RESUMEN

Influenza neuraminidase (NA) is a promising target for a broadly protective vaccine. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to develop N2 NA vaccine candidates. The unique wild type (WT) N2 sequences of human and swine influenza strains isolated between 1957 and 2019 were used to design the COBRA N2-A NA vaccine, while the unique WT N2 sequences of human influenza strains isolated between 2000 and 2019 were used to design the COBRA N2-B NA vaccine. Sera collected from COBRA N2 NA vaccinated mice showed more broadly reactive antibody responses against a broad panel of H×N2 influenza virus strains than sera collected from mice vaccinated with WT N2 NA vaccines. Antibodies elicited by COBRA or WT N2 NA antigens cross react with recent human H3N2 influenza viruses from different clades, while the antibodies elicited by A/Switzerland/9715293/2013 hemagglutinin (HA) reacted with viruses from the same clade. Furthermore, mice vaccinated with COBRA N2-B NA vaccine had lower viral lung titers compared to mock vaccinated mice when challenged with human H3N2 influenza viruses. Thus, the COBRA N2 NA vaccines elicit broadly protective murine anti-NA antibodies against multiple strains across subtypes and the viral loads were significantly decreased in the lungs of the mice in the COBRA N2 NA vaccine groups, compared to the mice in the mock vaccinated group, indicating that the COBRA-based N2 subtype NA vaccines have a potential to be a component in a universal influenza vaccine.

Asunto(s)

Anticuerpos Antivirales , Subtipo H3N2 del Virus de la Influenza A , Vacunas contra la Influenza , Ratones Endogámicos BALB C , Neuraminidasa , Infecciones por Orthomyxoviridae , Animales , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Neuraminidasa/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Ratones , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Humanos , Femenino , Subtipo H3N2 del Virus de la Influenza A/inmunología , Gripe Humana/prevención & control , Gripe Humana/inmunología , Reacciones Cruzadas/inmunología , Proteínas Virales/inmunología , Carga Viral , Pulmón/virología , Pulmón/inmunología

RESUMEN

The hooding behavior exhibited by cobras is a distinct defensive mechanism against predators, encompassing both visual and auditory displays. This behavior can be triggered by natural predators or humans. Considering that human provocation may potentially stimulate the hypothalamic-pituitary-adrenal (HPA) axis, the present study aimed to determine the pattern of the HPA axis response following human provocation-induced hooding behavior (PV) and provide a detailed analysis of the behavioral PV displays. Our primary hypothesis was that a 5-minute PV could activate the HPA axis to a degree comparable to that in the restraint-induced stress model (RS). The PV, RS-1 (1-minute), and RS-5 (5-minute) restraint models indeed activated the HPA axis. However, the pattern of plasma corticosteroid (CORT), but not arginine vasotocin, in the PV group differed from that in the RS-1 and RS-2 groups. The present study revealed the behavioral components of the PV. The first component appeared to be related to an increase in apparent size that is an intimidation display, while the second hissing and striking component consisted of a bluff charge. Moreover, no correlation was observed between the pattern of plasma CORT and any specific PV display. Finally, the body temperature (Tb) of cobras from RS-5 gradually increased, while the Tb of cobras from PV (5 min) remained unchanged. In conclusion, the activation of the HPA axis emerges as the main physiological response after human provocation. Within 5 min of provocation, the cobras' hooding behavior comprised two display components that were not related to the pattern of plasma CORT.

RESUMEN

Cellulose is a critical component of secondary cell walls and woody tissues of plants. Cellulose synthase (CESA) complexes (CSCs) produce cellulose as they move within the plasma membrane, extruding glucan chains into the cell wall that coalesce and crystallize into cellulose fibrils. Here we examine COBRA-LIKE4 (COBL4), a GPI-anchored protein on the outer leaflet of the plasma membrane that is required for normal cellulose deposition in secondary cell walls. Characterization of the Arabidopsis (Arabidopsis thaliana) cobl4 mutant alleles called irregular xylem6, irx6-2 and irx6-3, showed reduced ⍺-cellulose content and lower crystallinity, supporting a role for COBL4 in maintaining cellulose quantity and quality. In live-cell imaging, mNeon Green-tagged CESA7 moved in the plasma membrane at higher speeds in the irx6-2 background compared to wild type. To test conservation of COBL4 function between herbaceous and woody plants, poplar (Populus trichocarpa) COBL4 homologs PtCOBL4a and PtCOBL4b were transformed into, and rescued, the Arabidopsis irx6 mutants. Using the Arabidopsis secondary cell wall-inducible VND7-GR system to study poplar COBL4 dynamics, YFP-tagged PtCOBL4a localized to the plasma membrane in regions of high cellulose deposition in secondary cell wall bands. As predicted for a lipid-linked protein, COBL4 was more mobile in the plane of the plasma membrane than CESA7 or a control plasma membrane marker. Following programmed cell death, COBL4 anchored to the secondary cell wall bands. These data support a role for COBL4 as a modulator of cellulose organization in the secondary cell wall, influencing cellulose production and CSC velocity at the plasma membrane.

