Single-domain antibodies applied as antiviral immunotherapeutics.

Viral infections have been the cause of high mortality rates throughout different periods in history. Over the last two decades, outbreaks caused by zoonotic diseases and transmitted by arboviruses have had a significant impact on human health. The emergence of viral infections in different parts of the world encourages the search for new inputs to fight pathologies of viral origin. Antibodies represent the predominant class of new drugs developed in recent years and approved for the treatment of various human diseases, including cancer, autoimmune and infectious diseases. A promising group of antibodies are single-domain antibodies derived from camelid heavy chain immunoglobulins, or VHHs, are biomolecules with nanometric dimensions and unique pharmaceutical and biophysical properties that can be used in the diagnosis and immunotherapy of viral infections. For viral neutralization to occur, VHHs can act in different stages of the viral cycle, including the actual inhibition of infection, to hindering viral replication or assembly. This review article addresses advances involving the use of VHHs in therapeutic propositions aimed to battle different viruses that affect human health.

COVID-19 seroepidemiological survey among healthcare workers in the City of Ribeirão Preto, São Paulo, Brazil

ABSTRACT Background: Coronavirus disease (COVID-19) serology testing evaluates the prevalence of COVID-19 cases. Methods: A seroepidemiological survey of COVID-19 among healthcare workers was performed (June 2020 to November 2020) in Ribeirão Preto, São Paulo, Brazil. Overall, 10,172 and 2,129 workers participated in the first and second phases, respectively. Results: First phase: 12.7% tested positive for COVID-19 (73.5% females and 35.2% aged 30-39 years), and 29.6% were nursing technicians. Second phase: 12.1% tested positive for COVID-19 (65.5% females and 33.3% aged 40-49 years), and 24.8% were nursing assistants. Conclusions: In 2020, healthcare workers in Ribeirão Preto had COVID-19 in a similar way.

Antimicrobial peptidomes of Bothrops atrox and Bothrops jararacussu snake venoms.

The worrisome emergence of pathogens resistant to conventional drugs has stimulated the search for new classes of antimicrobial and antiparasitic agents from natural sources. Antimicrobial peptides (AMPs), acting through mechanisms that do not rely on the interaction with a specific receptor, provide new possibilities for the development of drugs against resistant organisms. This study sought to purify and proteomically characterize the antimicrobial and antiparasitic peptidomes of B. atrox and B. jararacussu snake venoms against Gram-positive (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus-MRSA), Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria, and the protozoan parasites Leishmania amazonensis and Plasmodium falciparum (clone W2, resistant to chloroquine). To this end, B. atrox and B. jararacussu venom peptides were purified by combination of 3 kDa cut-off Amicon® ultracentrifugal filters and reverse-phase high-performance liquid chromatography, and then identified by electrospray-ionization Ion-Trap/Time-of-Flight mass spectrometry. Fourteen distinct peptides, with masses ranging from 443.17 to 1383.73 Da and primary structure between 3 and 13 amino acid residues, were sequenced. Among them, 13 contained unique sequences, including 4 novel bradykinin-potentiating-like peptides (BPPs), and a snake venom metalloproteinase tripeptide inhibitor (SVMPi). Although commonly found in Viperidae venoms, except for Bax-12, the BPPs and SVMPi here reported had not been described in B. atrox and B. jararacussu venoms. Among the novel peptides, some exhibited bactericidal activity towards P. aeruginosa and S. aureus, had low hemolytic effect, and were devoid of antiparasitic activity. The identified novel antimicrobial peptides may be relevant in the development of new drugs for the management of multidrug-resistant Gram-negative and Gram-positive bacteria.

Camelid Single-Domain Antibodies for the Development of Potent Diagnosis Platforms.

