Development, Histological, and Ultrastructural Evaluation of Sheep Hyperimmune Serum Therapy for COVID-19 / Desarrollo, Evaluación Histológica y Ultraestructural de la Terapia con Suero Hiperinmune de Oveja para COVID-19

SUMMARY: In response to the threat posed by new variants of SARS-CoV-2 and the urgent need for effective treatments in the absence of vaccines, the aim of this study was to develop a rapid and cost-effective hyperimmune serum (HS) derived from sheep and assess its efficacy. The utilization of a halal-certified, easily maintained in certain geographic regions, easy-to-handle animal such as sheep could provide a viable alternative to the expensive option of horses. Sheep were immunized with a whole inactivated SARS-CoV- 2 antigen to produce HS, which was evaluated for neutralizing potency using the PRNT50 assay. K18-hACE2 transgenic mice (n=35) were divided into three groups: control, SARS-CoV-2 exposure through inhalation, and SARS-CoV-2 exposed mice treated with HS. HS efficacy was assessed through serum proinflammatory cytokine levels, qRT-PCR analysis, histopathological examination of lungs and hearts, and transmission electron microscopy. Purified HS exhibited significant neutralizing activity (1/24,576). The SARS-CoV-2+HS group showed lower levels of TNF-α, IL-10, and IL-6 (P<0.01) and relatively lower levels of MCP-1 compared to the SARS-CoV-2 group. HS prevented death, reduced viral RNA levels in the lungs and hearts, protected against severe interstitial pneumonia, preserved lung tissue integrity, and prevented myocyte damage, while the SARS-CoV-2 group exhibited viral presence in the lungs. This study successfully developed a sheep-derived HS against the entire SARS-CoV-2 virus, resulting in a significant reduction in infection severity, inflammation, and systemic cytokine production. The findings hold promise for treating severe COVID-19 cases, including emerging viral variants, and immunocompromised patients.

En respuesta a la amenaza que suponen las nuevas variantes del SARS-CoV-2 y la urgente necesidad de tratamientos eficaces en ausencia de vacunas, el objetivo de este estudio fue desarrollar un suero hiperinmune (HS) rápido y rentable derivado de ovejas. y evaluar su eficacia. La utilización de un animal con certificación halal, de fácil mantenimiento en determinadas regiones geográficas y de fácil manejo, como las ovejas, podría proporcionar una alternativa viable a la costosa opción de los caballos. Las ovejas fueron inmunizadas con un antígeno de SARS-CoV-2 completamente inactivado para producir HS, cuya potencia neutralizante se evaluó mediante el ensayo PRNT50. Los ratones transgénicos K18-hACE2 (n = 35) se dividieron en tres grupos: control, exposición al SARS-CoV-2 mediante inhalación y ratones expuestos al SARS-CoV-2 tratados con HS. La eficacia de HS se evaluó mediante niveles de citoquinas proinflamatorias en suero, análisis qRT-PCR, examen histopatológico de pulmones y corazones y microscopía electrónica de transmisión. El HS purificado exhibió una actividad neutralizante significativa (1/24,576). El grupo SARS-CoV-2+HS mostró niveles más bajos de TNF-α, IL-10 e IL-6 (P<0,01) y niveles relativamente más bajos de MCP-1 en comparación con el grupo SARS-CoV-2. HS evitó la muerte, redujo los niveles de ARN viral en los pulmones y el corazón, protegió contra la neumonía intersticial grave, preservó la integridad del tejido pulmonar y evitó el daño de los miocitos, mientras que el grupo SARS-CoV-2 exhibió presencia viral en los pulmones. Este estudio desarrolló con éxito un HS derivado de ovejas contra todo el virus SARS-CoV-2, lo que resultó en una reducción significativa de la gravedad de la infección, la inflamación y la producción sistémica de citocinas. Los hallazgos son prometedores para el tratamiento de casos graves de COVID- 19, incluidas las variantes virales emergentes y los pacientes inmunocomprometidos.

Development of polyclonal antisera to clone enterovirus 71 cellular receptor.

Purpose: Enterovirus 71 (ENV71) is a member of Picornaviridae family and was shown to be of public health concern in the Far East because of the notorious outbreaks it caused, with novel clinical features in the affected patients. In this study we assessed the use of virus capsid protein VP1 in viral receptor research. Material and Methods: The capsid protein (VP1) was cloned, expressed in a prokaryotic system, and purified for immunisation of rabbits. The immunisation was carried out according to the UK Home Office regulations. The polyclonal antisera were collected and tested for reactivity against recombinant and native VP1 of ENV71. Results: Both antisera were reactive against native and partially/fully denatured viral particles. Conclusion: The antisera are functional in receptor studies.

Development of a universal plate-agglutination test for detecting Haemophilus parasuis

Due to the serovar diversity in Haemophilus (H.) parasuis, it is difficult to develop a universal serological method for detection of this pathogen. Here, we report a universal plate-agglutination test for detecting H. parasuis. Diagnostic antisera were prepared by mixing antisera of serovars 4, 5, 12, 13 and 14 in the optimized ratio. The results of the plate-agglutination test showed that the diagnostic antisera could agglutinate with all 15 reference strains of H. parasuis and 74/75 clinical isolates. Further, the specificity of the method was validated with 22 bacterial strains from 12 related species.