RESUMEN

Development of next-generation influenza virus vaccines is crucial to improve protection against circulating and emerging viruses. Current vaccine formulations have to be updated annually due to mutations in seasonal strains and do not offer protection against strains with pandemic potential. Computationally optimized broadly reactive antigen (COBRA) methodology has been utilized by our group to generate broadly reactive immunogens for individual influenza subtypes, which elicit protective immune responses against a broad range of strains over numerous seasons. Octavalent mixtures of COBRA hemagglutinin (HA) (H1, H2, H3, H5, H7, and influenza B virus) plus neuraminidase (NA) (N1 and N2) recombinant proteins mixed with c-di-AMP adjuvant were administered intranasally to naive or pre-immune ferrets in prime-boost fashion. Four weeks after final vaccination, collected sera were analyzed for breadth of antibody response, and the animals were challenged with seasonal or pre-pandemic strains. The octavalent COBRA vaccine elicited antibodies that recognized a broad panel of strains representing different subtypes, and these vaccinated animals were protected against influenza virus challenges. Overall, this study demonstrated that the mixture of eight COBRA HA/NA proteins mixed with an intranasal adjuvant is a promising candidate for a universal influenza vaccine. IMPORTANCE: Influenza is a respiratory virus which infects around a billion people globally every year, with millions experiencing severe illness. Commercial vaccine efficacy varies year to year and can be low due to mismatch of circulating virus strains. Thus, the formulation of current vaccines has to be adapted accordingly every year. The development of a broadly reactive influenza vaccine would lessen the global economic and public health burden caused by the different types of influenza viruses. The significance of our research is producing a promising universal vaccine candidate which provides protection against a wider range of virus strains over a wider range of time.

Asunto(s)

Administración Intranasal , Anticuerpos Antivirales , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Animales , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Neuraminidasa/inmunología , Neuraminidasa/genética , Estaciones del Año , Adyuvantes Inmunológicos/administración & dosificación , Vacunación/métodos , Gripe Humana/prevención & control , Gripe Humana/inmunología , Gripe Humana/virología , Humanos , Femenino , Protección Cruzada/inmunología , Pandemias/prevención & control

RESUMEN

In this paper, we provide an overview of mitochondrial bioenergetics and specific conditions that lead to the formation of non-bilayer structures in mitochondria. Secondly, we provide a brief overview on the structure/function of cytotoxins and how snake venom cytotoxins have contributed to increasing our understanding of ATP synthesis via oxidative phosphorylation in mitochondria, to reconcile some controversial aspects of the chemiosmotic theory. Specifically, we provide an emphasis on the biochemical contribution of delocalized and localized proton movement, involving direct transport of protons though the Fo unit of ATP synthase or via the hydrophobic environment at the center of the inner mitochondrial membrane (proton circuit) on oxidative phosphorylation, and how this influences the rate of ATP synthesis. Importantly, we provide new insights on the molecular mechanisms through which cobra venom cytotoxins affect mitochondrial ATP synthesis, mitochondrial structure, and dynamics. Finally, we provide a perspective for the use of cytotoxins as novel pharmacological tools to study membrane bioenergetics and mitochondrial biology, how they can be used in translational research, and their potential therapeutic applications.

Asunto(s)

Venenos Elapídicos , Metabolismo Energético , Mitocondrias , Membranas Mitocondriales , Animales , Metabolismo Energético/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Humanos , Venenos Elapídicos/química , Venenos Elapídicos/toxicidad , Venenos Elapídicos/metabolismo , Citotoxinas/farmacología , Citotoxinas/toxicidad , Citotoxinas/química , Adenosina Trifosfato/metabolismo , Fosforilación Oxidativa/efectos de los fármacos