The distinct biophysical and pharmaceutical properties of camelid single-domain antibodies, referred to as VHHs or nanobodies, are associated with their nanometric dimensions, elevated stability, and antigen recognition capacity. These biomolecules can circumvent a number of diagnostic system limitations, especially those related to the size and stability of conventional immunoglobulins currently used in enzyme-linked immunosorbent assays and point-of-care, electrochemical, and imaging assays. In these formats, VHHs are directionally conjugated to different molecules, such as metallic nanoparticles, small peptides, and radioisotopes, which demonstrates their comprehensive versatility. Thus, the application of VHHs in diagnostic systems range from the identification of cancer cells to the detection of degenerative disease biomarkers, viral antigens, bacterial toxins, and insecticides. The improvements of sensitivity and specificity are among the central benefits resulting from the use of VHHs, which are indispensable parameters for high-quality diagnostics. Therefore, this review highlights the main biotechnological advances related to camelid single-domain antibodies and their use in in vitro and in vivo diagnostic approaches for human health.

Adjacent dimer epitope of envelope protein as an important region for Zika virus serum neutralization: a computational investigation.

The recent emergence of Zika virus (ZIKV) has affected many countries, with severe clinical manifestations such as fetal microcephaly and Guillain-Barré syndrome. However, even though it is a major public health concern, there is no approved treatment available. Structural knowledge of the main neutralization regions of the envelope (E) protein of ZIKV and its interactions with neutralizing antibodies (nAbs) are crucial for the rational development of subunit vaccines and establishment of antibody-based interventions. In this study we screened from public data hot spot epitopes in conserved regions of ZIKV E protein that are nAbs targets. The result points to a conserved epitope located at domain II of the ZIKV E protein, namely adjacent dimer epitope, which is the ZIKV-117 and Z20 nAbs target. Although these two nAbs have been isolated from different donors, we have demonstrated, from structural and energetic details obtained by molecular dynamics of native and mutants, that hot spots residues of the epitope are the same for these nAbs, thereby indicating that they may share similar binding and neutralization mechanism. This convergence of information between these nAbs is important because both are potential targets for the development of therapies against ZIKV and only Z20 has its sequence and its complex structure with ZIKV E protein determined. Finally, these findings also contribute to existing knowledge, by fine mapping of the epitope/paratope residue pairs that are important for biotechnological development of therapies such as epitope mimetics for subunit vaccines and the rational design for antibody-based interventions against ZIKV. Communicated by Ramaswamy H. Sarma.

Lectin isolated from Bothrops jararacussu venom induces IL-10 release by TCD4+ cells and TNF-α release by monocytes and natural killer cells.

BjcuL is a C-type lectin isolated from Bothrops jararacussu snake venom with specificity for binding ß-d-galactose units. BjcuL is not toxic to human peripheral blood mononuclear cells (PBMCs), but it inhibits PBMC proliferation and stimulates these cells to produce superoxide anions and hydrogen peroxide primarily via lymphocyte stimulation; it does not stimulate the production of nitric oxide and PGE2 . The purpose of this study was to investigate the effect of BjcuL on PBMC activation with a focus on cytokine release modulating PBMC proliferation. The results showed for the first time that BjcuL coupled to FITC interacted with monocytes, B cells, natural killer (NK) cells, and with subpopulations of T cells. These cell-cell interactions can lead to cell activation and inflammatory cytokines release, such as IL-6 and TNF-α, as well as the anti-inflammatory cytokine IL-10. In addition, TNF-α release was attributed to NK cells and monocytes, whereas IL-10 was attributed to TCD4+ and Treg cells when stimulated by BjcuL. The temporal cytokines profile produced by cells when stimulated with this lectin allows us to assert that BjcuL has immunomodulatory activity in this context.

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice

Abstract INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Epidemiological study of snakebite cases in Brazilian Western Amazonia.

