia , Limite de Detecção , Camundongos , Modelos MolecularesRESUMO
Spiking Neural Networks (SNNs) are fast becoming a promising candidate for brain-inspired neuromorphic computing because of their inherent power efficiency and impressive inference accuracy across several cognitive tasks such as image classification and speech recognition. The recent efforts in SNNs have been focused on implementing deeper networks with multiple hidden layers to incorporate exponentially more difficult functional representations. In this paper, we propose a pre-training scheme using biologically plausible unsupervised learning, namely Spike-Timing-Dependent-Plasticity (STDP), in order to better initialize the parameters in multi-layer systems prior to supervised optimization. The multi-layer SNN is comprised of alternating convolutional and pooling layers followed by fully-connected layers, which are populated with leaky integrate-and-fire spiking neurons. We train the deep SNNs in two phases wherein, first, convolutional kernels are pre-trained in a layer-wise manner with unsupervised learning followed by fine-tuning the synaptic weights with spike-based supervised gradient descent backpropagation. Our experiments on digit recognition demonstrate that the STDP-based pre-training with gradient-based optimization provides improved robustness, faster (~2.5 ×) training time and better generalization compared with purely gradient-based training without pre-training.
RESUMO
Concomitant rupture of the subclavian vessels and the left main bronchus caused by blunt trauma is a serious condition. Moreover, the diagnosis of a tracheobronchial injury with rupture of the subclavian vessels can be difficult. This report describes the case of a 33-year-old man who suffered from blunt trauma that resulted in the rupture of the left subclavian artery and vein. The patient underwent an operation for vascular control. On postoperative day 3, the left main bronchus was found to be transected on a computed tomography scan and bronchoscopy. The transected bronchus was anastomosed in an end-to-end fashion. He recovered without any notable problems. Although the bronchial injury was not detected early, this case of concomitant rupture of the great vessels and the airway was successfully treated after applying extracorporeal membrane oxygenation.