HIV-1 drug resistance and genetic diversity in people with HIV-1 in Cape Verde.

OBJECTIVES: To characterize the genetic diversity and drug resistance profiles of people with HIV-1 failing ART in Cape Verde (CV). DESIGN: Cross-sectional study conducted between January 2019 and December 2021 in 24 health centres on the islands of Santiago and São Vicente. METHODS: The HIV-1 pol gene was sequenced in individuals with a detectable viral load. HIV-1 genetic diversity was determined by phylogenetic analysis. Drug resistance mutation patterns and resistance phenotypes were estimated using the Stanford algorithm. RESULTS: Viral load was detected in 73 of 252 (29%) enrolled participants and sequencing data were produced for 58 (79%) participants. CRF02 AG strains predominated (46.5%), followed by subtype G (22.4%). Most patients (80%) had mutations conferring resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) (67%), nucleoside reverse transcriptase inhibitors (55%), integrase inhibitors (10%) and/or protease inhibitors (7%) used in Cape Verde, a significant increase compared with a study conducted in 2010-2011. The most common mutations were M184V/I (43%), K103N/S (36%) and G190A/S (19%). NNRTI resistance was associated with younger age and exposure to two or more drug regimens. CONCLUSION: The HIV-1 epidemic in Cape Verde is mainly driven by CRF02_AG and subtype G. Resistance to NNRTIs and/or NRTIs is highly prevalent and resistance to LPV/r and DTG is emerging. Our results support the use of DTG-based first-line ART and protease inhibitor-based regimens for patients with virological failure, but emerging resistance to LPV/r and DTG is a concern. Continued monitoring of drug resistance is essential to ensure adequate healthcare for PWH in Cape Verde.

An HIV-1/HIV-2 Chimeric Envelope Glycoprotein Generates Binding and Neutralising Antibodies against HIV-1 and HIV-2 Isolates.

The development of immunogens that elicit broadly reactive neutralising antibodies (bNAbs) is the highest priority for an HIV vaccine. We have shown that a prime-boost vaccination strategy with vaccinia virus expressing the envelope glycoprotein gp120 of HIV-2 and a polypeptide comprising the envelope regions C2, V3 and C3 elicits bNAbs against HIV-2. We hypothesised that a chimeric envelope gp120 containing the C2, V3 and C3 regions of HIV-2 and the remaining parts of HIV-1 would elicit a neutralising response against HIV-1 and HIV-2. This chimeric envelope was synthesised and expressed in vaccinia virus. Balb/c mice primed with the recombinant vaccinia virus and boosted with an HIV-2 C2V3C3 polypeptide or monomeric gp120 from a CRF01_AG HIV-1 isolate produced antibodies that neutralised >60% (serum dilution 1:40) of a primary HIV-2 isolate. Four out of nine mice also produced antibodies that neutralised at least one HIV-1 isolate. Neutralising epitope specificity was assessed using a panel of HIV-1 TRO.11 pseudoviruses with key neutralising epitopes disrupted by alanine substitution (N160A in V2; N278A in the CD4 binding site region; N332A in the high mannose patch). The neutralisation of the mutant pseudoviruses was reduced or abolished in one mouse, suggesting that neutralising antibodies target the three major neutralising epitopes in the HIV-1 envelope gp120. These results provide proof of concept for chimeric HIV-1/HIV-2 envelope glycoproteins as vaccine immunogens that can direct the antibody response against neutralising epitopes in the HIV-1 and HIV-2 surface glycoproteins.

Phylogeography of hepatitis B virus: The role of Portugal in the early dissemination of HBV worldwide.