INTRODUCTION: Brazil has the largest number of snakebite cases in South America, of which the large majority is concentrated in the Midwest and North. METHODS: In this descriptive observational study, we assessed the epidemiological and clinical snakebite cases referred to the Centro de Medicina Tropical de Rondônia from September 2008 to September 2010. RESULTS: We followed up 92 cases from admission until discharge, namely 81 (88%) men and 11 (12%) women, with a mean age of 37 years, and mainly from rural areas (91.3%). The snakebites occurred while performing work activities (63%) during the Amazon rainy season (78.3%). The vast majority of individuals presented from the Porto Velho microregion (84.7%). Approximately 95.6% of the snakebites were caused by snakes of the genus Bothrops, followed by two lachetics and two elapidics cases. Surgery was performed in 10 cases (9 fasciotomies in the lower limb and 1 amputation). No deaths were reported in this study, but 4 cases (4.3%) developed sequelae in the lower limb. CONCLUSIONS: This study can contribute to a better understanding of envenomation in the state of Rondônia and thus can be useful for identifying real conditions that can increase the incidence of snakebites in this region. Moreover, the study results can serve as a basis for improving educational campaigns designed to prevent these types of snakebites, as well as for preserving snakes.

Epidemiological study of snakebite cases in Brazilian Western Amazonia

Abstract INTRODUCTION Brazil has the largest number of snakebite cases in South America, of which the large majority is concentrated in the Midwest and North. METHODS In this descriptive observational study, we assessed the epidemiological and clinical snakebite cases referred to the Centro de Medicina Tropical de Rondônia from September 2008 to September 2010. RESULTS We followed up 92 cases from admission until discharge, namely 81 (88%) men and 11 (12%) women, with a mean age of 37 years, and mainly from rural areas (91.3%). The snakebites occurred while performing work activities (63%) during the Amazon rainy season (78.3%). The vast majority of individuals presented from the Porto Velho microregion (84.7%). Approximately 95.6% of the snakebites were caused by snakes of the genus Bothrops, followed by two lachetics and two elapidics cases. Surgery was performed in 10 cases (9 fasciotomies in the lower limb and 1 amputation). No deaths were reported in this study, but 4 cases (4.3%) developed sequelae in the lower limb. CONCLUSIONS This study can contribute to a better understanding of envenomation in the state of Rondônia and thus can be useful for identifying real conditions that can increase the incidence of snakebites in this region. Moreover, the study results can serve as a basis for improving educational campaigns designed to prevent these types of snakebites, as well as for preserving snakes.

Engenharia de anticorpos na busca por fragmentos funcionais para insumos de diagnóstico e aplicações terapêuticas / Antibody engineering in the search for functional fragments for diagnostic inputs and therapeutic applications

Anticorpos, agentes empregados no desenvolvimento de pesquisas biomédicas, no diagnóstico e na terapêutica, possuem elevada capacidade de interação aos mais variados ligantes, Estruturalmente são heterotetrameros constituídos por duas cadeias leves e duas cadeias pesadas com massa molecular de aproximadamente 150 kDa, Visando melhorar as características farmacocinéticas e minimizar possíveis reações adversas desencadeadas por imunoglobulinas de origem não humana, a engenharia molecular de anticorpos vem obtendo fragmentos de anticorpos como porções Fab, F(ab?)2, scFv e Fv, Em adição aos anticorpos convencionais, camelídeos produzem imunoglobulinas funcionais desprovidas de cadeia leve, onde o domínio variável da cadeia pesada, denominado VHH ou nanocorpo, é responsável pelo reconhecimento antigênico, Apresentando características adequadas ao desenvolvimento de fármacos com alta capacidade de neutralização, fragmentos VHHs vêm sendo propostos para uso em imunoterapia passiva ou em drug-delivery, No diagnóstico esses fragmentos podem ser aplicados na construção de biosensores ou na imagiologia, atuando na detecção de células cancerígenas, no monitoramento de tumores ou em alterações celulares...

