Hyperimmunized Chickens Produce Neutralizing Antibodies against SARS-CoV-2.

The novel severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV-2, is responsible for the global COVID-19 pandemic. Effective interventions are urgently needed to mitigate the effects of COVID-19 and likely require multiple strategies. Egg-extracted antibody therapies are a low-cost and scalable strategy to protect at-risk individuals from SARS-CoV-2 infection. Commercial laying hens were hyperimmunized against the SARS-CoV-2 S1 protein using three different S1 recombinant proteins and three different doses. Sera and egg yolk were collected at three and six weeks after the second immunization for enzyme-linked immunosorbent assay and plaque-reduction neutralization assay to determine antigen-specific antibody titers and neutralizing antibody titers, respectively. In this study we demonstrate that hens hyperimmunized against the SARS-CoV-2 recombinant S1 and receptor binding domain (RBD) proteins produced neutralizing antibodies against SARS-CoV-2. We further demonstrate that antibody production was dependent on the dose and type of antigen administered. Our data suggests that antibodies purified from the egg yolk of hyperimmunized hens can be used as immunoprophylaxis in humans at risk of exposure to SARS-CoV-2.

Oocyst wall formation and composition in coccidian parasites.

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90% protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.

Cloning and characterization of the 82 kDa tyrosine-rich sexual stage glycoprotein, GAM82, and its role in oocyst wall formation in the apicomplexan parasite, Eimeria maxima.

The sexual (macrogamete/macrogametocyte) stage antigen, GAM82, in the apicomplexan parasite Eimeria maxima, has an apparent molecular mass of 82 kDa, and has been implicated in protective immunity against coccidiosis in poultry. The gene encoding this protein, gam82, was cloned and sequenced. It is a single-copy, intronless gene, which localizes to a 2145 bp transcript, and is first detected at 130 h post-infection. The gene predicts two distinct domains rich in the residues tyrosine and serine, amino acids that have been implicated in oocyst wall formation in other Eimeria spp., and in the extraorganismic sclerotization of structural proteins throughout the animal kingdom. A high number of small amino acids, predominantly alanine and proline, were detected in the intervening sequence between these two domains. The inference that GAM82 is involved in oocyst wall formation in Eimeria was confirmed when it was shown that a specific antibody to a recombinant version of GAM82 recognized the wall forming bodies in macrogametes, and the walls of oocysts in E. maxima. A closer biochemical analysis of the role of GAM82 in oocyst wall formation by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting showed that the antibodies to the recombinant version of GAM82 recognized an 82 kDa protein in macrogametocyte extracts, and a 30 kDa protein in unsporulated and sporulated oocyst extracts, as well as in purified oocyst wall fragments. Together, these findings indicate that the 82 kDa macrogametocyte antigen, GAM82, is a tyrosine and serine rich precursor protein that is proteolytically processed during development to give rise to a 30 kDa protein, that is incorporated into the oocyst wall. In addition, these findings provide evidence that the oocyst wall of Eimeria species is composed of a family of tyrosine rich proteins, that arise from precursor proteins found in the wall forming bodies of macrogametes.

Functional genomics of gam56: characterisation of the role of a 56 kilodalton sexual stage antigen in oocyst wall formation in Eimeria maxima.

Gam56 (M(r) 56,000) is an antigen found in the sexual (macrogametocyte) stage of the intestinal parasite Eimeria maxima that is implicated in protective immunity. The gene (gam56) encoding this protein was cloned and sequenced. It is a single-copy, intronless gene, that localises to a 1,754 bp transcript, and is first detected at 120 h p.i. The gene predicts two distinct protein domains; a tyrosine-serine rich region, composed of amino acids implicated in oocyst wall formation in Eimeria spp., and a proline-methionine rich region often detected in extensins, protein components of plant cell walls. The tyrosine-serine rich region predicts a secondary structure commonly seen in the structural protein fibroin, a component of the cocoon of the caterpillar Bombyx mori. The inference that gam56 is a structural component of the oocyst wall was confirmed when a specific antibody to gam56 recognised the wall forming bodies in macrogametocytes, and the walls of oocysts and sporocysts. Together, these data identify a developmentally regulated, sexual stage gene in E. maxima that shares primary and secondary structure features in common with intrinsic structural proteins in other parasites such as Schistosoma mansoni and Fasciola hepatica, and other organisms across different phyla, including the caterpillar Bombyx mori. In addition, these findings provide evidence for the molecular mechanisms underlying oocyst wall formation in Eimeria and the role of gametocyte antigens in this process.

Molecular characterisation of EmTFP250: a novel member of the TRAP protein family in Eimeria maxima.