In Portugal, the genetic diversity, origin of HBV and the Portuguese role in the dissemination of HBV worldwide were never investigated. In this work, we studied the epidemic history and transmission dynamics of HBV genotypes that are endemic in Portugal. HBV pol gene was sequenced from 130 patients followed in Lisbon. HBV genotype A was the most prevalent (n = 54, 41.5%), followed by D (n = 44, 33.8%), and E (n = 32, 24.6%). Spatio-temporal evolutionary dynamics was reconstructed in BEAST using a Bayesian Markov Chain Monte Carlo method, with a GTR nucleotide substitution model, an uncorrelated lognormal relaxed molecular clock model, a Bayesian skyline plot, and a continuous diffusion model. HBV subgenotype D4 was the first to be introduced in Portugal around 1857 (HPD 95% 1699-1931) followed by D3 and A2 a few decades later. HBV genotype E and subgenotype A1 were introduced in Portugal later, almost simultaneously. Our results indicate a very important role of Portugal in the exportation of subgenotypes D4 and A2 to Brazil and Cape Verde, respectively, in the beginning of the XX century. This work clarifies the epidemiological history of HBV in Portugal and provides new insights in the early and global epidemic history of this virus.

Long-Term and Low-Level Envelope C2V3 Stimulation by Highly Diverse Virus Isolates Leads to Frequent Development of Broad and Elite Antibody Neutralization in HIV-1-Infected Individuals.

A minority of HIV-1-infected patients produce broadly neutralizing antibodies (bNAbs). Identification of viral and host correlates of bNAb production may help develop vaccines. We aimed to characterize the neutralizing response and viral and host-associated factors in Angola, which has one of the oldest, most dynamic, and most diverse HIV-1 epidemics in the world. Three hundred twenty-two HIV-1-infected adults from Angola were included in this retrospective study. Phylogenetic analysis of C2V3C3 env gene sequences was used for virus subtyping. Env-binding antibody reactivity was tested against polypeptides comprising the C2, V3, and C3 regions. Neutralizing-antibody responses were determined against a reference panel of tier 2 Env pseudoviruses in TZM-bl cells; neutralizing epitope specificities were predicted using ClustVis. All subtypes were found, along with untypeable strains and recombinant forms. Notably, 56% of the patients developed cross neutralizing, broadly neutralizing, or elite neutralizing responses. Broad and elite neutralization was associated with longer infection time, subtype C, lower CD4+ T cell counts, higher age, and higher titer of C2V3C3-specific antibodies relative to failure to develop bNAbs. Neutralizing antibodies targeted the V3-glycan supersite in most patients. V3 and C3 regions were significantly less variable in elite neutralizers than in weak neutralizers and nonneutralizers, suggesting an active role of V3C3-directed bNAbs in controlling HIV-1 replication and diversification. In conclusion, prolonged and low-level envelope V3C3 stimulation by highly diverse and ancestral HIV-1 isolates promotes the frequent elicitation of bNAbs. These results provide important clues for the development of an effective HIV-1 vaccine. IMPORTANCE Studies on neutralization by antibodies and their determinants in HIV-1-infected individuals have mostly been conducted in relatively recent epidemics caused by subtype B and C viruses. Results have suggested that elicitation of broadly neutralizing antibodies (bNAbs) is uncommon. The mechanisms underlying the elicitation of bNAbs are still largely unknown. We performed the first characterization of the plasma neutralizing response in a cohort of HIV-1-infected patients from Angola. Angola is characterized by an old and dynamic epidemic caused by highly diverse HIV-1 variants. Remarkably, more than half of the patients produced bNAbs, mostly targeting the V3-glycan supersite in HIV-1. This was associated with higher age, longer infection time, lower CD4+ T cell counts, subtype C infection, or higher titer of C2V3C3-specific antibodies relative to patients that did not develop bNAbs. These results may help develop the next generation of vaccine candidates for HIV-1.

Ceratocystiopsis quercina sp. nov. Associated with Platypus cylindrus on Declining Quercus suber in Portugal.