Antibodies, agents employed for the development of biomedical research, diagnostic and therapeutic, have high ability to interact with different ligands. Structurally are heterotetramers constituted by two light and two heavy chains, with molecular weight of approximately 150 kDa. Aiming to improve the pharmacokinetic properties and minimize possible adverse reactions triggered by immunoglobulins of non-human origin, the molecular engineering of antibodies has been obtaining fragments of antibodies, such as Fab, F(ab?)2, Fv and scFv. In addition to the conventional antibodies, camelids produce functional immunoglobulins devoid of light chain, in which the variable domain, named VHH or nanocorpo, is able to recognize the antigen. With appropriate characteristics for the development of drugs with high neutralizing capacity, VHH fragments have been proposed for use in passive immunotherapy or drug-delivery. To the diagnosis, these fragments can be used to construct biosensors, in the imagiology , acting in the detection of cancer cells, tumor monitoring or cell changes...

A lupane-triterpene isolated from Combretum leprosum Mart. fruit extracts that interferes with the intracellular development of Leishmania (L.) amazonensis in vitro.

BACKGROUND: 3beta,6beta,16beta-trihydroxylup-20(29)-ene is a lupane triterpene isolated from Combretum leprosum fruit. The lupane group has been extensively used in studies on anticancer effects; however, its possible activity against protozoa parasites is yet poorly known. The high toxicity of the compounds currently used in leishmaniasis chemotherapy stimulates the investigation of new molecules and drug targets for antileishmanial therapy. METHODS: The activity of 3beta,6beta,16beta-trihydroxylup-20(29)-ene was evaluated against Leishmania (L.) amazonensis by determining the cytotoxicity of the compound on murine peritoneal macrophages, as well as its effects on parasite survival inside host cells. To evaluate the effect of this compound on intracellular amastigotes, cultures of infected macrophages were treated for 24, 48 and 96 h and the percentage of infected macrophages and the number of intracellular parasites was scored using light microscopy. RESULTS: Lupane showed significant activity against the intracellular amastigotes of L. (L.) amazonensis. The treatment with 109 µM for 96 h reduced in 80 % the survival index of parasites in BALB/c peritoneal macrophages. At this concentration, the triterpene caused no cytotoxic effects against mouse peritoneal macrophages. Ultrastructural analyses of L. (L.) amazonensis intracellular amastigotes showed that lupane induced some morphological changes in parasites, such as cytosolic vacuolization, lipid body formation and mitochondrial swelling. Bioinformatic analyses through molecular docking suggest that this lupane has high-affinity binding with DNA topoisomerase. CONCLUSION: Taken together, our results have showed that the lupane triterpene from C. leprosum interferes with L. (L.) amazonensis amastigote replication and survival inside vertebrate host cells and bioinformatics analyses strongly indicate that this molecule may be a potential inhibitor of topoisomerase IB. Moreover, this study opens major prospects for the development of novel chemotherapeutic agents with leishmanicidal activity.

Structure-activity relationship of mastoparan analogs: Effects of the number and positioning of Lys residues on secondary structure, interaction with membrane-mimetic systems and biological activity.

In this study, a series of mastoparan analogs were engineered based on the strategies of Ala and Lys scanning in relation to the sequences of classical mastoparans. Ten analog mastoparans, presenting from zero to six Lys residues in their sequences were synthesized and assayed for some typical biological activities for this group of peptide: mast cell degranulation, hemolysis, and antibiosis. In relation to mast cell degranulation, the apparent structural requirement to optimize this activity was the existence of one or two Lys residues at positions 8 and/or 9. In relation to hemolysis, one structural feature that strongly correlated with the potency of this activity was the number of amino acid residues from the C-terminus of each peptide continuously embedded into the zwitterionic membrane of erythrocytes-mimicking liposomes, probably due to the contribution of this structural feature to the membrane perturbation. The antibiotic activity of mastoparan analogs was directly dependent on the apparent extension of their hydrophilic surface, i.e., their molecules must have from four to six Lys residues between positions 4 and 11 of the peptide chain to achieve activities comparable to or higher than the reference antibiotic compounds. The optimization of the antibacterial activity of the mastoparans must consider Lys residues at the positions 4, 5, 7, 8, 9, and 11 of the tetradecapeptide chain, with the other positions occupied by hydrophobic residues, and with the C-terminal residue in the amidated form. These requirements resulted in highly active AMPs with greatly reduced (or no) hemolytic and mast cell degranulating activities.