Falha na sexagem por inibição do desenvolvimento de embriões bovinos produzidos in vitro com anticorpos anti H-Y / Failure of sexing by developmental arrest of bovine embryos in vitro produced with H-Y antisera

Embriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1) embriões inibidos em estádio de mórula (classificados como machos) e 2) embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas). O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3 por cento, 5 por cento ou 7 por cento, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05) nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso.

In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1) embryos arrested at morula stage (classified as males); and 2) embryos that developed and formed a blastocoele (classified as female). The sex of 311 embryos, divided in three groups of concentration of H-Y antisera, 3 percent, 5 percent or 7 percent, was identified by polimerase chain reaction. The results showed no difference (P>0.05) on sexual deviation in groups in which the H-Y antisera was added, in relation to control group, in which no H-Y antisera was added. In contrast with results obtained with in vivo produced bovine embryos, the sexing of in vitro produced bovine embryos with high H-Y antisera titer did not succed.

Clinical significance of E. coli O26 isolates on urine specimen of urinary tract infection / 대한산부인과학회지

OBJECTIVE: Escherichia coli (E. coli) O26 has been the most common type of non-O157 human isolates and it has been related with urinary tract infection and its sequelae. So we investigated the clinical significance of E. coli O26 among the cases of urinary tract infection. METHODS: From January, 2005 to December, 2007, the 22 E. coli isolates that were related with urinary tract infection were analyzed. The isolates were identified biochemically by Vitek 1. We performed antisera test by O157, O26, O111 diagnostic antisera about the 22 E. coli isolates. We reviewed clinical history of the same patients retrospectively. RESULTS: 331 E. coli isolates in the urine specimen were isolated from January, 2005 to December, 2007. 175 E. coli isolates that were related with urinary tract infection were analyzed by O157, O26, O111 antisera test. As a result, 22 isolates (13.5%) were O26 antisera positive. There were 8, 3, and 2 cases of watery diarrhea, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura repectively. CONCLUSION: In our study, because E. coli O26 was pathogenic and developed major complications, we concluded that patients with urinary tract infection with E. coli. should examine the antisera test about E. coli O157 and O26.

Clinical Significance of Escherichia coli O26 Isolated from Clinical Specimens / 대한임상미생물학회지

BACKGROUND: Non-O157 human isolates among enterohemorrhagic Escherichia coli (EHEC) serogroup have been reported with increasing frequency in recent years; the serotype O26 is the most common among the non-O157 isolates. We performed serotyping of E. coli isolates with O157, O26, and O111 antisera at Ulsan University Hospital and identified 27 isolates of O26. The purpose of this study was to investigate the clinical significance of E. coli O26 isolates. METHODS: During the 24-month period from January 2002 to December 2003, E. coli isolates were serotyped when requested by the physician because of bloody diarrhea or when blood was noted in the stool specimen at the laboratory. The isolates were identified biochemically by Vitek 1 (BioMerieux Vitek Inc., Mo., USA) and serotyped using diagnostic antisera of O157, O26, and O111 (NIH, Korea). When a positive agglutination reaction was shown, the patient's was reviewed retrospectively. RESULTS: Of 4,921 isolates of E. coli during the 2-year period, 200 isolates were serotyped and 27 (13.5%) were identified as serotype O26. These were isolated from stool (13 isolates), urine (9), pus (1), blood (1), and bile (1). Among the 13 patients whose stool specimens grew E. coli O26, 12 had watery diarrhea and 7 bloody diarrhea; two patients had thrombocytopenia and purpura simultaneously. Two patients with watey diarrhea, two with bloody diarrhea, and one with TTP were among the 7 patients with E. coli O26 in the urine. Finally, one patient each with blood isolate and bile isolate of E. coli O26 both had acute gastroenteritis. CONCLUSIONS: Most of the patients infected with E. coli O26 had clinical manifestations consistent with EHEC infections. E. coli isolates from patients with boody diarrhea should be serotyped with O157 and O26 antisera.

Production of shigella antisera for diagnosis at IVAC

4 groups of antiserum were met the inquiry to make strongest immunized response and to produce most antibody against Sh.flexneri. Produced antiserum has a high specificity and a possible identificity to detect correspond antigene. It met the physical criteria, having good sterility, specificity and sensitivity. The preparation was stable after 24 months follow up. It can be used to differentiate the subgroups of Shigella

Generation of high titer antisera of a novel liver-specific gene FF456 in mice by DNA immunization and it’s application / 医学研究生学报

Objective: FF456 is a new gene cloned in our lab, which belongs to c-type G protein-coupled receptors and has high homology to the olfactory receptor family. Northern blotting and RT-PCR showed that the expression of FF456 was exclusively restricted in liver and was significantly down-regulated in hepatoma. In an effort to study the function of FF456 gene, high titer antibody is indispensable. Methods:Full-length cDNA of FF456 was reconstructed into pcDNA3.1(-) vector, which was used for immunizing 6- to 8- weeks old female BALB/c mice. The effects of different injection manners, solvents and dosages on the antibody production have been compared. For detecting the titer and specificity of the antisera produced by DNA immunization, a peptide containing FF456 specific sequence was expressed by E.coli expression system. Results:Finally specific antisera with titers as high as 1 ∶ 50 000 was obtained. Conclusion:Immunofluorescence assays showed FF456 was expressed in hepatocytes with high tissue specificity. The FF456 expression in hepatoma cells was decreased dramatically.

