Ovine IgG Research Articles

Post-exposure treatment of non-human primates lethally infected with Ebola virus with EBOTAb, a purified ovine IgG product

Despite sporadic outbreaks of Ebola virus (EBOV) over the last 4 decades and the recent public health emergency in West Africa, there are still no approved vaccines or therapeutics for the treatment of acute EBOV disease (EVD). In response to the 2014 outbreak, an ovine immunoglobulin therapy was developed, termed EBOTAb. After promising results in the guinea pig model of EBOV infection, EBOTAb was tested in the cynomolgus macaque non-human primate model of lethal EBOV infection. To ensure stringent therapeutic testing conditions to replicate likely clinical usage, EBOTAb was first delivered 1, 2 or 3 days post-challenge with a lethal dose of EBOV. Results showed a protective effect of EBOTAb given post-exposurally, with survival rates decreasing with increasing time after challenge. Viremia results demonstrated that EBOTAb resulted in a decreased circulation of EBOV in the bloodstream. Additionally, assay of liver enzymes and histology analysis of local tissues identified differences between EBOTAb-treated and untreated groups. The results presented demonstrate that EBOTAb conferred protection against EBOV when given post-exposure and should be explored and developed further as a potential intervention strategy for future outbreaks, which are likely to occur.

Multiplex Detection of IgG and IgM to Rift Valley Fever Virus Nucleoprotein, Nonstructural Proteins, and Glycoprotein in Ovine and Bovine.

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were assembled into a multiplex and tested in serum samples from infected wild-type RVFV or MP12, a modified live virus vaccine. As expected, the N protein was immunodominant and the best target for early detection of infection. Antibody activity against the other targets was also detected. The experimental results demonstrate the capabilities of FMIA for the detection of antibodies to RVFV structural and nonstructural proteins, which can be applied to future development and validation of diagnostic tests that can be used to differentiate vaccinated from infected animals.

Linear models of ovine IgG1 and IgG2 subclasses and predicted pepsin cleavage sites

Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab′)2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab′)2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the molecules and mapped onto the individual models. A theoretical order of digestion was subsequently constructed, which appeared to agree with the experimental data, suggesting an accurate prediction model for ovine IgG1 and IgG2 subclasses. These findings lay the foundations for a more detailed analysis of pepsin cleavage fragments in the future. Additionally, the F(ab′)2 generated following pepsin digestion were predicted to contain subclass unique C-terminal octapeptide neoepitopes, despite the high 89% sequence identity of the intact gamma-1 and gamma-2 chain constant regions. These neoepitopes have the potential to be utilised for identification purposes once confirmed experimentally.

An Intravaginal Ring for the Sustained Delivery of Antibodies

Human monoclonal antibodies (mAbs) based on IgG and IgA have shown promise as topical microbicide candidates to protect women from HIV infection. Application of mAbs has been limited, however, by the inability of vaginal gels and conventional intravaginal ring (IVR) designs, the predominant vaginal product formulations, to effectively deliver biomolecules in a coitally independent fashion with retention of bioactivity. We have developed a novel pod-IVR platform that delivers ovine IgG (ov-IgG) as a model for IgG and IgA human mAbs. In vitro release of ov-IgG from the pod-IVRs was sustained for 14 days. Facile control of release rate was achieved by changing the size of delivery channels in the ring structure, and the feasibility of ov-IgG delivery in the range 0.5-30 mg day(-1) from a 10-pod IVR was demonstrated. The activity of ov-IgG in pod-IVR formulations was maintained as confirmed by ELISA binding assay. Pod-IVRs delivering ov-IgG show promise for the effective sustained topical delivery of antibody-based microbicides. This significantly broadens the range of microbicides that can be delivered in a sustained fashion from IVRs and enables a new arsenal of topical biologic microbicide candidates beyond small molecule antiretrovirals.

Determination of IgG levels in bulk ewe's milk

Colostrum addition to milk results in a reduction of the yield during cheese manufacturing. Determination of IgG levels is accepted as an evidence of this troublesome habit due to the high level of this protein in colostrum. This study shows for the first time the IgG levels in bulk ewe's milk samples from Spain throughout the year. IgG quantification was carried out by a sandwich ELISA that recognizes specifically ovine IgG. The mean value of IgG concentration obtained from 481 samples was 0.271±0.253mg/ml. In general, this value is within the range considered acceptable for mature milk according to quality payment systems. Surprisingly, a high mean concentration was obtained in samples collected in winter, 0.742±0.378mg/ml, suggesting that births were grouped in that period. These results point up that standardization of ewe's milk production has not been achieved yet. For this reason, determination of immunoglobulin levels periodically would be a good parameter to control milk quality.