We have previously described a high molecular mass, asexual stage antigen from Eimeria maxima (EmTFP250), implicated as a target of maternal antibodies produced by breeding hens infected with this protozoan parasite. Following partial purification of the protein by ion exchange chromatography, N-terminal and internal peptide sequences were generated and used in the design of degenerate PCR primers. Using a rapid amplification of cDNA ends PCR-based strategy, the cDNA encoding EmTFP250 has been cloned and sequenced. Translation predicts a mature polypeptide with a molecular mass of 246kDa and an isoelectric point of 4.2. Analysis of the amino acid sequence has revealed a novel member of the TRAP (thrombospondin-related anonymous protein) family, containing 16 thrombospondin type-1 repeats and 31 epidermal growth factor-like calcium binding domains. EmTFP250 also contains two low complex, hydrophilic regions rich in glutamic acid and glycine residues, and a transmembrane domain/cytosolic tail associated with parasite gliding motility that is highly conserved within apicomplexan microneme proteins. The protein has 61% identity (71% similarity) with EtMIC4, a 218kDa microneme protein of Eimeria tenella also rich in epidermal growth factor-like and thrombospondin type-1 domains. Using Southern blotting, the gene encoding EmTFP250 has been determined to be present as a single copy within the genome, and reverse transcriptase-PCR has shown that expression is confined to the asexual stages of development. By employing a PCR-based method, a region of the E. maxima Houghton strain EmTFP250 gene was found conserved in Australian isolates of several (at least four) Eimeria species that parasitise chickens. The characterisation of EmTFP250 adds to the expanding apicomplexan TRAP family and suggests a functional significance for the protein.

Eimeria maxima TRAP family protein EmTFP250: subcellular localisation and induction of immune responses by immunisation with a recombinant C-terminal derivative.

EmTFP250 is a high molecular mass, asexual stage antigen from Eimeria maxima strongly associated with maternally derived immunity to this protozoan parasite in hatchling chickens. Cloning and sequence analysis has predicted the antigen to be a novel member of the thrombospondin-related anonymous protein (TRAP) family of apicomplexan parasites. Members of the TRAP family are microneme proteins and are associated with host cell invasion and apicomplexan gliding motility. In order to assess the immunogenicity of EmTFP250, a C-terminal derivative encoding a low complex, hydrophilic region and putative transmembrane domain/cytosolic tail was expressed in a bacterial host system. The recombinant protein was used to immunise mice and chickens and found to induce strong IgG responses in both animal models as determined by specific ELISAs. Using Western blotting, protective maternal IgG antibodies previously shown to recognise native EmTFP250 recognised the recombinant protein and, in addition, antibodies raised against the recombinant protein were shown to recognise native EmTFP250. Localisation studies employing immuno-light microscopy and immuno-electron microscopy showed that antibodies to the recombinant protein specifically labeled micronemes within merozoites of E. maxima. Furthermore, antibodies to the recombinant EmTFP250 derivative showed similar labeling of micronemes within merozoites of Eimeria tenella. This study is further suggestive of a functional importance for EmTFP250 and underscores its potential as a candidate for a recombinant vaccine targeting coccidiosis in chickens.

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima.

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.

Cross-protection of chicken immunoglobulin Y antibodies against H5N1 and H1N1 viruses passively administered in mice.

Influenza viruses remain a major threat to global health due to their ability to undergo change through antigenic drift and antigenic shift. We postulated that avian IgY antibodies represent a low-cost, effective, and well-tolerated approach that can easily be scaled up to produce enormous quantities of protective antibodies. These IgY antibodies can be administered passively in humans (orally and intranasally) and can be used quickly and safely to help in the fight against an influenza pandemic. In this study, we raised IgY antibodies against H1N1, H3N2, and H5N1 influenza viruses. We demonstrated that, using whole inactivated viruses alone and in combination to immunize hens, we were able to induce a high level of anti-influenza virus IgY in the sera and eggs, which lasted for at least 2 months after two immunizations. Furthermore, we found that by use of in vitro assays to test for the ability of IgY to inhibit hemagglutination (HI test) and virus infectivity (serum neutralization test), IgYs inhibited the homologous as well as in some cases heterologous clades and strains of viruses. Using an in vivo mouse model system, we found that, when administered intranasally 1 h prior to infection, IgY to H5N1 protected 100% of the mice against lethal challenge with H5N1. Of particular interest was the finding that IgY to H5N1 cross-protected against A/Puerto Rico/8/34 (H1N1) both in vitro and in vivo. Based on our results, we conclude that anti-influenza virus IgY can be used to help prevent influenza virus infection.

Peroxidase catalysed cross-linking of an intrinsically unstructured protein via dityrosine bonds in the oocyst wall of the apicomplexan parasite, Eimeria maxima.