RESUMEN

Influenza outbreaks are a major burden worldwide annually. While seasonal vaccines do provide protection against infection, they are limited in that they need to be updated every year to account for the constantly mutating virus. Recently, lipid nanoparticles (LNPs) encapsulating mRNA have seen major success as a vaccine platform for SARS-CoV-2. Herein, we applied LNPs to deliver an mRNA encoding a computationally optimized broadly active (COBRA) influenza immunogen. These COBRA mRNA LNPs induced a broadly active neutralizing antibody response and protection after lethal influenza challenge. To further increase the immunogenicity of the COBRA mRNA LNPs, we combined them with acetalated dextran microparticles encapsulating a STING agonist. Contrary to recent findings, the STING agonist decreased the immunogenicity of the COBRA mRNA LNPs which was likely due to a decrease in mRNA translation as shown in vitro. Overall, this work aids in future selection of adjuvants to use with mRNA LNP vaccines.

Asunto(s)

Vacunas contra la Influenza , Nanovacunas , Nucleótidos Cíclicos , Animales , Femenino , Ratones , Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Neutralizantes/inmunología , Dextranos/química , Dextranos/administración & dosificación , Inmunogenicidad Vacunal , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/inmunología , Lípidos/química , Lípidos/administración & dosificación , Liposomas , Ratones Endogámicos BALB C , Vacunas de ARNm , Nanopartículas/administración & dosificación , Nanopartículas/química , Nanovacunas/administración & dosificación , Nanovacunas/química , Nucleótidos Cíclicos/administración & dosificación , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Polímeros/química , Polímeros/administración & dosificación , ARN Mensajero/administración & dosificación , ARN Mensajero/inmunología

RESUMEN

In humans, seasonal influenza viruses cause epidemics. Avian influenza viruses are of particular concern because they can infect multiple species and lead to unpredictable and severe disease. Therefore, there is an urgent need for a universal influenza vaccine that provides protection against all influenza strains. The cyclic GMP-AMP (cGAMP) is a promising adjuvant for subunit vaccines, which promotes type I interferons' production through the stimulator of interferon genes (STING) pathway. The encapsulation of cGAMP in acetalated dextran (Ace-DEX) microparticles (MPs) enhances its intracellular delivery. In this study, the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology was used to generate H1, H3, and H5 vaccine candidates. Monovalent and multivalent COBRA HA vaccines formulated with cGAMP Ace-DEX MPs were evaluated in mice for protective antibody responses. cGAMP MPs adjuvanted COBRA HA vaccines elicited robust antigen-specific antibodies following vaccination. Compared with COBRA HA vaccine groups with no adjuvant or blank MPs, the cGAMP MPs enhanced HAI activity elicited by COBRA HA vaccines. The HAI activity was not significantly different between cGAMP MPs adjuvanted monovalent or multivalent COBRA HA vaccines. The cGAMP MPs adjuvanted COBRA vaccine groups had higher antigen-specific IgG2a-binding titers than the COBRA vaccine groups with no adjuvant or blank MPs. The COBRA vaccines formulated with cGAMP MPs mitigated diseases caused by influenza viral challenge and decreased pulmonary viral titers in mice. Therefore, the formulation of COBRA vaccines plus cGAMP MPs is a promising universal influenza vaccine that elicits protective immune responses against human seasonal and pre-pandemic strains. IMPORTANCE: Influenza viruses cause severe respiratory disease, particularly in the very young and the elderly. Next-generation influenza vaccines are needed to protect against new influenza variants. This report used a promising adjuvant, cyclic GMP-AMP (cGAMP), to enhance the elicited antibodies by an improved influenza hemagglutinin candidate and protect against influenza virus infection. Overall, adding adjuvants to influenza vaccines is an effective method to improve vaccines.

Asunto(s)

Adyuvantes Inmunológicos , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas contra la Influenza , Nucleótidos Cíclicos , Infecciones por Orthomyxoviridae , Animales , Femenino , Humanos , Ratones , Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Ratones Endogámicos BALB C , Nucleótidos Cíclicos/inmunología , Nucleótidos Cíclicos/administración & dosificación , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología

RESUMEN

The influenza viruses cause seasonal respiratory illness that affect millions of people globally every year. Prophylactic vaccines are the recommended method to prevent the breakout of influenza epidemics. One of the current commercial influenza vaccines consists of inactivated viruses that are selected months prior to the start of a new influenza season. In many seasons, the vaccine effectiveness (VE) of these vaccines can be relatively low. Therefore, there is an urgent need to develop an improved, more universal influenza vaccine (UIV) that can provide broad protection against various drifted strains in all age groups. To meet this need, the computationally optimized broadly reactive antigen (COBRA) methodology was developed to design a hemagglutinin (HA) molecule as a new influenza vaccine. In this study, COBRA HA-based inactivated influenza viruses (IIV) expressing the COBRA HA from H1 or H3 influenza viruses were developed and characterized for the elicitation of immediate and long-term protective immunity in both immunologically naïve or influenza pre-immune animal models. These results were compared to animals vaccinated with IIV vaccines expressing wild-type H1 or H3 HA proteins (WT-IIV). The COBRA-IIV elicited long-lasting broadly reactive antibodies that had hemagglutination-inhibition (HAI) activity against drifted influenza variants.

