Serological Profile for Major Respiratory Viruses in Unvaccinated Cows from High-Yielding Dairy Herds.

This study aims to determine the serological profile of high-yielding dairy cows for four main viruses (bovine alphaherpesvirus 1 (BoAHV1), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV3), and bovine respiratory syncytial virus (BRSV)) related to bovine respiratory disease (BRD) in cattle herds worldwide. In this survey, 497 blood serum samples were collected from non-vaccinated dairy cows without clinical respiratory signs in 39 herds in the central-eastern mesoregion of Paraná State, South Brazil. The presence of neutralizing antibodies was determined by virus neutralization (VN) tests. VN antibodies against BoAHV1, BVDV, BPIV3, and BRSV were detected in 355 (71.4%), 280 (56.3%), 481 (96.8%), and 315 (63.4%) serum samples, respectively. The frequencies of seropositive herds for BoAHV1, BVDV, BPIV3, and BRSV were 79.5 (n = 31), 82.0 (n = 32), 100 (n = 39), and 84.6% (n = 33), respectively. The frequencies of seropositive cows varied according to the type of herd management and the number of cows in the herd. The detection of VN antibodies in unvaccinated dairy cattle herds demonstrated the endemic circulation of the four viruses in the herds evaluated. For BRD prevention, it is recommended to implement a vaccination program for cows that provides passive immunity in calves and active immunity in cows.

Humoral Immunity across the SARS-CoV-2 Spike after Sputnik V (Gam-COVID-Vac) Vaccination.

SARS-CoV-2 vaccines have contributed to attenuating the burden of the COVID-19 pandemic by promoting the development of effective immune responses, thus reducing the spread and severity of the pandemic. A clinical trial with the Sputnik-V vaccine was conducted in Venezuela from December 2020 to July 2021. The aim of this study was to explore the antibody reactivity of vaccinated individuals towards different regions of the spike protein (S). Neutralizing antibody (NAb) activity was assessed using a commercial surrogate assay, detecting NAbs against the receptor-binding domain (RBD), and a plaque reduction neutralization test. NAb levels were correlated with the reactivity of the antibodies to the spike regions over time. The presence of Abs against nucleoprotein was also determined to rule out the effect of exposure to the virus during the clinical trial in the serological response. A high serological reactivity was observed to S and specifically to S1 and the RBD. S2, although recognized with lower intensity by vaccinated individuals, was the subunit exhibiting the highest cross-reactivity in prepandemic sera. This study is in agreement with the high efficacy reported for the Sputnik V vaccine and shows that this vaccine is able to induce an immunity lasting for at least 180 days. The dissection of the Ab reactivity to different regions of S allowed us to identify the relevance of epitopes outside the RBD that are able to induce NAbs. This research may contribute to the understanding of vaccine immunity against SARS-CoV-2, which could contribute to the design of future vaccine strategies.

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom.

Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean Artemia salina could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of A. salina as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub-Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in A. salina. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and A. salina was investigated. There was a positive correlation between the i.v. median lethal doses (LD50s) and the i.p. LD50s in mice (r = 0.804; p < 0.0001), a moderate correlation between the i.v. LD50s in mice and the median lethal concentrations (LC50s) in A. salina (r = 0.606; p = 0.003), and a moderate correlation between the i.p. LD50s in mice and the LC50s in A. salina (r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED50s) and the i.v. ED50s in mice (r = 0.941, p < 0.0001), between the i.p. ED50s in mice and the ED50s in A. salina (r = 0.818, p < 0.0001), and between the i.v. ED50s in mice and the ED50s in A. salina (r = 0.972, p < 0.0001). These findings present A. salina as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of A. salina in venom research and antivenom development.

Second booster dose improves antibody neutralization against BA.1, BA.5 and BQ.1.1 in individuals previously immunized with CoronaVac plus BNT162B2 booster protocol.

