Equine Immunoglobulin Research Articles

Monoclonal antibody development advances immunological research in horses

Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

Therapeutic equine hyperimmune antibodies with high and broad-spectrum neutralizing activity protect rodents against SARS-CoV-2 infection.

The emergence of SARS-CoV-2 variants stresses the continued need for broad-spectrum therapeutic antibodies. Several therapeutic monoclonal antibodies or cocktails have been introduced for clinical use. However, unremitting emerging SARS-CoV-2 variants showed reduced neutralizing efficacy by vaccine induced polyclonal antibodies or therapeutic monoclonal antibodies. In our study, polyclonal antibodies and F(ab')2 fragments with strong affinity produced after equine immunization with RBD proteins produced strong affinity. Notably, specific equine IgG and F(ab')2 have broad and high neutralizing activity against parental virus, all SARS-CoV-2 variants of concern (VOCs), including B.1.1,7, B.1.351, B.1.617.2, P.1, B.1.1.529 and BA.2, and all variants of interest (VOIs) including B.1.429, P.2, B.1.525, P.3, B.1.526, B.1.617.1, C.37 and B.1.621. Although some variants weaken the neutralizing ability of equine IgG and F(ab')2 fragments, they still exhibited superior neutralization ability against mutants compared to some reported monoclonal antibodies. Furthermore, we tested the pre-exposure and post-exposure protective efficacy of the equine immunoglobulin IgG and F(ab')2 fragments in lethal mouse and susceptible golden hamster models. Equine immunoglobulin IgG and F(ab')2 fragments effectively neutralized SARS-CoV-2 in vitro, fully protected BALB/c mice from the lethal challenge, and reduced golden hamster's lung pathological change. Therefore, equine pAbs are an adequate, broad coverage, affordable and scalable potential clinical immunotherapy for COVID-19, particularly for SARS-CoV-2 VOCs or VOIs.

Decomplexation proteomic analysis and purity assessment of a biologic for snakebite envenoming: Philippine Cobra Antivenom.

Philippine Cobra Antivenom (PCAV) is the only snake antivenom manufactured in the Philippines. It is used clinically to treat envenoming caused by the Philippine Spitting Cobra (Naja philippinensis). While PCAV is effective pharmacologically, it is crucial to ensure the safety profile of this biologic that is derived from animal plasma. This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS). SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions. Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.

Preparation of Equine Immunoglobulin F(ab′)2 against Smallpox and Evaluation of its Immunoprotective Effect

Objective: Smallpox, a severe infectious disease caused by the smallpox virus, causes a death rate as high as 30% within 15-20 days after infection. Therefore, development of anti-Smallpox product as a strategic reserve is urgently needed. Methods: We prepared and tested pepsin-digested F(ab′)2 fragments of serum IgG from horses. Results: Transmission electron microscopy indicated that the purified virus showed morphology consistent with VVTT. The titer was above 1.0 × 107 PFU/mL. The purity of the antigen exceeded 90%, according to HPLC. After purification and cleavage, the yield of the purified product F(ab′)2 was approximately 1.3%, its purity exceeded 90%, and the neutralizing antibody titer exceeded 1:3200. F(ab′)2 fragments had good preventive and therapeutic effects in mice at antibody doses of 5.2 mg/mL and 2.6 mg/mL. The viral loads of the drug-treated mice were suppressed to varying degrees, and the higher dose groups (5.2 and 2.6 mg/mL) showed a 2-3 fold lower viral load than that in the control group. Conclusion: A process for producing equine immunoglobulin F(ab′)2 against VVTT was established. The prepared horse anti-smallpox immunoglobulin product had good neutralizing antibody effects on VVTT. The highly purified preparation may serve as a potential candidate for smallpox treatment.