Asunto(s)

Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Vacunas de Productos Inactivados , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Animales , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Ratones , Femenino , Ratones Endogámicos BALB C , Humanos , Gripe Humana/prevención & control , Gripe Humana/inmunología , Eficacia de las Vacunas , Pruebas de Inhibición de Hemaglutinación

RESUMEN

African cobras (Naja species) represent one of the most encountered medically important snakes in Africa. They are classified as African spitting (Afronaja subgenus) and non-spitting cobras (Uraeus and Boulengerina subgenera) with similar and different characteristics. Snake venom toxins including three-finger toxin (3FTx), phospholipase A2 (PLA2), and snake venom metalloproteinase (SVMP) cause snakebite envenomation leading to morbidity and mortality. The profile of the proteome of African cobra venoms will help to develop safer and more effective antivenoms. The approval of Captopril by the US Food and Drug Administration (FDA) for the treatment of cardiovascular diseases, has led to intensified research towards possible use of venom toxins as therapeutics. In this review, we compare the venom proteome profile of 3 African Naja subgenera. In both Afronaja and Boulengerina subgenera, 3FTx (Afronaja-69.79%; Boulengerina-60.56%) followed by PLA2 (Afronaja-21.15%; Boulengerina-20.21%) dominated the venoms compared to the Uraeus subgenus dominated by 3FTx (84.55%) with little to no PLA2 abundance (0.8%). The venom of subgenus Uraeus was distinct from the other two subgenera by the almost total absence of PLA2, thus indicating little or no contribution of PLA2 in the envenomation caused by Uraeus compared to Afronaja and Boulengerina. Furthermore, we report studies on the experimental testing of African cobra venoms and toxins against diseases including anti-cancer properties.

Asunto(s)

Venenos Elapídicos , Proteoma , Animales , Venenos Elapídicos/química , Antivenenos/uso terapéutico , Naja , Fosfolipasas A2

RESUMEN

Cytotoxins (CTs) are three-finger membrane-active toxins present mainly in cobra venom. Our analysis of the available CT amino acid sequences, literature data on their membrane activity, and conformational equilibria in aqueous solution and detergent micelles allowed us to identify specific amino acid residues which interfere with CT incorporation into membranes. They include Pro9, Ser28, and Asn/Asp45 within the N-terminal, central, and C-terminal loops, respectively. There is a hierarchy in the effect of these residues on membrane activity: Pro9 > Ser28 > Asn/Asp45. Taking into account all the possible combinations of special residues, we propose to divide CTs into eight groups. Group 1 includes toxins containing all of the above residues. Their representatives demonstrated the lowest membrane activity. Group 8 combines CTs that lack these residues. For the toxins from this group, the greatest membrane activity was observed. We predict that when solely membrane activity determines the cytotoxic effects, the activity of CTs from a group with a higher number should exceed that of CTs from a group with a lower number. This classification is supported by the available data on the cytotoxicity and membranotropic properties of CTs. We hypothesize that the special amino acid residues within the loops of the CT molecule may indicate their involvement in the interaction with non-lipid targets.

Asunto(s)

Membrana Celular , Citotoxinas , Membrana Celular/efectos de los fármacos , Animales , Citotoxinas/química , Citotoxinas/toxicidad , Venenos Elapídicos/química , Venenos Elapídicos/toxicidad , Aminoácidos/química , Secuencia de Aminoácidos , Humanos

RESUMEN

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells. Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system. Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC50 0.25 µg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%). Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment.