Introduction: SARS-CoV-2 vaccines production and distribution enabled the return to normalcy worldwide, but it was not fast enough to avoid the emergence of variants capable of evading immune response induced by prior infections and vaccination. This study evaluated, against Omicron sublineages BA.1, BA.5 and BQ.1.1, the antibody response of a cohort vaccinated with a two doses CoronaVac protocol and followed by two heterologous booster doses. Methods: To assess vaccination effectiveness, serum samples were collected from 160 individuals, in 3 different time points (9, 12 and 18 months after CoronaVac protocol). For each time point, individuals were divided into 3 subgroups, based on the number of additional doses received (No booster, 1 booster and 2 boosters), and a viral microneutralization assay was performed to evaluate neutralization titers and seroconvertion rate. Results: The findings presented here show that, despite the first booster, at 9m time point, improved neutralization level against omicron ancestor BA.1 (133.1 to 663.3), this trend was significantly lower for BQ.1.1 and BA.5 (132.4 to 199.1, 63.2 to 100.2, respectively). However, at 18m time point, the administration of a second booster dose considerably improved the antibody neutralization, and this was observed not only against BA.1 (2361.5), but also against subvariants BQ.1.1 (726.1) and BA.5 (659.1). Additionally, our data showed that, after first booster, seroconvertion rate for BA.5 decayed over time (93.3% at 12m to 68.4% at 18m), but after the second booster, seroconvertion was completely recovered (95% at 18m). Discussion: Our study reinforces the concerns about immunity evasion of the SARS-CoV-2 omicron subvariants, where BA.5 and BQ.1.1 were less neutralized by vaccine induced antibodies than BA.1. On the other hand, the administration of a second booster significantly enhanced antibody neutralization capacity against these subvariants. It is likely that, as new SARS-CoV-2 subvariants continue to emerge, additional immunizations will be needed over time.

High-Content Imaging-Based Assay for SARS-CoV-2-Neutralizing Antibodies.

The COVID-19 pandemic and the consequent emergence of new SARS-CoV-2 variants of concern necessitates the determination of populational serum potency against the virus. Here, we standardized and validated an imaging-based method to quantify neutralizing antibodies against lentiviral particles expressing the spike glycoprotein (pseudovirus). This method was found to efficiently quantify viral titers based on ZsGreen-positive cells and detect changes in human serum neutralization capacity induced by vaccination with up to two doses of CoronaVac, Comirnaty, or Covishield vaccines. The imaging-based protocol was also used to quantify serum potency against pseudoviruses expressing spikes from Delta, Omicron BA.1.1.529, and BA.4/5. Our results revealed increases in serum potency after one and two doses of the vaccines evaluated and demonstrated that Delta and Omicron variants escape from antibody neutralization. The method presented herein represents a valuable tool for the screening of antibodies and small molecules capable of blocking viral entry and could be used to evaluate humoral immunity developed by different populations and for vaccine development.

Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.

Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

Thio-Induced Organophosphate Breakdown Promoted by Methimazole: an Experimental and Theoretical Study.

Investigating the reactivity of small nucleophilic scaffolds is a strategic approach for the design of new catalysts aiming at effective detoxification processes of organophosphorus compounds. The drug methimazole (MMZ) is an interesting candidate featuring two non-equivalent nucleophilic centers. Herein, phosphoryl transfer reactions mediated by MMZ were assessed by means of spectrophotometric kinetic studies, mass spectrometry (MS) analyses, and density functional theory (DFT) calculations using the multi-electrophilic compound O,O-diethyl 2,4-dinitrophenyl phosphate (DEDNPP). MMZ anion acts primarily as an S-nucleophile, exhibiting a nucleophilic activity comparable to that of certain oximes featuring alpha-effect. Selective nucleophilic aromatic substitution was observed, consistent with the DFT prediction of a low energy barrier. Overall, the results bring important advances regarding the mechanistic understanding of nucleophilic dephosphorylation reactions, which comprises a strategic tool for neutralizing toxic organophosphates, hence promoting chemical security.

SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Antimicrobial, toxicological, and antigenic characteristics of three scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus.