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

The IgG N-glycome provides sufficient complexity and information content to serve as an excellent source for biomarker discovery in mammalian health. Since oligosaccharides play a significant role in many biological processes it is very important to understand their structure. The glycosylation is cell type specific as well as highly variable depending on the species producing the IgG. We evaluated the variation of N-linked glycosylation of human, bovine, ovine, equine, canine and feline IgG using three orthogonal glycan separation techniques: hydrophilic interaction liquid chromatography (HILIC)–UPLC, reversed phase (RP)–UPLC and capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The separation of the glycans by these high resolution methods yielded different profiles due to diverse chemistries. However, the % abundance of structures obtained by CE-LIF and HILIC–UPLC were similar, whereas the analysis by RP-UPLC was difficult to compare as the structures were separated by classes of glycans (highly mannosylated, fucosylated, bisected, fucosylated and bisected) resulting in the co-elution of many structures. The IgGs from various species were selected due to the complexity and variation in their N-glycan composition thereby highlighting the complementarity of these separation techniques.

Single-reagent one-step procedures for the purification of ovine IgG, F(ab′)2 and Fab antivenoms by caprylic acid

Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab′)2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab′)2 and Fab fragments, respectively. A “single-reagent” method was developed for the production of F(ab′)2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the “single reagent” comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18–20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab′)2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

Envenomation by African adders (Bitis ssp.) induces complement activation

An antivenom should be stable under the conditions that it will be both transferred and stored. Thus instability may lead to a loss of efficacy and an increased incidence and severity of adverse effects. Stability is a particular problem in countries where the temperatures and humidity are high. Here we investigate the stability of a liquid-formulated, intact ovine immunoglobulin-based antivenom, EchiTAbG™, which is used extensively in Nigeria to treat envenoming by the West African saw-scaled viper, Echis ocellatus. Ampoules of antivenom were assessed as to their specific antibody content by small scale affinity chromatography and their purity by size exclusion gel filtration and turbidity. Three different batches of the antivenom revealed no significant changes, using these assessment techniques, during 42 months storage at 4 °C or at ambient temperature, followed by one month at 37 °C. These real-time studies indicate that the antivenom remains stable for a minimum of 3.5 years and that it can be exposed to tropical temperatures without any loss in immunoglobulin binding activity. This further highlights the clinical utility of liquid formulated ovine IgG antivenoms by demonstrating their retention of potency in the event of a short term failing in the cold chain.

Recovery of intact IgG in the gastrointestinal tract of the growing rat following ingestion of an ovine serum immunoglobulin

The aim of this study was to determine whether orally ingested ovine serum IgG partly resists digestion in the growing rat. Fifteen Sprague-Dawley male rats were allocated to one of three diets for a 3-week study: a control diet (CON) and two test diets containing either freeze-dried ovine serum immunoglobulin (FDOI) or inactivated ovine serum immunoglobulin (IOI). Samples of stomach chyme and intestinal digesta from the ad libitum-fed rats were subjected to ELISA and Western blot analysis. Amounts of intact ovine IgG for the FDOI diet were found to be 13.9, 20.0, 34.1, 13.0 and 36.9 μg in the total wet digesta from the stomach chyme, duodenal, jejunal, ileal and colonic digesta respectively. Qualitative detection by Western blot revealed the presence of intact ovine serum IgG with a ~150 kDa MW. This was detected in all of the gut segments (stomach chyme, duodenal, jejunal, ileal and colonic digesta) for growing rats fed the FDOI diet. No ovine IgG was detected in the chyme or digesta from rats fed the CON or the IOI diets. Ovine serum IgG partly resisted digestion in the growing rat fed the FDOI diet and was found throughout the digestive tract. These results provide a basis to explain the reported biological effects of orally administered immunoglobulin.