Platypus cylindrus is the most common ambrosia beetle in stands of Quercus suber in Portugal. This insect farms specialized fungi in sapwood galleries, using its mycangia to carry and store these organisms. Some ectosymbiotic fungi carried by P. cylindrus are phytopathogenic and cause extensive tree mortality and severe economic losses. To understand the role of P. cylindrus fungal symbionts in stands of Q. suber we examined beetle galleries present in declining and/or dying cork oak trees during field surveys. Logs with active galleries were obtained in situ and from captured emerging beetles. Insects were aseptically dissected, and their mycangia and intestine were retrieved. Morphological and molecular profiles of fungal isolates obtained from cultured insect parts were carried out to accurately characterize and identify isolated fungi. Molecular characterizations were performed with DNA sequence data from four loci, i.e., LSU, SSU, 5.8S-ITS2-28S, and TUB. Morphological results consistently showed a collection of Ophiostoma-like fungal axenic isolates, while phylogenies inferred that this collection constitutes an undescribed taxon reported herein for the first time in association with P. cylindrus in Portuguese cork oak stands. The novel species was erected as Ceratocystiopsis quercina sp. nov. and constitutes a new phytopathogenic fungal species associated with symptoms of vegetative cork oak decline.

Study of inorganic elements in different organs and tissues of Amazonian manatee (Trichechus inunguis) from Brazil.

Multielement concentrations (P, S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Rh) and total mercury (T-Hg) were analyzed in different organs and tissues of Amazonian manatee (Trichechus inunguis). Samples of 27 T. inunguis specimens, maintained in the collection of the Amazonian Center for the Research and Preservation of Aquatic Mammals, were used, situated in an area highly impacted by gold mining in the northern region of the Brazilian Amazon. Samples of aquatic plants used as food by the animals were also analyzed. The elements S, Cl, K, Cr, and Mn accumulated mainly in the musculature, while Fe and Cu were more concentrated in the liver. Trace elements, such as rubidium (Rb) and rhodium (Rh), not previously reported in the organs of animals of the family Trichechidae, were also identified. The averages for T-Hg in the skin, muscle, encephalon, liver, kidney, and lung samples were, respectively, 0.1540 ± 0.1332, 0.0593 ± 0.1044, 0.0517 ± 0.0467, 0.0486 ± 0.0543, 0.0237 ± 0.0336, and 0.0013 ± 0.0032 µg.g-1. The values obtained for the vibrissae samples were below the limit of quantification, which allows for the conclusion that this tissue cannot be used as a contamination marker. It was observed that even when kept in a conservation breeding site, these animals were exposed to non-essential trace elements. Differences in the accumulation of elements were observed between the different organs and tissues analyzed. The presence of contaminants in animals that live in a preservation center, even at low levels, deserves attention.

Antibody response against selected epitopes in the HIV-1 envelope gp41 ectodomain contributes to reduce viral burden in HIV-1 infected patients.

The ectodomain of gp41 is the target of potent binding and neutralizing antibodies (NAbs) and is being explored in new strategies for antibody-based HIV vaccines. Previous studies have suggested that the W164A-3S (3S) and EC26-2A4 (EC26) peptides located in the gp41 ectodomain may be potential HIV vaccine candidates. We assessed 3S- and EC26-specific binding antibody responses and related neutralizing activity in a large panel of chronic HIV-1-infected Portuguese individuals on ART. A similar proportion of participants had antibodies binding to 3S (9.6%) and EC26 (9.9%) peptides but the level of reactivity against 3S was significantly higher compared to EC26, except in the rare patients with double peptide reactivity. The higher antigenicity of 3S was unrelated with disease stage, as assessed by CD4+ T cell counts, but it was directly related with plasma viral load. Most patients that were tested (89.9%, N = 268) showed tier 1 neutralizing activity, the potency being inversely associated with plasma viral load. In the subset of patients that were tested for neutralization of tier 2 isolates, neutralization breadth was inversely correlated with plasma viral load and directly correlated with CD4+ T cell counts. These results are consistent with a role for neutralizing antibodies in controlling viral replication and preventing the decline of CD4+ T lymphocytes. Importantly, in patients with 3S-specific antibodies, neutralizing titers were inversely correlated with viral RNA levels and proviral DNA levels. Moreover, patients with 3S and/or EC26-specific antibodies showed a 1.9-fold higher tier 2 neutralization score than patients without antibodies suggesting that 3S and/or EC26-specific antibodies contribute to neutralization breadth and potency in HIV-1 infected patients. Overall, these results suggest that antibodies targeting the S3 and EC26 epitopes may contribute to reduce viral burden and provide further support for the inclusion of 3S and EC26 epitopes in HIV-1 vaccine candidates.