The venom fractions of three buthid scorpion species from Colombia, C. margaritatus, T. pachyurus and T. n. sp. aff. metuendus, were examined for antimicrobial and toxicity toward mice and insects. The three venoms were separated into individual fractions using RP-HPLC, resulting in 85 fractions from C. margaritatus, 106 from T. pachyurus, and 70 from T. n. sp. aff. metuendus. The major fractions from the three scorpion venoms, which were eluted between 35 and 50 min, were tested for antimicrobial activity and toxicity. It was confirmed that the venom of the three species contains fractions with antimicrobial peptides that were evaluated against two bacterial strains of public health importance, Pseudomonas aeruginosa and Staphylococcus aureus. The venom of C. margaritatus had two antimicrobial fractions that showed activity against the named tested strains. The venom of T. pachyurus had three fractions that showed activity against S. aureus and two against both bacterial strains. Finally, the venom of T. n. sp. aff. metuendus had one fraction that showed activity against S. aureus, and five fractions showed activity against both bacterial strains. Also, some peptide fractions from the three venoms were toxic to mice. Last, the venoms of C. margaritatus and T. pachyurus were used as immunogens to obtain neutralizing antibodies against its respective venoms and to observe antibody recognition to related and unrelated scorpion venoms. A total of 15 mg of lyophilized antibodies were able to neutralize 1.5⋅LD50 of the venoms from T. n. sp. aff. metuendus, T. pachyurus and C. margaritatus, respectively. This information provides valuable insights into the diversity of each species' venom and their potential role in antimicrobial and venom toxicity.

Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Anamnestic response against bovine viral diarrhea virus and bovine herpesvirus type 1 in young Holstein heifers vaccinated with four different commercial formulations / Resposta anamnéstica contra o vírus da diarreia viral bovina e herpesvírus bovino tipo 1 em novilhas Holandesas jovens vacinadas com quatro diferentes formulações comerciais

This study evaluated the vaccine-induced serological response after administering four commercial formulations containing Bovine Viral Diarrhea Virus (BVDV) type-1, BVDV-2, and Bovine Herpesvirus type 1 (BoHV-1) to young heifers with circulating maternal antibody titers. The study also determined the anamnestic response to vaccinations after the complete metabolization of maternal antibodies when the calves reached six months. Eighty-seven Holstein heifers were selected and randomly distributed into four experimental groups based on the vaccine administered. The four vaccine-based experimental groups were as follows: vaccine A (n = 22), vaccine B (n = 22), vaccine C (n = 24), and vaccine D (n = 19), given on the 60th, 90th, and 180th day of life, respectively. Virus-neutralization (VN) tests were performed at baseline (day 60) and 30 days after administering the second (day 120) and third doses (day 210). We analyzed the effect of vaccine group (P ≤ 0.001), day of vaccination (P ≤ 0.001), and group versus vaccine day interaction (P ≤ 0.001) for antibody titers produced against BVDV-1, BVDV-2, and BoHV-1 using the PROC MIXED method (Statistical Analysis System -SAS 9.4). Antibody titers against BVDV-1, BVDV-2, and BoHV-1 were similar at baseline and on day 60 of life. The mean antibody titers were constant and persisted against BVDV-1 in heifers immunized with vaccines A and C. Heifers immunized with vaccine A alone had a similar effect against BVDV-2. Regarding BoHV-1, the antibody titers decreased between days 60 and 210 in groups B, C, and D. The antibody titer for heifers in group A also decreased between days 60 and day 120, and an intense increase in titers was observed on day 210. After being immunized with formulations B, C, and D, the frequency of animals with titers above protective levels for BVDV-1, BVDV-2, and BoHV-1 was very low or null. Indicators of anamnestic response were observed in heifers vaccinated with formulation A only. Therefore, it can be concluded that passive immunity negatively interferes with antibody production after vaccination.