Long-term physicochemical and immunological stability of a liquid formulated intact ovine immunoglobulin-based antivenom

An antivenom should be stable under the conditions that it will be both transferred and stored. Thus instability may lead to a loss of efficacy and an increased incidence and severity of adverse effects. Stability is a particular problem in countries where the temperatures and humidity are high. Here we investigate the stability of a liquid-formulated, intact ovine immunoglobulin-based antivenom, EchiTAbG™, which is used extensively in Nigeria to treat envenoming by the West African saw-scaled viper, Echis ocellatus. Ampoules of antivenom were assessed as to their specific antibody content by small scale affinity chromatography and their purity by size exclusion gel filtration and turbidity. Three different batches of the antivenom revealed no significant changes, using these assessment techniques, during 42 months storage at 4 °C or at ambient temperature, followed by one month at 37 °C. These real-time studies indicate that the antivenom remains stable for a minimum of 3.5 years and that it can be exposed to tropical temperatures without any loss in immunoglobulin binding activity. This further highlights the clinical utility of liquid formulated ovine IgG antivenoms by demonstrating their retention of potency in the event of a short term failing in the cold chain.

Immune potential of a novel multiple-epitope vaccine to FMDV type Asia 1 in guinea pigs and sheep

To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus (FMDV) type Asia 1 in sheep, a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized. The biologically functional molecule, the ovine IgG heavy constant region (oIgG) as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed. The recombinant proteins, RE and RE-oIgG, were expressed as a formation of inclusion bodies in E. coli. The immune potential of this vaccine regime in guinea pigs and sheep was evaluated. The results showed that IgG could significantly enhance the immune potential of antigenic epitopes. The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs, but confer complete protection in guinea pigs against virus challenge. Although the recombinant protein RE could not confer protection in the vaccinated animals, it could delay the appearance of the clinical signs and reduce the severity of disease. Inspiringly, the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination. Therefore, we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.

Analysis of camelid IgG for antivenom development: Immunoreactivity and preclinical neutralisation of venom-induced pathology by IgG subclasses, and the effect of heat treatment

Antivenom is the most effective treatment of snake envenoming and is manufactured from the IgG of venom-immunised horses and sheep. Camelids have a unique IgG structure which may account for the report that camel IgG is less immunogenic and less likely to activate complement than equine or ovine IgG. Camelid IgG therefore offers potential safety advantages over conventional IgGs used for antivenom manufacture. The reported thermostability of camelid IgG also holds promise in the inclusion of a relatively inexpensive anti-microbial heat step in antivenom manufacture. However, these potential benefits of camelid IgG would be much reduced if any one of the three camel IgG subclasses dominated, or under-performed, the serological response of camels to venom immunisation because of the prohibitive manufacturing costs of having to purify, or exclude, one or more IgG subclasses. This study compared the titre, antigen-specificity, relative avidity and ability to neutralise the haemorrhagic and coagulopathic effects of Echis ocellatus venom of each IgG subclass from the venom-immunised camels. The results demonstrated that no one IgG subclass consistently out-performed or under-performed the others in their immunoreactivity to venom proteins and ability to neutralise venom-induced pathologies. We concluded therefore that IgG taken from a pool of immunised camels could be processed into antivenom without requiring the implementation of expensive chromatographic separations to select, or indeed to exclude, a specific IgG subclass. The immunoreactivity of the heavy and light chain, IgG1 subclass, was markedly more vulnerable to extreme heat treatment than the heavy chain-only IgG2 and IgG3 subclasses.

A means to study details of complex antigen mixture neutralizations when limited to antibodies unresponsive to Fc region dependent technologies for immunoprecipitation. (144.6)

Abstract The Crofab™ antivenin, an ovine Fab IgG fragment product, thus lacking an Fc region for typical immunochemical physical separations, is the sole antivenin used in American for treatment of the thousands of life threatening Crotalid envenomations each year, but has unexplained clinical failures. We postulated that by removing the immune complexes and unreacted antibody from neutralization mixtures, thus leaving only the unreacted venom components, that high resolution chromatographic analysis of in vitro binding studies could be developed. So we evaluated biotinylation of the ovine Fab antivenin, although successful biotin conjugation of Crofab™ or any ovine Fab fragments has not been reported, and reduced activity of biotinylated ovine Fab2 had been reported. Our results show with varying coupling reaction mass ratios tested, up to a 75% coupling yield and a ratio of 2-3 biotin per Fab. By SE-HPLC analysis, the biotinylated antivenin did not lose binding activity. RP-HPLC showed differential neutralization of various venom components. This innovative method provides a means to conduct a much more detailed analysis of any ovine Fab antivenin neutralization or similar antibody-complex antigen mixtures, and suggests that Fab forms of other species of antivenin, or other species’ antibodies which cannot be immunoprecipitated with Fc region dependent technologies might be responsive to this approach, leading to detailed analyses of neutralizations.