Asunto(s)

Antineoplásicos , Venenos Elapídicos , Liposomas , Liposomas/química , Concentración de Iones de Hidrógeno , Animales , Venenos Elapídicos/química , Venenos Elapídicos/farmacología , Humanos , Línea Celular Tumoral , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Ratones , Apoptosis/efectos de los fármacos , Liberación de Fármacos , Citotoxinas/química , Citotoxinas/farmacología , Citotoxinas/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Distribución Tisular , Microambiente Tumoral/efectos de los fármacos , Nanopartículas/química

RESUMEN

Introduction: There is a significant shortage of observational studies on neurotoxic snakebite envenomation in the Philippines. This lack of data, especially concerning treatment using Purified Cobra Antivenom (PCAV), has prompted the initiation of this foundational study. Methods: The target population included snakebite patients admitted to the Eastern Visayas Medical Center and treated with PCAV between 2016 and 2020. A retrospective chart review was conducted for data collection. The investigation analyzed the hospital stay and patient features of individuals who were administered either lower or higher doses of PCAV. Results: Eighty-two patients were identified during the study. Of these, 27 (33%) were under 20 years of age and 50 (61%) were male. Most patients, totalling 75 (92%) were hailed from rural areas. Of the 82 patients, 59 (72%) received one or two ampoules of PCAV during the course. However, patients who received more than two ampoules had a longer median hospital stay than those who received less than three ampoules [96 h (interquartile range, IQR 66-122) vs. 125 h (IQR 96-218), P = 0.038]. The study reported five in-hospital mortalities (6.1%). Conclusions: The individuals who needed a high dosage of PCAV tended to have more extended hospital stays, yet over 70% of the patient population required a lower dosage. To gain a clearer understanding of the burden of neurotoxic snakebites and determine the optimal PCAV dosage based on disease severity in the area, a more comprehensive, prospective study is recommended.

RESUMEN

Adjuvants enhance, prolong, and modulate immune responses by vaccine antigens to maximize protective immunity and enable more effective immunization in the young and elderly. Most adjuvants are formulated with injectable vaccines. However, an intranasal route of vaccination may induce mucosal and systemic immune responses for enhancing protective immunity in individuals and be easier to administer compared to injectable vaccines. In this study, a next generation of broadly-reactive influenza hemagglutinin (HA) vaccines were developed using the Computationally Optimized Broadly Reactive Antigen (COBRA) methodology. These HA vaccines were formulated with Mastoparan 7 (M7-NH2) mast cell degranulating peptide adjuvant and administered intranasally to determine vaccine-induced seroconversion of antibodies against a panel of influenza viruses and protection following infection with H1N1 and H3N2 viruses in mice. Mice vaccinated intranasally with M7-NH2-adjuvanted COBRA HA vaccines had high HAIs against a panel of H1N1 and H3N2 influenza viruses and were protected against both morbidity and mortality, with reduced viral lung titers, following challenge with an H1N1 influenza virus. Additionally, M7-NH2 adjuvanted COBRA HA vaccines induced Th2 skewed immune responses with robust IgG and isotype antibodies in the serum and mucosal lung lavages. Overall, this intranasally delivered M7-NH2 -adjuvanted COBRA HA vaccine provides effective protection against drifted H1N1 and H3N2 viruses.

Asunto(s)

Adyuvantes Inmunológicos , Administración Intranasal , Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Subtipo H1N1 del Virus de la Influenza A , Subtipo H3N2 del Virus de la Influenza A , Vacunas contra la Influenza , Infecciones por Orthomyxoviridae , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/administración & dosificación , Animales , Ratones , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antivirales/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/inmunología , Femenino , Ratones Endogámicos BALB C , Péptidos y Proteínas de Señalización Intercelular/inmunología , Adyuvantes de Vacunas/administración & dosificación

RESUMEN

Snake venoms, comprising a complex array of protein-rich components, an important part of which are snake venom metalloproteinases (SVMPs). These SVMPs, which are predominantly isolated from viperid venoms, are integral to the pathology of snakebites. However, SVMPs derived from elapid venoms have not been extensively explored, and only a handful of SVMPs have been characterized to date. Atrase A, a nonhemorrhagic P-III class metalloproteinase from Naja atra venom, exhibits weak proteolytic activity against fibrinogen in vitro but has pronounced anticoagulant effects in vivo. This contrast spurred investigations into its anticoagulant mechanisms. Research findings indicate that atrase A notably extends the activated partial thromboplastin time, diminishes fibrinogen levels, and impedes platelet aggregation. The anticoagulant action of atrase A primarily involves inhibiting coagulation factor VIII and activating the endogenous fibrinolytic system, which in turn lowers fibrinogen levels. Additionally, its effect on platelet aggregation further contributes to its anticoagulant profile. This study unveils a novel anticoagulant mechanism of atrase A, significantly enriching the understanding of the roles of cobra venom metalloproteinases in snake venom. Furthermore, these findings underscore the potential of atrase A as a novel anticoagulant drug, offering insights into the functional evolutions of cobra venom metalloproteinases.