Development and Validation of an ELISA to Evaluate Neutralizing Equine Anti Shiga Toxin Antibodies in Preclinical Studies

Background: Hemolytic uremic syndrome associated with Shiga-toxin produced by Escherichia coli is a serious worldwide foodborne disease. Nowadays, no treatment is available, only supportive care can be provided, and 50 % of the patients require a period of dialysis. Recently, a therapy based on Neutralizing Equine Anti Shiga Toxin (NEAST) antibodies has been developed. NEAST is composed of F(ab’)2 fragments from equine immunoglobulins. Objective: The purpose of this study was to develop an ELISA to measure serum concentrations of NEAST in mice and rabbits, and to validate it according to international recommendations. The validated method was further used to analyze the NEAST PK during preclinical studies. Methods: A sandwich ELISA was developed, the performance of the calibration curve was assessed, and it was validated based on the parameters as accuracy, precision, specificity, selectivity, stability of the analyte, and dilutional linearity. Results: This immunoassay was specific, sensitive, accurate and precise in a dynamic range from 7.81 to 500 ng/mL and from 15.63 to 500 ng/mL for mice and rabbits, respectively. This method was successfully applied to PK studies of NEAST after intravenous administration. conclusion: The results obtained are expected for a robust ELISA used for macromolecule analysis. Since NEAST is an equine F(ab′)2, this immunoassay would serve for the evaluation of the PK profile of any biological product based on molecules with similar characteristics. This immunoassay may be useful for current and future preclinical trials conducted for registration purposes.

Equine Anti-SARS-CoV-2 Serum (ECIG) Binds to Mutated RBDs and N Proteins of Variants of Concern and Inhibits the Binding of RBDs to ACE-2 Receptor.

The COVID-19 pandemic caused by the severe acute syndrome virus 2 (SARS-CoV-2) has been around since November 2019. As of early June 2022, more than 527 million cases were diagnosed, with more than 6.0 million deaths due to this disease. Coronaviruses accumulate mutations and generate greater diversity through recombination when variants with different mutations infect the same host. Consequently, this virus is predisposed to constant and diverse mutations. The SARS-CoV-2 variants of concern/interest (VOCs/VOIs) such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28/P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) have quickly spread across the world. These VOCs and VOIs have accumulated mutations within the spike protein receptor-binding domain (RBD) which interacts with the angiotensin-2 converting enzyme (ACE-2) receptor, increasing cell entry and infection. The RBD region is the main target for neutralizing antibodies; however, other notable mutations have been reported to enhance COVID-19 infectivity and lethality. Considering the urgent need for alternative therapies against this virus, an anti-SARS-CoV-2 equine immunoglobulin F(ab’)2, called ECIG, was developed by the Butantan Institute using the whole gamma-irradiated SARS-CoV-2 virus. Surface plasmon resonance experiments revealed that ECIG binds to wild-type and mutated RBD, S1+S2 domains, and nucleocapsid proteins of known VOCs, including Alpha, Gamma, Beta, Delta, Delta Plus, and Omicron. Additionally, it was observed that ECIG attenuates the binding of RBD (wild-type, Beta, and Omicron) to human ACE-2, suggesting that it could prevent viral entry into the host cell. Furthermore, the ability to concomitantly bind to the wild-type and mutated nucleocapsid protein likely enhances its neutralizing activity of SARS-CoV-2. We postulate that ECIG benefits COVID-19 patients by reducing the infectivity of the original virus and existing variants and may be effective against future ones. Impacting the course of the disease, mainly in the more vulnerable, reduces infection time and limits the appearance of new variants by new recombination.

Ensuring Viral Safety of Equine Immunoglobulins during Production.

Equine blood plasma/serum and intermediates must be monitored for the presence of live viruses pathogenic in humans during production of equine immunoglobulins. Information concerning low-cost and simple methods for the detection of live horse viruses pathogenic and non-pathogenic to humans was gained using data of modern domestic and foreign literature. These methods are based on cultivation of these viruses on sensitive biosystems. The presented information can be used to set up blood plasma/serum control of horses at different stages of immunoglobulin production, i.e., when taking blood from horses during their quarantine period, when collecting blood from immunized horses, and before bottling the medicinal intermediate in the primary package.