Relationships between immune indicators of parasitic gastroenteritis, nematode burdens and faecal dry matter in sheep

Immune-mediated scouring in sheep is a mucosal hypersensitivity response caused by ingesting infective parasite larvae. In this experiment we tested the hypothesis that levels of parasite-specific antibodies and also interleukin-5 (IL-5) would be negatively correlated with worm numbers, but also faecal dry matter (FDM), in parasite-resistant Merino sheep. Forty Merino rams were challenged with Trichostrongylus colubriformis and Teladorsagia circumcincta larvae every day for 6 weeks, after which they were euthanised and total worm burdens determined. Faecal samples were taken for measurement of worm egg counts and FDM. Serum was collected from the rams before and after the larval challenge commenced, and levels of ovine immunoglobulin light chain, IgG, IgM, IgA and IgE specific for T. colubriformis and T. circumcincta as well as IL-5 were determined by sandwich enzyme-linked immunosorbent assay. IL-5 and all serum antibodies apart from T. colubriformis-specific light chain were significantly increased by the larval challenge. However, none of the antibodies, or IL-5, was correlated with FDM. Negative correlations were observed between the number of adult T. circumcincta and antibody levels; however, there was little relationship between antibodies and numbers of T. colubriformis. It was concluded that serum antibody levels are a poor indicator of the susceptibility of sheep to immune-mediated scouring. Because of these results, sheep breeders should continue to select for low worm egg count and focus on phenotypic indicators of scouring, such as dags, as a means to reduce diarrhoea.

An in vivo comparison of the efficacy of CSL box jellyfish antivenom with antibodies raised against nematocyst-derived Chironex fleckeri venom

Although CSL box jellyfish antivenom (AV) remains the primary treatment for Chironex fleckeri envenoming, there has been considerable debate regarding its clinical effectiveness. Animal studies have shown that AV is largely ineffective in preventing C. fleckeri-induced cardiovascular collapse. This study examined the effectiveness of CSL box jellyfish AV (ovine IgG), raised against 'milked' venom, and polyclonal rabbit IgG antibodies (Ab) raised against nematocyst-derived venom. A venom dose of 30microg/kg, i.v., which causes an initial presser response (34+/-5mmHg; n=7) followed by cardiovascular collapse, was used in all experiments. A bolus dose of AV (3000U/kg, i.v.) or Ab (12mg; i.e. an equivalent protein 'load' to 3000U/kg AV), administered 15min prior to a bolus dose of venom, did not significantly attenuate the effects of venom. The venom response was also not significantly attenuated when AV (3000U/kg) was given as a bolus dose 10-60min prior to venom infusion. However, when the venom was incubated with either AV (3000U/kg) or Ab (12mg) for 3h prior to infusion, the effect of the venom was almost abolished. The results of this study demonstrate that antibodies raised against both 'milked' and nematocyst-derived venom are able to neutralise the cardiovascular collapse produced by the venom. However, large amounts of AV are required and must be preincubated with the venom to be protective. This indicates a very rapid action of the toxin(s) and that AV is unlikely to be clinically effective because it cannot be administered early enough.

PRODUÇÃO DE ANTÍGENO SOMÁTICO DE Haemonchus contortus ADULTOS E SEU USO EM ENSAIO IMUNOENZIMÁTICO INDIRETO PARA DETECÇÃO DE IMUNOGLOBULINA G OVINA

Um dos fatores limitantes à criação de pequenos ruminantes é a verminose gastrintestinal, sobretudo em regiões de clima ameno, onde o Haemonchus contortus é o parasita mais prevalente. Para compreender a resposta imune do ovino a este parasita e identificar os animais mais responsivos, é necessária a adoção de metodologias adequadas para mensurar a resposta imune do hospedeiro. O presente trabalho foi desenvolvido com o objetivo de produzir um antígeno somático de H. contortus e utilizá-lo em técnicas de ensaio imunoenzimático indireto (ELISA) para detecção de IgG ovina. Para a obtenção do antígeno, cerca de 1000 helmintos adultos (machos e fêmeas) foram lavados, submetidos à maceração, ciclos de congelamento e descongelamento, sonicação e centrifugação. Foram utilizados como soros controle-positivo 20 amostras oriundas de ovelhas adultas submetidas à infecção natural. Como controle-negativos, foram utilizadas amostras pré-colostrais de 15 cordeiros, bem como de dois borregos mantidos livres de parasitas. Foram testadas várias diluições de antígeno, soros e um conjugado comercial anti-IgG ovina a fim de obter as concentrações ideais. A concentração ótima de antígeno foi de 5 μg.mL-1, e as diluições de soro e de conjugado que permitiram melhor discriminação entre positivos e negativos foram, respectivamente, 1:80 e 1:1000.