O objetivo desse estudo foi avaliar a resposta sorológica induzida após vacinação com quatro formulações comerciais contendo o BVDV-1, BVDV-2 e BoHV-1 em novilhas jovens com títulos de anticorpos maternos circulantes, assim como analisar uma possível resposta anamnéstica em vacinações, após a completa metabolização dos anticorpos maternais aos seis meses de idade. Foram selecionadas 87 novilhas Holandesas, distribuídas aleatoriamente em quatro grupos experimentais de acordo com as vacinas: vacina A (n = 22), vacina B (n = 22), vacina C (n = 24) e vacina D (n = 19), aplicadas aos dias 60, 90 e 180 de vida. Testes de vírus-neutralização (VN) foram realizados no momento basal (D60) e 30 dias após a aplicação da 2ª dose (D120) e 3ª dose (D210). Observou-se efeito do grupo (P ≤ 0,001), dia da vacinação (P ≤ 0,001), e interação grupo versus dia (P ≤ 0,001) para os títulos de anticorpos produzidos contra o BVDV-1, BVDV-2 e BoHV-1, por meio do comando PROC MIXED (Statistical Analysis System, versão 9,4). Os títulos de anticorpos contra o BVDV-1, BVDV-2 e BoHV-1 eram semelhantes no momento basal aos 60 dias de vida. Os títulos médios de anticorpos foram constantes e persistentes contra o BVDV-1 nas novilhas imunizadas com as vacinas A e C, porém apenas a vacina A teve este mesmo perfil contra o BVDV-2. Em relação ao BoHV-1, os grupos B, C e D apresentaram queda nos títulos de anticorpos do D60 ao D210, enquanto as novilhas do grupo A apresentaram queda do D60 ao D120, com aumento intenso dos títulos no D210. A frequência de animais com títulos acima dos protetores contra o BVDV-1, BVDV-2 e BoHV-1 foram muito baixos ou nulos após as vacinações nos animais imunizados com as formulações B, C e D. Sugere-se que a imunidade passiva interferiu negativamente na indução de anticorpos pelas vacinas, observando-se indicadores de resposta anamnéstica apenas para as novilhas vacinadas com a formulação A.

Extraction and quantification of antivenomous antibodies in chicken eggs against scorpion venom / Extração e quantificação de anticorpos entivenenos em ovos de galinha contra veneno de escorpião

Avian-derived IgY is thought to be the best therapy for scorpion bites concerning low-level side effects. The present study analyzed a hypothesis about the neutralization of scorpion venom Androcotonus australis through antibodies produced in the egg yolks of chickens. The venom used for inoculation was obtained from Androctonus australis (yellow fat-tailed scorpion) from southern Punjab, Pakistan. The lethal dose of LD50 against scorpion venom was calculated in chickens and mice. Safe doses were given to egg-laying chickens to produce IgY antibodies. The antivenom IgY antibodies were extracted from the egg yolks of immunized chicken using the polyethylene glycol (PEG) method. Moreover, IgY was confirmed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Ouchterlony double immunodiffusion assay test. The antibody titers were evaluated by the enzyme-linked immunosorbent assay (ELISA). The neutralisation capacity of extracted anti-scorpion antibodies was tested on mice. The calculated LD50 of scorpion venom for chicken and mice was 4 mg/kg and 2.5 mg/kg, respectively. SDS-PAGE and Ouchterlony double immunodiffusion confirmed the presence of IgY against scorpion venom. The maximum titer value of specific IgY produced against scorpion venom was 3.5 ug/ml. A concentration of 220 ul/LD50 was effective to neutralize 1 mg of scorpion venom. It is suggested that IgY obtained from egg yolks is safe against targeted venom and can be used as an effective alternative to equine IgG antibodies against scorpion envenoming.