Epidemiological Analysis of the Effectiveness of Rabies Post-Exposure Prophylaxis in the Russian Federation (2001–2018)

Relevance. Post-exposure prophylaxis (PEP) is the most effective method for reducing the morbidity and mortality of human rabies. Since 2000, in Russia, domestically produced cultural purified and concentrated rabies vaccine (COCAV) and equine immunoglobulin (AIG) are used as the main drugs for PEP.Aims. To assess the epidemiological effectiveness of post-exposure rabies prophylaxis and possible causes of failure in the Russian Federation in 2001–2018.Materials & methods. A comprehensive retrospective epidemiological study of 167 cases of rabies in humans and PEP statistics in the Russian Federation was carried out. To test the hypotheses about the relationship between PEP failures and risk factors, namely (gender, age, place of residence of the sick, nature of the injuries received, source of infection), two main comparison groups were formed: «vaccinated» (n = 28) and «not vaccinated» (n = 139). In addition, we compared the groups who received PEP correctly and those vaccinated with violations of instructions. The failure rate was assessed in relation to the total number of vaccinated and the number of vaccinated after contact with rabid animals.Results. During the analyzed period, the incidence of human rabies decreased from 0.015 to 0.0013 per 100 thousand population. Of the patients, 83.2% did not receive PEP (did not apply, vaccinations were not prescribed, refused). In the group «vaccinated» in 53.6% (n = 15), the schemes of PEP prescribing and administering were violated, including in 35.7% (n = 10) of cases AIG was not administered. The rest 46.4% (n = 13), received vaccinations in accordance with the instructions. Before the end of the vaccination course, 85.7% (n = 24) fell ill; the rest fell ill with an incubation period of more than three months. Only one person received AIG and all 6 vaccine injections. The group «vaccinated» differed from the group «not vaccinated» by a higher frequency of category III injuries (χ2 = 9.99, p = 0.019) and injuries caused by wild animals, especially wolves (χ2 = 22.24, p < 0.001).Conclusions. Among people who developed rabies in 2001–2018, 16.8% received PEP. The proportion of the number of cases and the total number of those who received PEP was 1: 240.0 thousand. The failure rate of PEP after contact with rabid animals was 0.03% (excluding the nature of the contact and the animal species). More than 70% of all failures are associated with wolf and fox bites, more than 85% with category III injuries.

Production of equine sera as a potential immunotherapy against COVID-19

Emerging viruses such as the COVID-19-inducing virus, SARSCoV- 2, represent a threat to human health, unless effective vaccines, drugs or alternative treatments, such as passive immunization, become accessible. Animal-derived immunoglobulins, such as equine immunoglobulins might be useful as immunoprophylaxis or immunotherapy against this viral disease. Therapeutic antibodies (Abs) for SARS-CoV-2 were obtained from hyperimmune equine plasma using the Spike protein receptor binding domain (RBD) as an immunogen. The presence of anti-RBD antibodies was evaluated by ELISA and the titres of neutralizing antibodies were determined in viral cell culture. Immunized horses generated high-titre of anti-RBD antibodies with antiviral neutralizing activity on Vero-E6 cells of 1/1,000. To minimize potential adverse effects, the immunoglobulins were digested with pepsin, and purified to obtain the F(ab’)2 fragments with the protocol standardized by Biotecfar C.A for the production of snake antivenom. Pre-immune serum displayed an unexpected anti-RBD reactivity by ELISA (titre up to 1/900) and Western Blot, but no angioneutralizing activity. Modelling of the RBD of equine coronavirus showed that some of the known epitopes of SARS-CoV-2 RBD were structurally conserved in the equine coronavirus protein. This might suggest that some of the reactivity observed in the pre-immune serum to the SARS-CoV-2 RBD might be due to a previous exposure to equine coronavirus.

