Immunosuppressed Hamsters Research Articles

Development and characterization of anti-SARS-CoV-2 intravenous immunoglobulin from COVID-19 convalescent plasma.

Background: The authors describe the developmental process of intravenous anti-COVID-19 hyperimmune immunoglobulin from anti-SARS-CoV-2 neutralizing antibody-containing plasma. Furthermore, the authors investigated its safety and protective activity in animal models. Materials & methods: The manufacturing process included standard ethanol fractionation, chromatographic purification steps and virus removal or inactivation. Results: The authors produced pure and safe immunoglobulin for intravenous administration, with 98.1 ± 6.5 mg/ml protein content, of which 97.6 ± 0.7% was IgG. The concentration factor of SARS-CoV-2 neutralizing antibodies was 9.4 ± 1.4-times. Safety studies in animals showed no signs of acute/chronic toxicity or allergenic or thrombogenic properties. Intravenous anti-COVID-19 hyperimmune immunoglobulin protected immunosuppressed hamsters against SARS-Cov-2. Conclusion: The obtained results can allow the start of clinical trials to study the safety and efficacy in healthy adults.

Development and Characterization of Hyperimmune Anti-SARS-CoV-2 Intravenous Immunoglobulin From COVID-19 Convalescent Plasma

Background: Polyclonal immunoglobulin G (IgG) medications have successfully treated different bacterial and viral infections for several decades. Herein, we describe the development process of hyperimmune anti-Coronavirus disease 2019 (COVID-19) intravenous immunoglobulin (COVID-globulin) from anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody (nAb)-containing plasma. Furthermore, we investigate the safety and protective activity of the COVID-globulin in animal models.Methods: COVID-19 convalescent plasma samples collected from Russian blood service organizations with known anti-SARS-CoV-2 nAb titers were used for COVID-globulin production. The manufacturing process included standard ethanol fractionation, chromatographic purification steps, and virus removal or inactivation. Immunoglobulin batches were characterized based on Russian and EU Pharmacopeia guidelines, with the anti-SARS-CoV-2 nAb titers determined using a virus neutralization test (performed in Vero E6 cells) and enzyme-linked immunosorbent assay (ELISA). COVID-globulin safety was assessed by evaluating its toxicity and thrombogenic potential. COVID-globulin protective efficacy was evaluated in immunosuppressed Syrian hamsters via the intraperitoneal administration of COVID-globulin or a placebo Changes in animal body weight and health status were observed daily. Lungs were subjected to macroscopic, histological, and virological studies. The lung virus load was determined using titration of 10% lung tissue homogenate in Vero E6 cells, and the cytopathic effect was visually evaluated.Findings: We produced pure and safe immunoglobulin for intravenous administration, with 98·1±6·5 mg/mL protein content.. The concentration factor of SARS-CoV-2 nAbs in ten COVID-globulin lots was 9·4±1·4 times. Safety studies in animals showed no signs of acute/chronic toxicity or allergenic or thrombogenic properties. COVID-globulin demonstrated protective effects against SARS-CoV-2 in immunosuppressed hamsters. The survival rate in the COVID-globulin groups was 66·6–100% compared with total mortality in the control groups.Interpretation: COVID-globulin, manufactured in accordance with the Ph. Eur. requirements, protected immunosuppressed hamsters against SARS-Cov-2.Funding Information: This work was sponsored and performed by JSC Nacimbio.Declaration of Interests: We declare no competing interests.Ethics Approval Statement: Studies were performed at The National Research Center for epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation, after receiving bioethical approval №26 from 08.10.2020 and №28 from 16.11.2020.

Protective efficacy of a SARS-CoV-2 DNA vaccine in wild-type and immunosuppressed Syrian hamsters

A worldwide effort to counter the COVID-19 pandemic has resulted in hundreds of candidate vaccines moving through various stages of research and development, including several vaccines in phase 1, 2 and 3 clinical trials. A relatively small number of these vaccines have been evaluated in SARS-CoV-2 disease models, and fewer in a severe disease model. Here, a SARS-CoV-2 DNA targeting the spike protein and delivered by jet injection, nCoV-S(JET), elicited neutralizing antibodies in hamsters and was protective in both wild-type and transiently immunosuppressed hamster models. This study highlights the DNA vaccine, nCoV-S(JET), we developed has a great potential to move to next stage of preclinical studies, and it also demonstrates that the transiently-immunosuppressed Syrian hamsters, which recapitulate severe and prolonged COVID-19 disease, can be used for preclinical evaluation of the protective efficacy of spike-based COVID-19 vaccines.

Disruption of Adaptive Immunity Enhances Disease in SARS-CoV-2-Infected Syrian Hamsters.