Acredita-se que a IgY derivada de aves seja a melhor terapia para picadas de escorpião em relação aos efeitos colaterais. O presente estudo teve como objetivo analisar uma hipótese sobre a neutralização do veneno do escorpião Androcotonus australis através de anticorpos produzidos na gema de ovos de galinhas. O veneno usado para inoculação foi obtido de Androctonus australis (escorpião amarelo de cauda gorda) do sul de Punjab, Paquistão. A dose letal de LD50 contra veneno de escorpião foi calculada em galinhas e camundongos. Doses seguras foram dadas a galinhas poedeiras para produzir anticorpos IgY. Os anticorpos antiveneno IgY foram extraídos das gemas de ovos de galinhas imunizadas pelo método do polietilenoglicol (PEG). Além disso, a IgY foi confirmada por eletroforese em gel de poliacrilamida e dodecil sulfato de sódio (SDS-PAGE) e pelo teste de imunodifusão dupla de Ouchterlony. Os títulos de anticorpos foram avaliados pelo ensaio imunoenzimático (ELISA). A capacidade de neutralização dos anticorpos anti-escorpião extraídos foi testada em camundongos. A LD50 calculada do veneno de escorpião para galinhas e camundongos foi de 4 mg/kg e 2,5 mg/kg, respectivamente. SDS-PAGE e imunodifusão dupla Ouchterlony confirmaram a presença de IgY contra veneno de escorpião. O valor máximo do título de IgY específico produzido contra veneno de escorpião foi de 3,5 ug/ml. Uma concentração de 220 ul/LD50 foi considerada eficaz para neutralizar 1 mg de veneno de escorpião. Sugere-se que a IgY obtida da gema do ovo seja segura contra o veneno direcionado e possa ser usada como uma alternativa eficaz aos anticorpos IgG equinos contra o envenenamento por escorpiões.

Fibrinogenolytic potential of venoms of medically important Brazilian snakes

One of the main clinical manifestations presented by victims of snake bite envenoming are coagulation disorders. Considering that fibrinogen is a key molecule for crosslinked fibrin clot formation, the objective of this work was the quantitative analysis of the fibrinogenolytic activity of snakes of medical importance in Brazil and neutralization by specific antivenom. For this, pools of three genera of medical importance (Bothrops, Crotalus and Lachesis) that are used for the production of antivenom were used, and three pools of species of the genus Bothrops that are not part of the pool for the production of antivenom. The Lachesis pool had the highest fibrinogenolytic activity, even demonstrating partial cleavage (42.9 % consumption) of the fibrinogen gamma chain. The Bothrops genus venom pools have shown subtle variations between them. The Crotalus pool, despite not showing total cleavage of any fibrinogen chain, began cleavage of fibrinogen by the beta chain. The specific antivenoms used were able to delay the cleavage of fibrinogen in all the venoms used, which could be the first step towards implementing previous in vitro tests to analyze the quality of the batches of antivenoms produced, thus potentially reducing the use of animals used in this process.

Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 μg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Towards better antivenoms: navigating the road to new types of snakebite envenoming therapies.

Snakebite envenoming is a significant global health challenge, and for over a century, traditional plasma-derived antivenoms from hyperimmunized animals have been the primary treatment against this infliction. However, these antivenoms have several inherent limitations, including the risk of causing adverse reactions when administered to patients, batch-to-batch variation, and high production costs. To address these issues and improve treatment outcomes, the development of new types of antivenoms is crucial. During this development, key aspects such as improved clinical efficacy, enhanced safety profiles, and greater affordability should be in focus. To achieve these goals, modern biotechnological methods can be applied to the discovery and development of therapeutic agents that can neutralize medically important toxins from multiple snake species. This review highlights some of these agents, including monoclonal antibodies, nanobodies, and selected small molecules, that can achieve broad toxin neutralization, have favorable safety profiles, and can be produced on a large scale with standardized manufacturing processes. Considering the inherent strengths and limitations related to the pharmacokinetics of these different agents, a combination of them might be beneficial in the development of new types of antivenom products with improved therapeutic properties. While the implementation of new therapies requires time, it is foreseeable that the application of biotechnological advancements represents a promising trajectory toward the development of improved therapies for snakebite envenoming. As research and development continue to advance, these new products could emerge as the mainstay treatment in the future.

Neutralization of Centruroides tecomanus scorpion venom by the use of two human recombinant antibody fragments.