Standardised arthropod (Arthropoda) inventory across natural and anthropogenic impacted habitats in the Azores archipelago.

BACKGROUND: In this paper, we present an extensive checklist of selected arthropods and their distribution in five Islands of the Azores (Santa Maria. São Miguel, Terceira, Flores and Pico). Habitat surveys included five herbaceous and four arboreal habitat types, scaling up from native to anthropogenic managed habitats. We aimed to contribute to the ongoing effort to document the terrestrial biodiversity of the world, in particular the Portuguese archipelago of the Azores, as islands harbour a significant portion of unique terrestrial biodiversity. Selection of Arthropoda groups for the current checklist was based on their known richness and abundance (Arachnida, Collembola, Hemiptera, Neuroptera, Coleoptera, Hymenoptera), in almost all terrestrial ecosystems, as well as their importance in current Integrated Pest Management and alternative Biocontrol protocols at large (i.e. hymenopteran parasitoids and beneficial Coleoptera). In addition, we include the list of Dermaptera, Orthoptera, Psocoptera and Thysanoptera species. These assembled groups represent part of the monitoring programme EDEN Azores (2008-2014), where all Arthropod fauna, at all strata, within nine representative habitats of the abovementioned five Islands of the Azores was recorded. NEW INFORMATION: In this study, a total of 116,523 specimens, belonging to 483 species and subspecies of selected groups of arthropods, are reported by order, family and, when possible, genus and species. Hymenopteran, mostly parasitoids, accounted for the most represented taxa across all the monitoring and sampling phase of EDEN Azores (193 species and mophospecies), followed by Coleoptera (95 species); Collembola (89 species); and Araneae (72 species).A total of 37 non-native species are reported for the first time in the Azores. Coleoptera: Asaphidion flavipes (Linnaeus, 1761) (Carabidae); Tachyporus dispar (Paykull, 1789) (Staphylinidae). Hemiptera: Acrosternum heegeri Fieber, 1861 (Pentatomidae). Collembola: Entomobrya regularis Stach, 1963 (Entomobryidae); Lepidocyrtus lusitanicus piezoensis (Simón-Benito, 2007) (Entomobryidae); Jordanathrix articulata (Ellis, 1974) (Sminthuridae); Sminthurinus quadrimaculatus (Ryder, 1879) (Katiannidae); Himalanura sp. (Entomobryidae); Protophorura sp. (Onychiuridae). Hymenoptera, parasitoids: Aphidius colemani Viereck, 1912 (Braconidae); Aphidius ervi Haliday, 1834 (Braconidae); Aphidius matricariae Viereck, 1912 (Braconidae); Aphidius rhopalosiphi Stefani-Perez, 1902 (Braconidae); Aphidius rosae (Haliday, 1834) (Braconidae); Aphidius urticae Haliday, 1834 (Braconidae); Centistidea ectoedemiae Rohwer, 1914 (Braconidae); Meteorus unicolor (Wesmael, 1835) (Braconidae); Meteorus collaris (Spin.) Hal. - Ruschka, Fulmek, 1915 (Braconidae); Orthostigma cratospilum (Thomson, 1895) (Braconidae); Orthostigma latriventris Ratzeburg, 1844 (Braconidae); two other species of Orthostigma sp.; Pseudopezomachus bituberculatus (Marshall, 1905) (Braconidae); Tanycarpa punctata (van Achterberg, 1976) (Braconidae); Gonatopus clavipes (Thunberg, 1827) (Dryinidae). New genera not previously recorded for the Azores include: Pycnetron sp. (Chalcidoidea: Pteromalidae); four species of Aspilota sp. (Braconidae: Alysiinae); four species of Chorebus sp. (Braconidae: Aphidiinae: Alysiinae); Microgaster sp. (Braconidae: Microgastrinae); Homolobus sp. (Braconidae: Homolobinae); Lodbrokia sp. (Braconidae: Alysiinae).These 37 taxa were found in several Islands and five are new species for Flores Island, 10 species are new for Pico Island, 12 species are new for Terceira Island, 19 species are new for S. Miguel Island and five species are new for S. Maria Island.Additional species records for the Islands included: Flores (5 Collembola, 9 Araneae; 2 Hemiptera; 8 Coleoptera, 8 Hymenoptera), Pico (4 Collembola; 7 Araneae; 4 Hemiptera; 11 Coleoptera; 9 Hymenoptera), Terceira (4 Collembola; 1 Araneae; 3 Hymenoptera), S. Miguel (1 Araneae; 2 Coleoptera; 3 Hymenoptera), S. Maria (5 Collembola; 3 Araneae; 2 Hemiptera; 2 Hymenoptera).