Echis coloratus envenomation in children: a retrospective case series

Background Antivenom has been used successfully to treat systemic and progressive, local manifestations of envenomations inflicted by Echis coloratus, the second most common cause of snake envenomation in Israel. There is a paucity of published data regarding the use of monovalent (equine) immunoglobulin G antivenom in children. This study describes outcomes from the regimen used in two large, tertiary care pediatric centers. Methods A retrospective chart review of children admitted with definite or probable signs of Echis coloratus envenomation to Sourasky (Tel Aviv) and Soroka (Be’er Sheva) Medical Centers from January 1st 2008–to June 1st 2019. Extracted data included age, location of bite, time to hospital arrival, laboratory test results, complications, time to antivenom administration if indicated, adverse effects of the antivenom, and outcomes. Indications for antivenom were: diagnosis of Echis coloratus as the etiology of envenomation, local and systemic signs e.g. skin puncture wounds, swelling of the involved limb, local hematoma, and abnormal coagulation blood test results. Results During the study period, 11 children were treated with intravenous Echis coloratus antivenom. Median age was 9 years and 10 of 11 patients were male. Two patients underwent fasciotomy; in one, compartment syndrome was diagnosed by pressure measurement, and in the second, clinically. One patient developed mild urticaria 30 min after initiation of the antivenom; the treatment was stopped and then restarted at a slower rate after he was treated with hydrocortisone and diphenhydramine. No further adverse reactions were observed. Conclusions In children, Echis coloratus antivenom appeared to be effective and safe for the treatment of systemic and progressive local manifestations of envenomation by Echis coloratus.

Prophylactic Efficacy of Equine Immunoglobulin F(ab')2 Fragments Against Feline Parvovirus.

Feline parvovirus (FPV), a type of parvovirus prevalent worldwide, can cause foetal death and acute enteritis in adult cats with severe leukopenia, and yet there are no effective drugs to prevent or treat FPV. Here, the immune effects of two FPV vaccines on horses were compared. IgG was extracted from FPV-immunized horse sera. Equine F(ab′)2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. The results showed that the inactivated FPV oil vaccine was more effective than the inactivated FPV propolis vaccine in helping healthy horses to produce hyper-immune serum. Four methods were tested, among which the optimized octanoic acid-ammonium sulphate precipitation method was proved to be the best process for extracting IgG. The optimal condition for preparing F(ab′)2 by pepsin digestion was 30 °C for 3.5 h, and the content, purity and recovery of F(ab′)2 were 8.64 mg/mL, 90.36% and 93.24%, respectively. Our equine immunoglobulin F(ab′)2 fragments effectively neutralized activity in vitro against FPV, alleviated the clinical symptoms of FPV-infected cats, reduced the viral loads in the intestine and had prophylactic effects in FPV-infected cats. These results indicate that the F(ab′)2 fragment prepared from inactivated FPV-immunized horses may be used as a prophylactic agent for diseases caused by FPV.

The Development of Mucosal Immunization as an Alternative Approach for Production of Antisera Against Saw Scale Viper from Sindh


 The reported cases of snake bite are 5.4 million per year, making the serious envenoming issue so, that WHO categorises cases of snake bite in neglected tropical diseases. Anti snake venom is produced by intramuscular injection of crude venom with different formulation of adjuvants to equine animals, but these intramuscular injections have several serious health impact in equine. Snake species categorised according to geographical locations, and there is a different composition of the snake venom at different locations. So, this research had been designed and evaluated new oral adjuvant formulation for viper family of Sindh, Pakistan. This is a animal immune based analysis consisting two Saw scale viper from Thar and costal area of Sindh because both have different habitat and morphological appearance. The venom toxin protein, combined with a mineral oil adjuvant for oral immunization. The adjuvant with venom via oral route produced of specific IgG and horse give maximum response with three doses of venom. The antibody titer was measured by ELISA showed significant results in both groups. For confirmation of specific antibodies isolated from horse serum, serum neutalization assay and immunodiffusion test were carried out. The snake venom with oral adjuvant immunization, is safe, efficient and time saving for production of equine Immunoglobulins IgG.
 