Evaluation of a biosensor assay to quantify polyclonal IgG in ovine serum used for the production of biotherapeutic antibody fragments

Here, we evaluate an automated, direct binding surface plasmon resonance (Biacore ® C) biosensor assay for the determination of total IgG in hyper-immunised ovine serum for use within the biopharmaceutical industry for production of polyclonal Fab fragments, including the FDA approved polyclonal biotherapeutic CroFab™ (Crotalidae Polyvalent Immune Fab). Serum IgG concentrations were evaluated using recombinant Streptococcus protein G immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. Ligand immobilisation, sensor surface regeneration and ovine IgG binding responses were evaluated and the suitability of the method for the screening of large numbers of ovine serum samples within a quality control analytical laboratory discussed. The report describes the evaluation and application of a simple, sensitive and rapid assay for determination of total ovine polyclonal IgG that may also be applicable for the determination of antibody concentration within serum or crude supernatants produced by recombinant or transgenic technologies used for biotherapeutic antibody production.

Optimal conditions for the papain digestion of polyclonal ovine IgG for the production of biotherapeutic Fab fragments

In the present paper, we describe a rapid method for the determination of optimum conditions for papain digestion of polyclonal ovine IgG (purified by Na(2)SO(4) precipitation) for the production of bio-therapeutic Fabs (antigen-binding fragments). To determine the optimum conditions for digestion, a factorial approach to the design of experiments was undertaken. The resulting experimental data were used to construct the mathematical models using Design Expert 6.06(R) (Stat-Ease, Minneapolis, MN, U.S.A.) to predict the optimum conditions for a robust IgG digestion step. Optimum conditions were evaluated experimentally, and the applicability of the conditions for large-scale manufacture of bio-therapeutic Fab fragments was assessed. The results and methods described in the present paper suggest that, provided the time and temperature are maintained at the high settings evaluated (24 h, 40 degrees C), the modelled data predict IgG digestion close to 100% for all the papain concentrations used. Provided papain is used at >2.5% (w/w), either time and/or temperature may be reduced. The results and methods described in the present paper may also be applicable to the generation of therapeutic Fab fragments from other immunoglobulins, including monoclonal antibodies purified from mammalian cell culture.

Jellyfish Antivenoms: Past, Present, and Future

If snake antivenoms are considered orphan drugs, then jellyfish antivenoms are the poorest of the orphans. Despite the diversity, ubiquity and toxicity of the venomous cnidarians, only a single antivenom is available for jellyfish stings worldwide. That antivenom, an ovine whole IgG product, is directed against the ‘box’ jellyfish, Chironex fleckeri, and is manufactured by CSL Limited (Melbourne, Australia). It also neutralises the venom of closely related cubozoans such as Chiropsalmus quadrigatus. The recognition of the life‐threatening effects of various other jellyfish demonstrates the need for broadening the specificity of the existing product and/or developing additional specific jellyfish antivenoms. These emerging threats include the irukandji syndrome, due to Carukia barnesi and other carybdeids, as well as those from scyphozoans such as Stomolophus spp. The role of ancillary drug therapy, in addition to, or instead of, antivenoms remains controversial. This review will consider the development o...

Localisation and characterisation of ovine immunglobulin within the sheep scab mite, Psoroptes ovis

Ovine IgG was detected in homogenates of repeatedly washed Psoroptes ovis. Some of the immunoglobulin in the homogenates was fragmented although the host IgG present in mite washings was largely intact. The host immunoglobulin was immuno-localised to the surface or cytoplasm of the gut cells of feeding stages of freshly harvested P. ovis examined by cryosectioning. A similar distribution of rabbit IgG was detected in P. cuniculi. The IgG demonstrated in the mite gut represented partially digested as well as intact immunoglobulin. The presence of intact host immunoglobulin suggests that P. ovis may be susceptible to vaccination by the gut antigen approach, a method used successfully for blood-feeding ectoparasites like Boophilus microplus.