A Prime-Boost Immunization Strategy with Vaccinia Virus Expressing Novel gp120 Envelope Glycoprotein from a CRF02_AG Isolate Elicits Cross-Clade Tier 2 HIV-1 Neutralizing Antibodies.

Development of new immunogens eliciting broadly neutralizing antibodies (bNAbs) is a main priority for the HIV-1 vaccine field. Envelope glycoproteins from non-B-non-C HIV-1clades have not been fully explored as components of a vaccine. We produced Vaccinia viruses expressing a truncated version of gp120 (gp120t) from HIV-1 clades CRF02_AG, H, J, B, and C and examined their immunogenicity in mice and rabbits. Mice primed with the recombinant Vaccinia viruses and boosted with the homologous gp120t or C2V3C3 polypeptides developed antibodies that bind potently to homologous and heterologous envelope glycoproteins. Notably, a subset of mice immunized with the CRF02_AG-based envelope immunogens developed a cross-reactive neutralizing response against tier 2 HIV-1 Env-pseudoviruses and primary isolates. Rabbits vaccinated with the CRF02_AG-based envelope immunogens also generated potent binding antibodies, and one animal elicited antibodies that neutralized almost all (13 of 16, 81.3%) tier 2 HIV-1 isolates tested. Overall, the results suggest that the novel CRF02_AG-based envelope immunogens and prime-boost immunization strategy elicit the type of immune responses required for a preventive HIV-1 vaccine.

Soybean aphid biotype 1 genome: Insights into the invasive biology and adaptive evolution of a major agricultural pest.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae) is a serious pest of the soybean plant, Glycine max, a major world-wide agricultural crop. We assembled a de novo genome sequence of Ap. glycines Biotype 1, from a culture established shortly after this species invaded North America. 20.4% of the Ap. glycines proteome is duplicated. These in-paralogs are enriched with Gene Ontology (GO) categories mostly related to apoptosis, a possible adaptation to plant chemistry and other environmental stressors. Approximately one-third of these genes show parallel duplication in other aphids. But Ap. gossypii, its closest related species, has the lowest number of these duplicated genes. An Illumina GoldenGate assay of 2380 SNPs was used to determine the world-wide population structure of Ap. Glycines. China and South Korean aphids are the closest to those in North America. China is the likely origin of other Asian aphid populations. The most distantly related aphids to those in North America are from Australia. The diversity of Ap. glycines in North America has decreased over time since its arrival. The genetic diversity of Ap. glycines North American population sampled shortly after its first detection in 2001 up to 2012 does not appear to correlate with geography. However, aphids collected on soybean Rag experimental varieties in Minnesota (MN), Iowa (IA), and Wisconsin (WI), closer to high density Rhamnus cathartica stands, appear to have higher capacity to colonize resistant soybean plants than aphids sampled in Ohio (OH), North Dakota (ND), and South Dakota (SD). Samples from the former states have SNP alleles with high FST values and frequencies, that overlap with genes involved in iron metabolism, a crucial metabolic pathway that may be affected by the Rag-associated soybean plant response. The Ap. glycines Biotype 1 genome will provide needed information for future analyses of mechanisms of aphid virulence and pesticide resistance as well as facilitate comparative analyses between aphids with differing natural history and host plant range.