Quality-Related Properties of Equine Immunoglobulins Purified by Different Approaches.

Whole IgG antivenoms are prepared from hyperimmune animal plasma by various refinement strategies. The ones most commonly used at industrial scale are precipitation by sodium or ammonium sulphate (ASP), and caprylic acid precipitation (CAP) of non-immunoglobulin proteins. The additional procedures, which have so far been used for experimental purposes only, are anion-exchange (AEX) and cation-exchange chromatography (CEX), as well as affinity chromatography (AC) using IgG’s Fc-binding ligands. These protocols extract the whole IgG fraction from plasma, which contains both venom-specific and therapeutically irrelevant antibodies. Such preparations represent a complex mixture of various IgG subclasses whose functional and/or structural properties, as well as relative distribution, might be affected differently, depending on employed purification procedure. The aim of this work was to compare the influence of aforementioned refinement strategies on the IgG subclass distribution, venom-specific protective efficacy, thermal stability, aggregate formation and retained impurity profile of the final products. A unique sample of Vipera ammodytes ammodytes specific hyperimmune horse plasma was used as a starting material, enabling direct comparison of five purification approaches. The highest purity was achieved by CAP and AC (above 90% in a single step), while the lowest aggregate content was present in samples from AEX processing. Albumin was the main contaminant in IgG preparations obtained by ASP and CEX, while transferrin dominantly contaminated IgG sample from AEX processing. Alpha-1B-glycoprotein was present in CAP IgG fraction, as well as in those from ASP- and AEX-based procedures. AC approach induced the highest loss of IgG(T) subclass. CEX and AEX showed the same tendency, while CAP and ASP had almost no impact on subclass distribution. The shift in IgG subclass composition influenced the specific protective efficacy of the respective final preparation as measured in vivo. AC and CEX remarkably affected drug’s venom-neutralization activity, in contrary to the CAP procedure, that preserved protective efficacy of the IgG fraction. Presented data might improve the process of designing and establishing novel downstream processing strategies and give guidance for optimization of the current ones by providing information on potency-protecting and purity-increasing properties of each purification principle.

Discrepancies in the bilateral intradermal test and serum tests in atopic horses.

BackgroundIn equine atopic patients intradermal testing (IDT) and immunoglobulin (Ig)E serology are used frequently. There is little evidence regarding the reproducibility of the IDT and IgE serology in horses.ObjectivesTo compare the results of a simultaneously performed IDT on the left and right side of the neck in atopic horses, and to compare these results with allergen‐specific IgE serology.AnimalsTen equine patients from a university hospital population with chronic urticaria and/or pruritus.Methods and materialsThe IDT was performed using 16 allergens and the results were evaluated after 30 min, 1, 4 and 24 h. Thirteen allergens also were analysed in duplicate with two monoclonal allergen‐specific IgE enzyme‐linked immunosorbent assays (ELISA).ResultsGood agreement (Kappa > 0.6) between left and right IDT was found only for Dermatophagoides farinae, Lepidoglyphus destructor, birch pollen mixture and perennial rye at 30 min, birch pollen mixture at 1 h, and Acarus siro and nettle and common mugwort mixture at 4 h. The bilateral comparison of the other allergens and even the same allergens at other time points showed little or no concordance between left and right IDT. The interlaboratory comparison between both ELISAs, and the comparison between the ELISAs and IDT, showed a good agreement for two of 13 allergens: D. farinae and Dermatophagoides pteronyssinus.Conclusions and clinical importanceBased on these preliminary data, IDT and IgE serological test results should be interpreted with great care and further studies are needed to indicate the clinical relevance of these findings.