Apicomplexan parasites such as Eimeria maxima possess a resilient oocyst wall that protects them upon excretion in host faeces and in the outside world, allowing them to survive between hosts. The wall is formed from the contents of specialised organelles - wall-forming bodies - found in macrogametes of the parasites. The presence of dityrosine in the oocyst wall suggests that peroxidase-catalysed dityrosine cross-linking of tyrosine-rich proteins from wall-forming bodies forms a matrix that is a crucial component of oocyst walls. Bioinformatic analyses showed that one of these tyrosine-rich proteins, EmGAM56, is an intrinsically unstructured protein, dominated by random coil (52-70%), with some α-helix (28-43%) but a relatively low percentage of ß-sheet (1-11%); this was confirmed by nuclear magnetic resonance and circular dichroism. Furthermore, the structural integrity of EmGAM56 under extreme temperatures and pH indicated its disordered nature. The intrinsic lack of structure in EmGAM56 could facilitate its incorporation into the oocyst wall in two ways: first, intrinsically unstructured proteins are highly susceptible to proteolysis, explaining the several differently-sized oocyst wall proteins derived from EmGAM56; and, second, its flexibility could facilitate cross-linking between these tyrosine-rich derivatives. An in vitro cross-linking assay was developed using a recombinant 42kDa truncation of EmGAM56. Peroxides, in combination with plant or fungal peroxidases, catalysed the rapid formation of dityrosine cross-linked polymers of the truncated EmGAM56, as determined by western blotting and HPLC, confirming this protein's propensity to form dityrosine bonds.

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina.

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.

Field application of a subunit vaccine against an enteric protozoan disease.

BACKGROUND: Coccidiosis is a major global veterinary health problem in intensively reared chickens. It is caused by apicomplexan parasites of the genus Eimeria. PRINCIPAL FINDINGS: A subunit vaccine composed of purified antigens from the gametocytes of Eimeria maxima was used to stimulate the production and transfer of maternal antibodies between breeding hens and their hatchlings. The vaccine was injected into hens twice before they began laying eggs. Immunization had no adverse affects on egg laying or health of the hens and resulted in high antibody levels throughout the life of the hens. Progeny of immunized hens excreted significantly less oocysts of various species of Eimeria in their faeces than chicks from unvaccinated hens. Furthermore, the offspring of vaccinated hens developed stronger natural immunity to Eimeria, so that they were resistant to challenge infection even at 8 weeks of age, well after all maternal antibodies had left their circulation. Field trials were conducted in South Africa, Brazil and Thailand, involving at least 1 million progeny of vaccinated hens and at least 1 million positive control birds (raised on feed containing anticoccidial drugs or immunized with a live vaccine) in each country. Additionally, trials were carried out in Israel involving 60 million progeny of vaccinated hens and 112 million positive control birds. There were no significant differences in growth rate, feed conversion ratios or mortality in the offspring of vaccinated hens compared with the positive control chickens in any of these countries regardless of different management practices, different breeds of chickens or climate. CONCLUSIONS: These results demonstrate that a vaccine composed of antigens purified from the gametocytes of Eimeria can be used safely and effectively to prevent the deleterious effects of coccidiosis. It is the first subunit vaccine against any protozoan parasite to be successfully applied on a commercial scale.

Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima.

The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.

Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima.

Coccidiosis in poultry is caused by the intestinal parasite Eimeria; it causes significant financial losses to the commercial poultry industry worldwide. CoxAbic is the first commercially available subunit vaccine against coccidiosis. The vaccine consists of affinity purified sexual stage (gametocyte) antigens (APGA) isolated from Eimeria maxima. Production of this vaccine is time-consuming and laborious and, therefore, a recombinant subunit vaccine substitute for CoxAbic is desirable. The genes encoding the two immunodominant components of CoxAbic, gam56 and gam82, were cloned into the bacterial expression vector, pTRCHisB, and the proteins expressed and purified. Both recombinant proteins were recognised by protective chicken antibodies that were raised to APGA, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of anti-APGA antibodies to APGA by 76%, which was comparable to the inhibition of 98% observed when APGA was used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised APGA by ELISA, and gametocytes by immunoblotting. Together, the results suggested that the development of a recombinant subunit vaccine that maintains the antigenic and immunogenic properties of the native protein vaccine, CoxAbic, is feasible.

Oocyst wall formation and composition in coccidian parasites

The oocyst wall of coccidian parasites is a robust structure that is resistant to a variety of environmental and chemical insults. This resilience allows oocysts to survive for long periods, facilitating transmission from host to host. The wall is bilayered and is formed by the sequential release of the contents of two specialized organelles - wall forming body 1 and wall forming body 2 - found in the macrogametocyte stage of Coccidia. The oocyst wall is over 90 percent protein but few of these proteins have been studied. One group is cysteine-rich and may be presumed to crosslink via disulphide bridges, though this is yet to be investigated. Another group of wall proteins is rich in tyrosine. These proteins, which range in size from 8-31 kDa, are derived from larger precursors of 56 and 82 kDa found in the wall forming bodies. Proteases may catalyze processing of the precursors into tyrosine-rich peptides, which are then oxidatively crosslinked in a reaction catalyzed by peroxidases. In support of this hypothesis, the oocyst wall has high levels of dityrosine bonds. These dityrosine crosslinked proteins may provide a structural matrix for assembly of the oocyst wall and contribute to its resilience.