Positive Rate of HLA Class I Antibodies in Multiparous Korean Women / 대한임상병리학회지

BACKGROUND: Anti-HLA antibodies are most frequently induced by transfusion or pregnancy, and these anibodies can be used as antisera for HLA typing. However these antibodies may elicit adverse reactions such as transfusion reaction or rejection of transplanted organs. In this study, frequency and specificities of antibodies against HLA class I antigens were determined in multiparous Korean women. METHODS: Sera from 671 multiparous women were tested for anti-HLA antibody screening by standard microlymphocytotoxicity test using 49~50 lymphocyte panels. PRA(panel reactive antibody) values were calculated as percentage of postive panels among total lymphocyte panels tested. HLA antibody specificities and reaction strengths were determined by analysis of serologic reaction patterns. RESULTS: A total of 671 sera were tested and 124 sera(18.5%) were positive for HLA antibodies. Among HLA antibody-positive sera(n=124), 117(94.4%) showed PRA values of 50%. Specificities of HLA antibodies were identified in 51 sera(41.1%) and 18 sera(14.5%) contained reagent quality antibodies(r> or =0.8, SI> or =70%), corresponding to 2.7% of total multiparous women. Among these, 4 sera had monospecific HLA antibodies and 14 sera had HLA antibodies against two or more antigens: 4 sera containing HLA antibodies against 7 CREG(cross reactive group), 5 sera containing antibodies against 5 CREG. CONCLUSION: Through the analysis of frequency and specificity of HLA antibodies in 671 multiparous women, it is concluded that HLA antisera can be obtained from multiparous women as effectively as from pregnant women. The frequency of high level of sensitization(PRA>50%), which can elicit problems in relation to transfusion or organ transplantation, is very low(1.0%).

A Study the Procurement of HLA Class I Typing Trays Using Gushed Out Blood During Placental Delivery

BACKGROUND: Microlymphocytotoxicity test is most widely used for HLA Class l typing but almost all laboratories depend on imported HLA Class 1 typing trays. Matching criteria for the selection of HLA- matched platelets to treat platelet refractoriness is not as strict as for bone marrow transplantation. Therefore, with the acquisition of various antisera against high frequency HLA antigens, self-made HLA typing trays can be used for HLA typing of HLA-matched platelet donors. METHODS: 140 samples obtained during placental delivery were tested for the presence of HLA antibodies against a well-characterized panel of 90 cells. Specificity of HLA antisera were determined by evaluating the correlation coefficient r of the 2 x 2 table, x2 test. Antisera strength was evaluated by the strength index. RESULTS: HLA antibodies were detected in 25 samples by primary screening and 23 samples also showed a positive reaction in secondary screening(16%). Among 23 samples, 1 1 antisera were of reagent grade quality and 7 were monospecific antisera. DISCUSSION: Imported HLA typing trays can be replaced by harvesting HLA antisera against HLA antigens which are relatively common in Koreans through continuous HLA antibody screening using gushed out blood during placental delivery. (Korean J Blood Transfusion 10(1): 53-60, 1999)

Procurement of HLA Class I Antisera from Multiparous Blood Donors / 대한임상병리학회지

BACKGROUND: HLA antisera are procured mainly from placental blood or blood of multiparous women. The latter has a merit that a large volume of antisera could be obtained, once the antisera are found to be of good quality. METHODS: A total of 1,437 multiparous blood donors were screened for the presence of anti- HLA antibodies. After the first screening with 20 panel cells, initially reactive sera were re- screened with 30 panel cells. RESULTS: Of 1,437 sera, 50 sera (3.5%) were reactive to both the first and the second screening panel cells. Among 50 sera, 25 (50.0%) sera could be assigned for their antibody specificity with r value of 0.8 or more. Only 14 samples (1.0%) showed reactivity to two or more panels with same antigen specificity and strength index of 80% or more. Four donors repeatedly donated blood with specificities of A24, A26, B7, and B7+B40, respectively. CONCLUSIONS: Screening of HLA class I antibodies in multiparous blood donors showed that HLA antisera of good quality could be obtained in about 1% of the donors in Korea.

Radioimmunoassay of polypeptide hormones using immunochemically coated plastic tubes.

A method has been developed for immobilisation of antisera on fresh plastic tubes through an immunochemical bridge. This type of immobilisation has been shown to be more consistent than direct adsorption on plastic. Such immunochemically coated antisera on plastic tube has been used in the development of a noncentrifugation radioimmunoassay. This assay system has been found to be technically as sound as the conventional method.