Estudo randomizado de intervenção com dieta hiperproteica vs dieta de alto teor de carboidrato em idosas com excesso de peso submetidas a treino de força / Randomized intervention study with high protein vs. high carbohydrate diet in overweight older women undergoing strength training

INTRODUÇÃO: Estudos sugerem que dietas hipocalóricas ricas em proteínas podem ser mais eficazes na perda de peso e de gordura corporal do que dietas hipocalóricas com quantidades normais de proteínas. OBJETIVO: Avaliar o impacto de intervenções dietéticas isocalóricas com modificações nas quantidades de proteínas e carboidratos sobre o peso e a composição corporal de idosas. MÉTODOS: 25 mulheres idosas com excesso de peso (índice de massa corporal > 25Kg/m2) foram submetidas a treino de força e dietas com redução de 300Kcal, modificações nas quantidades de proteínas (1,8g/Kg/dia X 1,0g/Kg/dia) e carboidratos (2,0g/Kg/dia X 3,0g/Kg/dia) e quantidades similares de lipídios e fibras durante oito semanas. RESULTADOS: O grupo carboidrato apresentou uma perda ponderal clinicamente significativa comparado ao grupo controle (-2,5±2,3 X -0,4 ±2,1 p = 0,086). Quanto à perda de gordura corporal, os grupos carboidrato e proteína apresentaram valores superiores ao dobro do grupo controle, com diferencial clínico importante, principalmente entre os grupos controle e carboidrato (714±1701 X -2061±2297). DISCUSSÃO: Apesar de alguns estudos relatarem os efeitos benéficos de uma dieta rica em proteína no emagrecimento, como saciedade e maior efeito termogênico induzido pela dieta, no presente estudo, a restrição calórica foi mais importante do que a manipulação na quantidade dos macronutrientes. CONCLUSÃO: A dieta hipocalórica com padrões diferentes de ingestão de proteínas parece não ser superior à simples restrição calórica na perda de peso e mudança de composição corporal

INTRODUCTION: Studies suggest that low protein calorie diets may be more effective in losing weight and body fat than low calorie diets with normal amounts of protein. OBJECTIVE: To evaluate the impact of isocaloric dietary interventions with changes in the amounts of proteins and carbohydrates on the weight and body composition of elderly women. mass index> 25 kg / m2) were submitted to strength training and diets with a reduction of 300 kg, changes in the amounts of proteins (1.8 g / kg / day X 1.0 g / Kg / day) and carbohydrates (2.0g / kg / day X 3.0g / kg / day) and similar amounts of lipids and fibers for eight weeks. RESULTS: The carbohydrate group had a clinically significant weight loss compared to the control group (-2.5 ± 2.3 X -0.4 ± 2.1 p = 0.086). As for the loss of body fat, the carbohydrate and protein groups showed values higher than twice the control group, with an important clinical differential, especially between the control and carbohydrate groups (714 ± 1701 X -2061 ± 2297). DISCUSSION: Although some studies report the beneficial effects of a protein-rich diet on weight loss, such as satiety and a greater thermogenic effect induced by the diet, in the present study, caloric restriction was more important than manipulation in the amount of macronutrients. CONCLUSION: The low-calorie diet with different patterns of protein intake does not seem to be superior to the simple caloric restriction in weight loss and changes in body composition

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Tetanus antitoxin potency assessment by surface plasmon resonance and ToBI test.

