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.

Correlation between ToBI test and in vivo titration for diphtheria and tetanus.

Alternatives to animal testing for quality control of biologicals have been a goal since 1959. Instituto Butantan has been developing such methods for quality control of biologicals for human use (vaccines and hyperimmune equine sera) for the last 13 years. In this paper we compare the modified ToBI test and the in vivo seroneutralization test to assess immunogenicity of diphtheria and tetanus vaccines and hyperimmune sera. Data from the last 10 years were statistically analyzed to compare the results for in vivo and in vitro titrations (diphtheria, n = 525 and tetanus, n = 455). The agreement between the tests depended on the serum titer range. For both diphtheria and tetanus components, the correlation and concordance coefficient was higher as the serum titer increased. Overall, the in vitro/in vivo titer ratio did not vary systematically over the range of measurements. These results indicate that although the in vitro ToBI test is not completely able to replace the in vivo serum titration, it is a useful tool to guide the tests during the production process, which can reduce the number of animals used for lot release.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Anti-SARS-CoV-2 equine F (Ab′)2 immunoglobulin as a possible therapy for COVID-19

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab′)2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab′)2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

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.

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.

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.