Immunoglobulin fragment F(ab’)2 against RBD potently neutralizes SARS-CoV-2 in vitro

COVID-19, which is caused by the emerging human coronavirus SARS-CoV-2, has become a global pandemic that poses a serious threat to human health. To date, no vaccines or specific antiviral drugs have been approved for the treatment of this disease in clinic. Herein, therapeutic antibodies for SARS-CoV-2 were obtained from hyperimmune equine plasma. First, a recombinant SARS-CoV-2 spike protein receptor-binding domain (RBD) was obtained in gram-level quantities through high-cell density fermentation of Chinese hamster ovary cells. Then, the binding of the RBD to the SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, was verified by several biochemical methods. The efficacy of the RBD in triggering antibody response in vivo was subsequently tested in both mice and equines, and the results showed that the RBD triggered high-titer neutralizing antibody production in vivo. Immunoglobulin F(ab’)2 fragments were prepared from equine antisera via removal of the Fc region from the immunoglobulins. Finally, a neutralization test with live virus demonstrated that RBD-specific F(ab’)2 inhibited SARS-CoV-2 with an EC50 of 0.07 μg/ml and an EC80 of 0.18 μg/ml, showing a potent inhibitory effect on SARS-CoV-2. These results highlight RBD-specific equine immunoglobulin F(ab’)2 fragment as a candidate for the treatment of SARS-CoV-2.

Properties of Heterologous anti-Ebola Immunoglobulin after Long Storage

Ebola outbreak in eastern parts of the Democratic Republic of the Congo in 2018–2020 proved that the virus remains highly hazardous for humans, and the outbreak in West Africa in 2014–2016, which was the largest Ebola outbreak in history, showed that it could be imported to other continents, including Russia. In 1993 the Federal State Budgetary Institution “48th Central Scientific Research Institute” of the Russian Ministry of Defence developed a specific equine immunoglobulin for emergency prophylaxis of Ebola in risk groups. The evaluation and improvement of the product’s properties is an important area in the development of biological defence technologies.The aim of the studywas to examine the properties of the equine anti-Ebola immunoglobulin which had been stored for a long time at 2–8 °C.Materials and methods:the authors studied batches of heterologous anti-Ebola immunoglobulin that had been stored for 17–22 years. The properties of the product were evaluated according to the requirements of the State Pharmacopoeia of the Russian Federation, 14th ed. (Ph. Rus. 14 ed.). The specific activity of the product was determined in a plaque reduction neutralisation test using Ebola virus and African green monkey kidney cells (GMK-AH-1(D)). Immunoglobulin molecular parameters were determined by size-exclusion high-performance liquid chromatography using the test methods described in the European Pharmacopoeia 9.6 and Ph. Rus. 14 ed.Results:the storage of anti-Ebola immunoglobulin for 17–22 years at 2–8 °C resulted in a four-fold reduction of the level of virus-neutralising antibodies against Ebola, decrease in the proportion of monomers from 98 to 74–90%, increase in the proportion of dimers and polymers, and formation of immunoglobulin molecules’ fragments. Signs of toxicity for mice were observed in one of the three product batches.Conclusions:the obtained results suggest the need to perform more studies to test the quality of antiEbola immunoglobulin batches that were stored for shorter periods of time in order to assess the stability of their initial characteristics.

Experimental Testing of a Size-Exclusion Chromatography Method Used for Evaluation of Molecular Parameters of Equine Anti-Ebola Immunoglobulin