Potency testing of tetanus antitoxin must be performed in vivo, in a very painful, stressful and prone to high variability assay. It is, therefore, mandatory to find alternatives to this kind of potency assessment. Immunochemical tests as ELISA or ToBI test are already available but usually results in a poor correlation to the in vivo protection. Considering research and development of mono and oligoclonal antibodies against tetanus and the improvement of equine polyclonal antitoxin production and control, we developed an alternative instrumental test for tetanus antitoxin by using surface plasmon resonance. Tetanus antitoxin from hyperimmune equine sera (16 batches) were tested and the results indicated excellent concordance and correlation to the in vivo test (Lin's ρ = 0.9). This innovative approach should now be improved in order to extend it to oligoclonal and monoclonal human antibodies aiming to replace mice for the potency assessment of tetanus antitoxin especially during research and development steps.

Tetanus antitoxin potency assessment by surface plasmon resonance and ToBI test

Potency testing of tetanus antitoxin must be performed in vivo, in a very painful, stressful and prone to high variability assay. It is, therefore, mandatory to find alternatives to this kind of potency assessment. Immunochemical tests as ELISA or ToBI test are already available but usually results in a poor correlation to the in vivo protection. Considering research and development of mono and oligoclonal antibodies against tetanus and the improvement of equine polyclonal antitoxin production and control, we developed an alternative instrumental test for tetanus antitoxin by using surface plasmon resonance. Tetanus antitoxin from hyperimmune equine sera (16 batches) were tested and the results indicated excellent concordance and correlation to the in vivo test (Lin's Ró=0.9). This innovative approach should now be improved in order to extend it to oligoclonal and monoclonal human antibodies aiming to replace mice for the potency assessment of tetanus antitoxin especially during research and development steps.

Tetanus antitoxin potency assessment by surface plasmon resonance and ToBI test

Potency testing of tetanus antitoxin must be performed in vivo, in a very painful, stressful and prone to high variability assay. It is, therefore, mandatory to find alternatives to this kind of potency assessment. Immunochemical tests as ELISA or ToBI test are already available but usually results in a poor correlation to the in vivo protection. Considering research and development of mono and oligoclonal antibodies against tetanus and the improvement of equine polyclonal antitoxin production and control, we developed an alternative instrumental test for tetanus antitoxin by using surface plasmon resonance. Tetanus antitoxin from hyperimmune equine sera (16 batches) were tested and the results indicated excellent concordance and correlation to the in vivo test (Lin's Ró=0.9). This innovative approach should now be improved in order to extend it to oligoclonal and monoclonal human antibodies aiming to replace mice for the potency assessment of tetanus antitoxin especially during research and development steps.

Quality of horse F(ab')2 antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity.

BACKGROUND: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. METHODS: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. RESULTS: Horse F(ab')2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. CONCLUSIONS: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

The history of antivenoms development: Beyond Calmette and Vital Brazil.

This review presents the main contributions to our knowledge regarding the development of antivenoms for therapeutic use in victims of venomous animal bites. We cover the progress of serum therapy since tetanus and diphtheria antitoxins in Germany and France until the current scenario of antivenom production worldwide. During these more than 120 years of antivenom development, many researchers contributed to establish what are nowadays the antivenoms used for therapeutic purpose. The history of antivenoms development is fascinating! This review aims to recognize all those who contributed to the establishment of new sera, new methodologies and saving lives: much more than Calmette and Vital Brazil.

Does the administration of pilocarpine prior to venom milking influence the composition of Micrurus corallinus venom?