Structure-function relationships of the peptide Paulistine: a novel toxin from the venom of the social wasp Polybia paulista.

BACKGROUND: The peptide Paulistine was isolated from the venom of wasp Polybia paulista. This peptide exists under a natural equilibrium between the forms: oxidised - with an intra-molecular disulphide bridge; and reduced - in which the thiol groups of the cysteine residues do not form the disulphide bridge. The biological activities of both forms of the peptide are unknown up to now. METHODS: Both forms of Paulistine were synthesised and the thiol groups of the reduced form were protected with the acetamidemethyl group [Acm-Paulistine] to prevent re-oxidation. The structure/activity relationships of the two forms were investigated, taking into account the importance of the disulphide bridge. RESULTS: Paulistine has a more compact structure, while Acm-Paulistine has a more expanded conformation. Bioassays reported that Paulistine caused hyperalgesia by interacting with the receptors of lipid mediators involved in the cyclooxygenase type II pathway, while Acm-Paullistine also caused hyperalgesia, but mediated by receptors involved in the participation of prostanoids in the cyclooxygenase type II pathway. CONCLUSION: The acetamidemethylation of the thiol groups of cysteine residues caused small structural changes, which in turn may have affected some physicochemical properties of the Paulistine. Thus, the dissociation of the hyperalgesy from the edematogenic effect when the actions of Paulistine and Acm-Paulistine are compared to each other may be resulting from the influence of the introduction of Acm-group in the structure of Paulistine. GENERAL SIGNIFICANCE: The peptides Paulistine and Acm-Paulistine may be used as interesting tools to investigate the mechanisms of pain and inflammation in future studies.

Effect of Bothrops bilineata snake venom on neutrophil function.

The aim of the study was to evaluate the in vitro effects of Bothrops bilineata crude venom (BbV) on isolated human neutrophil function. We proved that BbV isn't toxic towards human neutrophils. During an incubation of human neutrophils with BbV hydrogen peroxide was produced. Moreover, BbV was able to stimulate neutrophil release of proinflammatory mediators such as IL-8 and IL-6 as well as PGE2 and NETs liberation. There is no data in the literature showing the effect of BbV on the production of IL-6 and IL-8 or NETs liberation by isolated human neutrophils. Taken together our results testify that BbV triggers relevant proinflammatory events in human neutrophils.

Copaifera multijuga ethanolic extracts, oil-resin, and its derivatives display larvicidal activity against Anopheles darlingi and Aedes aegypti (Diptera: Culicidae)

Copaifera spp. is a common tree species found in the tropical region of Latin America, popularly known as copaiba or pau-d'alho. Oil-resin from different Copaifera species and its components present several biological activities such as antimicrobial, anti-inflammatory, antioxidant and insecticidal, including larvicidal activity against mosquitoes. Thus, bark and leaf ethanolic extracts, oil-resin, essential oil and alepterolic acid from Copaifera multijuga Hayne, Fabaceae, were tested as larvicides against the main malaria vector in the north of Brazil, Anopheles darlingi and also Aedes aegypti, the dengue vector. A. darlingi larval mortality was significantly higher than A. aegypti for most tested compounds. Bark and leaf extracts resulted in lower Lethal Concentrations (LC50) values for A. darlingi, 3 and 13 ppm, respectively, while the essential oil provided the lowest LC50 value for A. aegypti, 18 ppm. Despite of that, the lowest LC values were from the alepterolic acid for both species, i.e. 0.9 and 0.7 ppm for A. darlingi and A. aegypti, respectively.