RESUMEN

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Asunto(s)

Anticuerpos Antivirales , Glicoproteínas Hemaglutininas del Virus de la Influenza , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Humanos , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Modelos Moleculares , Antígenos Virales/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Epítopos/inmunología , Epítopos/química , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/química , Gripe Humana/inmunología , Gripe Humana/virología , Cristalografía por Rayos X , Unión Proteica

RESUMEN

Adjuvants enhance immune responses stimulated by vaccines. To date, many seasonal influenza vaccines are not formulated with an adjuvant. In the present study, the adjuvant Advax-SM™ was combined with next generation, broadly reactive influenza hemagglutinin (HA) vaccines that were designed using a computationally optimized broadly reactive antigen (COBRA) methodology. Advax-SM™ is a novel adjuvant comprising inulin polysaccharide and CpG55.2, a TLR9 agonist. COBRA HA vaccines were combined with Advax-SM™ or a comparator squalene emulsion (SE) adjuvant and administered to mice intramuscularly. Mice vaccinated with Advax-SM™ adjuvanted COBRA HA vaccines had increased serum levels of anti-influenza IgG and IgA, high hemagglutination inhibition activity against a panel of H1N1 and H3N2 influenza viruses, and increased anti-influenza antibody secreting cells isolated from spleens. COBRA HA plus Advax-SM™ immunized mice were protected against both morbidity and mortality following viral challenge and, at postmortem, had no detectable lung viral titers or lung inflammation. Overall, the Advax-SM™-adjuvanted COBRA HA formulation provided effective protection against drifted H1N1 and H3N2 influenza viruses.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN , Desarrollo Embrionario , Morfogénesis , Animales , Humanos

RESUMEN

This report details a documented case of fatal King cobra (Ophiophagus hannah) envenomation in the Philippines. A 46-year-old woman from a mountainous town in Leyte was bitten on her left thigh by a snake. Despite receiving prompt medical attention, including administration of fluids and oxygen, she went into arrest and succumbed within 2.5 hours of the bite. Inadequate pre-hospital care, including endotracheal intubation and assisted ventilation, highlights a notable gap in emergency medical services. Photographic evidence, verified by a herpetologist, confirmed the involvement of a King cobra, with venom presenting with a swift and lethal systemic effect that led to the patient's demise, despite minimal local manifestations. This incident accentuates the urgent need for accessible, effective antivenom and improved snakebite management protocols in the Philippines. It also calls for heightened awareness and preparedness among pre-hospital healthcare providers and the public, alongside advocating for more research into snakebite envenomation.

Asunto(s)

Venenos Elapídicos , Elapidae , Mordeduras de Serpientes , Animales , Femenino , Persona de Mediana Edad , Humanos , Filipinas , Resultado Fatal , Antivenenos/uso terapéutico

RESUMEN

Barley (Hordeum vulgare L.), a diverse cereal crop, exhibits remarkable versatility in its applications, ranging from food and fodder to industrial uses. The content of cellulose in barley is significantly influenced by the COBRA genes, which encode the plant glycosylphosphatidylinositol (GPI)-anchored protein (GAP) that plays a pivotal role in the deposition of cellulose within the cell wall. The COBL (COBRA-Like) gene family has been discovered across numerous species, yet the specific members of this family in barley remain undetermined. In this study, we discovered 13 COBL genes within the barley genome using bioinformatics methods, subcellular localization, and protein structure analysis, finding that most of the barley COBL proteins have a signal peptide structure and are localized on the plasma membrane. Simultaneously, we constructed a phylogenetic tree and undertook a comprehensive analysis of the evolutionary relationships. Other characteristics of HvCOBL family members, including intraspecific collinearity, gene structure, conserved motifs, and cis-acting elements, were thoroughly characterized in detail. The assessment of HvCOBL gene expression in barley under various hormone treatments was conducted through qRT-PCR analysis, revealing jasmonic acid (JA) as the predominant hormonal regulator of HvCOBL gene expression. In summary, this study comprehensively identified and analyzed the barley COBL gene family, aiming to provide basic information for exploring the members of the HvCOBL gene family and to propose directions for further research.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Hordeum , Familia de Multigenes , Filogenia , Reguladores del Crecimiento de las Plantas , Proteínas de Plantas , Hordeum/genética , Hordeum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/genética , Genoma de Planta , Oxilipinas/metabolismo , Ciclopentanos/metabolismo

RESUMEN

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.