The first toxic component identified against mammals in the venom from Centruroides tecomanus scorpion from Colima, Mexico was Ct1a toxin, which was neutralized by human single chain variable fragment (scFv) RAS27. Venom characterization from these scorpions collected on the Pacific coast of Colima, enabled the identification of a second component of medical importance named Ct71 toxin. Amino acid sequence of Ct71 shares a high identity with Chui5 toxin from C. huichol scorpion, which was neutralized by scFv HV. For this reason, the kinetic parameters of interaction between Ct71 toxin and scFv HV were determined by surface plasmon resonance. Results showed a significantly higher affinity for Ct71 as compared to Chui5. As expected, this toxin was neutralized by scFv HV. The injection of a mixture of scFvs HV and RAS27, resulted in the neutralization of C. tecomanus venom, corroborating that human recombinant antibody fragments can efficiently contribute to the neutralization of medically important toxins and their respective venoms from Mexican scorpions.

The fourth COVID-19 vaccine dose increased the neutralizing antibody response against the SARS-CoV-2 Omicron (B.1.1.529) variant in a diverse Brazilian population.

IMPORTANCE: Several additional COVID-19 vaccine doses were administered in the Brazilian population to prevent the disease caused by the B.1.1.529 (Omicron) variant. The efficacy of a third dose as a booster is already well described. However, it is important to clarify the humoral immune response gain induced by a fourth dose. In this study, we evaluate the effect of the fourth COVID-19 vaccine dose in a diverse Brazilian population, considering a real-life context. Our study reveals that the fourth dose of the COVID-19 vaccine increased the neutralizing antibody response against SARS-CoV-2 Omicron and significantly contributed in the reduction of the disease caused by this variant.

Serological profile of respiratory viruses in unvaccinated steers upon their arrival at Brazilian feedlot facilities.

Bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoAHV1), bovine respiratory syncytial virus (BRSV), and bovine parainfluenza virus 3 (BPIV-3) are involved in bovine respiratory disease. These viruses can infect the respiratory system and cause considerable economic losses to beef and dairy cattle herds. This study aimed to determine the serological profiles of steers for BVDV, BoAHV1, BRSV, and BPIV-3 upon their arrival at Brazilian feedlot facilities. A total of 1,282 serum samples from unvaccinated steers were obtained on the first day of feeding. Samples were collected from 31 beef cattle herds reared in an extensive rearing system in six Brazilian states. Antibodies against BVDV, BoAHV1, BRSV, and BPIV-3 were detected using a virus neutralization test. The steers were distributed in agreement with their age and the Brazilian state of origin. The highest seropositivity was for BoAHV1 and BPIV-3 at 92.1% (1,154/1,253) and 86.6% (1,100/1,270), respectively. The seropositivity of BRSV was 77.1% (959/1,244). BVDV presented a lower rate, at slightly more than 50% (51.8%; 656/1,266). Age was a risk factor for the presence of antibodies against BVDV, BoAHV1, and BPIV-3 but not BRSV. A positive correlation was identified between BoAHV1 and BPIV-3 (P = 0.85) and between BRSV and BPIV-3 (P = 0.47). The high rate of seropositive steers for these four respiratory viruses on the first day of confinement identified in this serological survey provides important epidemiological information on respiratory infections, as the seropositivity of the four main bovine respiratory viruses in Brazilian beef cattle herds in an extensive rearing system.

Customizably designed multibodies neutralize SARS-CoV-2 in a variant-insensitive manner.

The COVID-19 pandemic evolves constantly, requiring adaptable solutions to combat emerging SARS-CoV-2 variants. To address this, we created a pentameric scaffold based on a mammalian protein, which can be customized with up to 10 protein binding modules. This molecular scaffold spans roughly 20 nm and can simultaneously neutralize SARS-CoV-2 Spike proteins from one or multiple viral particles. Using only two different modules targeting the Spike's RBD domain, this construct outcompetes human antibodies from vaccinated individuals' serum and blocks in vitro cell attachment and pseudotyped virus entry. Additionally, the multibodies inhibit viral replication at low picomolar concentrations, regardless of the variant. This customizable multibody can be easily produced in procaryote systems, providing a new avenue for therapeutic development and detection devices, and contributing to preparedness against rapidly evolving pathogens.