Haemorrhagic fever caused by the Ebola virus is a highly hazardous infectious disease with a mortality rate of 50– 90 %. Heterologous immunoglobulins with a high virus-neutralizing titer are an important element of the WHO-endorsed set of measures for emergency prevention and treatment of the disease. Specific activity of these products is largely determined by their fractional composition, and, in particular, by molecular mass distribution (MMD). The size-exclusion-high-performance liquid chromatography (SEC-HPLC) has traditionally been used for determination of the MMD of the target protein in human immunoglobulin-based products. The use of this method for evaluation of molecular parameters of heterologous immunoglobulin requires confirmation of its specificity, accuracy and precision, and establishment of the chromatographic system suitability criteria in the context of a new test object.The aimof the study was to test the applicability of the SEC-HPLC method to the assessment of molecular parameters of anti-Ebola immunoglobulin derived from horse serum.Materials and methods: three batches of purified equine anti-Ebola immunoglobulin were used in the study. Normal equine and human immunoglobulins of the IgG isotype were used as reference standards. The HPLC test procedures described in the European Pharmacopoeia 9.6 and State Pharmacopoeia of the Russian Federation, 14th ed., were used for determination of monomers and other immunoglobulin fractions. An Agilent 1260 Infinity (Agilent, USA) HPLC system with a diode array detector and an Agilent Bio SEC-3 HPLC column were used for quality evaluation of the tested products.Results: the resolution factor between IgG monomer and dimer peaks (1.69 and 2.10), and the chromatographic column efficiency (>2000) make it possible to use the SEC-HPLC system for evaluation of molecular parameters of heterologous immunoglobulin. The study demonstrated reproducibility of the test procedure.Conclusions: the study confirmed the applicability of the SEC-HPLC procedure for evaluation of molecular parameters of anti-Ebola immunoglobulin derived from horse serum. It demonstrated the compliance of the purified immunoglobulin to the national and international quality requirements in terms of «Molecular parameters».

Equine immunoglobulin F(ab')2 fragments protect cats against feline calicivirus infection

Feline calicivirus (FCV) causes upper respiratory tract infections in felines and threatens the health of wild and domestic felines. Clinically, specific drugs to treat FCV have not yet been developed. Here, IgG was extracted from inactivated FCV-immunized horse sera. Equine F(ab')2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. In our study, equine immunoglobulin F(ab')2 fragments showed efficient neutralizing activity in vitro against FCV and had therapeutic and prophylactic effects in FCV-infected cats. The anti-FCV-specific F(ab')2 fragment can significantly alleviate the clinical symptoms of FCV-infected cats and reduce the viral loads of the trachea, lung and spleen. These results indicate that the F(ab')2 fragment prepared from inactivated FCV-immunized horses may be used as a prophylactic and therapeutic agent for diseases caused by FCV.

Equine immunoglobulin F(ab′)2 fragments protect mice from Rift Valley fever virus infection

BackgroundRift Valley fever virus (RVFV) is an emerging arbovirus in Africa and the Arabian Peninsula, in which infection with RVFV poses a serious threat to humans and livestock globally. Approved treatments for RVFV infection, especially for use in humans, have not yet been developed. There is an urgent need for effective drugs to prevent RVFV disease. MethodsIn previous study, we developed RVFV virus like particles (VLPs) expressing the surface glycoproteins Gn and Gc. The morphology was shown to be similar to live RVFV under electron microscopy. In this study, we immunized horses with RVFV VLPs, prepared the immunoglobulin F(ab′)2 fragments, and characterized its in vitro neutralization and in vivo efficacy in mice. ResultsF(ab′)2 was found to potently neutralize RVFV in VeroE6 cells, and passive transfer of immunoglobulin F(ab′)2 fragments resulting in reduced mortality in RVFV infected mice. ConclusionOur results show that passive immunotherapy with equine immunoglobulin F(ab′)2 fragments is a promising strategy to treat RVFV infections.

Recombinant monoclonal antibodies for rabies post-exposure prophylaxis

Rabies virus is a prototypical neurotropic virus that causes one of the most dangerous zoonotic diseases in humans. Humanized or fully human monoclonal antibodies (mAb) that neutralize rabies virus would be the basis for powerful post-exposure prophylaxis of rabies in humans, having several significant benefits in comparison with human or equine rabies polyclonal immunoglobulins. The most advanced antibodies should broadly neutralize natural rabies virus isolates, bind with conserved antigenic determinants of the rabies virus glycoprotein, and show high neutralizing potency in assays in vivo. The antibodies should recognize nonoverlapping epitopes if they are used in combination. This review focuses on basic requirements for anti-rabies therapeutic antibodies. The urgency in the search for novel rabies post-exposure prophylaxis and methods of development of anti-rabies human mAb cocktail are discussed. The rabies virus structure and pathways of its penetration into the nervous system are also briefly described.