Considering that the scarcity of venom represents a huge challenge for biochemical and functional studies of Micrurus species (coral snakes), in this report we describe for the first time the influence of pilocarpine administration prior to venom milking on the yield and protein composition of Micrurus corallinus venom. The administration of pilocarpine resulted in an increase of about 127% in the volume of venom milked, with similar protein content. Venoms showed similar protein bands distribution and intensity by SDS-PAGE and equivalents RP-HPLC profiles. Our proteomic analysis showed that venoms milked in the presence and absence of pilocarpine presented comparable protein profiles, in terms of protein composition and relative abundance. The toxins identified were assigned to 13 protein families and represent the most complete M. corallinus venom proteome described so far, in terms of number of protein families identified. Our data indicate that the administration of pilocarpine prior to venom milking increases the venom yield and does not change significantly the venom composition of M. corallinus. The employment of pilocarpine represents a useful approach to increase the yield of venom not only for Micrurus species, but also for other genera of snakes with limitations regarding the amount of venom available. SIGNIFICANCE: In this report, we evaluated the influence of pilocarpine administration prior to venom milking in the overall composition of M. corallinus venom. We showed that the use of pilocarpine 10min before M. corallinus venom milking increases venom yield by ~127%. Not only the volume of venom obtained is higher, but also the protein concentration of both venoms is similar, opposing the idea that a more diluted venom is obtained as a result of pilocarpine administration, observed in non-front-fanged snakes. Shotgun proteomics analysis revealed that venom milked with and without the use of this drug showed similar overall protein composition and relative abundances. In addition, our proteomic approach allowed the identification of 13 toxin families in M. corallinus venom, representing the most complete M. corallinus venom proteome described so far. Moreover, two of these toxin families were identified for the first time in the venom of this species. Thus, considering the scarcity of Micrurus venom for biochemical and functional studies, we highlighted the usefulness of pilocarpine administration prior to venom milking to increase the venom yield of these snakes.

The history of antivenoms development: beyond Calmette and Vital Brazil

This review presents the main contributions to our knowledge regarding the development of antivenoms for therapeutic use in victims of venomous animal bites. We cover the progress of serum therapy since tetanus and diphtheria antitoxins in Germany and France until the current scenario of antivenom production worldwide. During these more than 120 years of antivenom development, many researchers contributed to establish what are nowadays the antivenoms used for therapeutic purpose. The history of antivenoms development is fascinating! This review aims to recognize all those who contributed to the establishment of new sera, new methodologies and saving lives: much more than Calmette and Vital Brazil.

Quality of horse F(ab')2 antitoxins and anti-rabies immunoglobulins: protein content and anticomplementary activity

Background: Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab')2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods: Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results: Horse F(ab')2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions: Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab')(2) immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab')2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.

A functional and thromboelastometric-based micromethod for assessing crotoxin anticoagulant activity and antiserum relative potency against Crotalus durissus terrificus venom

The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies on traditional rodent in vivo lethality assay. ED50 and LD50 assays require large quantities of venoms and antivenoms, and besides leading to animal suffering. Therefore, in vitro tests should be introduced for assessing antivenom neutralizing capacity in intermediary steps of antivenom production. This task is facilitated when one key lethal toxin is identified. A good example is crotoxin, a P-neurotoxin phospholipase A(2)-like toxin that presents anticoagulant activity in vitro and is responsible for the lethality of venoms of Crotalus durissus snakes. By using rotational thromboelastometry, we reported recently one sensitive coagulation assay for assessing relative potency of the anti-bothropic serum in neutralizing procoagulant activity of Bothrops jararaca venom upon recalcified factor-XII-deficient chicken plasma samples (CPS). In this study, we stablished conditions for determining relative potency of four batches of the anti-crotalic serum (ACS) (antagonist) in inactivating crotoxin anticoagulant activity in CPS (target) simultaneously treated with one classical activator of coagulation (agonists). The correlation coefficient (r) between values related the ACS potency in inactivating both in vitro crotoxin anticoagulant activity and the in vivo lethality of whole venom (ED50) was 0.94 (p value < 0.05). In conclusion, slowness in spontaneous thrombin/fibrin generation even after recalcification elicit time lapse sufficient for elaboration of one dose-response curve to pro-or anti-coagulant agonists in CPS. We propose this methodology as an alternative and sensitive assay for assessing antivenom neutralizing ability in plasma of immunized horses as well as for in-process quality control.