Animal models recapitulating human COVID-19 disease, especially severe disease, are urgently needed to understand pathogenesis and to evaluate candidate vaccines and therapeutics. Here, we develop novel severe-disease animal models for COVID-19 involving disruption of adaptive immunity in Syrian hamsters. Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the respiratory route. Both the CyP-treated and RAG2 KO hamsters developed clinical signs of disease that were more severe than those in immunocompetent hamsters, notably weight loss, viral loads, and fatality (RAG2 KO only). Disease was prolonged in transiently immunosuppressed hamsters and was uniformly lethal in RAG2 KO hamsters. We evaluated the protective efficacy of a neutralizing monoclonal antibody and found that pretreatment, even in immunosuppressed animals, limited infection. Our results suggest that functional B and/or T cells are not only important for the clearance of SARS-CoV-2 but also play an early role in protection from acute disease.IMPORTANCE Syrian hamsters are in use as a model of disease caused by SARS-CoV-2. Pathology is pronounced in the upper and lower respiratory tract, and disease signs and endpoints include weight loss and viral RNA and/or infectious virus in swabs and organs (e.g., lungs). However, a high dose of virus is needed to produce disease, and the disease resolves rapidly. Here, we demonstrate that immunosuppressed hamsters are susceptible to low doses of virus and develop more severe and prolonged disease. We demonstrate the efficacy of a novel neutralizing monoclonal antibody using the cyclophosphamide transient suppression model. Furthermore, we demonstrate that RAG2 knockout hamsters develop severe/fatal disease when exposed to SARS-CoV-2. These immunosuppressed hamster models provide researchers with new tools for evaluating therapies and vaccines and understanding COVID-19 pathogenesis.

A lethal disease model for New World hantaviruses using immunosuppressed Syrian hamsters.

BackgroundHantavirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaviruses are found on the American continent where the most prevalent viruses like Andes virus and Sin Nombre virus are known to cause hantavirus pulmonary syndrome. New World hantavirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes virus and Maporal virus; the only hantaviruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantavirus pulmonary syndrome in humans.Methodology/Principal findingsHamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantavirus strains (Bayou virus, Black Creek Canal virus, Caño Delgadito virus, Choclo virus, Laguna Negra virus, and Maporal virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantavirus results in an acute disease that precisely mimics both hantavirus disease in humans and Andes virus infection of hamsters.Conclusions/ SignificanceInfected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaviruses reaching a lethal outcome with signs of disease mimicking human disease.

Pre-existing Immunity and Passive Immunity to Adenovirus 5 Prevents Toxicity Caused by an Oncolytic Adenovirus Vector in the Syrian Hamster Model

We have used Syrian hamsters to examine the role of pre-existing immunity to adenovirus (Ad) 5 in the toxicity of the oncolytic Ad vector INGN 007. Groups of hamsters were or were not immunized with Ad5. Half the hamsters were immunosuppressed using cyclophosphamide (CP), then injected intravenously (i.v.) with 3x the maximum tolerated dose (MTD) of INGN 007 (in immunocompetent hamsters), and toxicity and vector replication in the liver were quantitated. In nonimmunized immunocompetent hamsters, toxicity was observed early but the hamsters recovered by day 6 after vector injection. In nonimmunized immunosuppressed hamsters, the vector was lethal by 3 days. Pre-existing neutralizing antibody (NAb) prevented liver infection and hepatotoxicity in both immunocompetent and immunosuppressed hamsters. In another study, passive immunization of immunosuppressed hamsters 1 day before a lethal dose (1x MTD) of INGN 007 prevented liver infection and replication, but immunization 1 day after vector administration was barely effective. When immunosuppressed hamsters were passively immunized 1 day after injection of 1/3rd the MTD of INGN 007, then significant protection was observed against liver infection and toxicity. Therefore, serum NAb are sufficient to prevent oncolytic Ad vector liver infection and toxicity. We saw no evidence that pre-existing immunity was associated with increased vector toxicity.

Evaluation of the Efficacy of a Recombinant Subunit West Nile Vaccine in Syrian Golden Hamsters

The efficacy of a recombinant subunit West Nile (WN) vaccine candidate was determined in a hamster model of encephalitis. Animals included young, aged, and immunocompromised animals in an effort to simulate key groups at risk of WN virus-induced disease. Groups of aged (12 month old), weanling, and adult hamsters rendered leukopenic after immunization were immunized subcutaneously with a WN virus recombinant envelope protein (WN-80E) with or without WN virus non-structural protein 1 (NS1) mixed with adjuvant or adjuvant alone. A challenge dose of wild-type WN virus was administered to produce 40-100% mortality in the control hamsters. The recombinant antigen preparations containing WN-80E with or without WN NS1 gave similar results. Hamsters in both groups had a strong antibody response after immunization, and none of the aged or weanling animals became ill or developed detectable viremia after challenge with WN virus at 2 weeks after booster vaccination. However, mortality among the control animals (administered adjuvant without antigen) at 2 weeks after booster challenge was 40-60%. In hamsters rendered leukopenic after immunization, survival rates up to 80% were observed, and a low-level viremia was detected in the vaccinated and challenged hamsters. The survival rate was significantly (P<0.05) higher in animals vaccinated with a higher dose of WN-80E than a lower dose. The addition of NS1 did not significantly affect survival after challenge. In contrast, all of the control animals that received adjuvant only developed a very high level of viremia, and the mortality rate was 100%. These findings indicate that the recombinant WN vaccines induced antibody in and afforded protection to young and aged hamsters and immunosuppressed hamsters.

An immunosuppressed Syrian golden hamster model for SARS-CoV infection

Several small animal models have been developed for the study of severe acute respiratory syndrome coronavirus (SARS-CoV) replication and pathogenesis. Syrian golden hamsters are among the best small animal models, though little clinical illness and no mortality are observed after virus infection. Cyclophosphamide was used to immunosuppress hamsters leading to a prolonged disease course and higher mortality after SARS-CoV infection. In addition, there was a significant weight loss, expanded tissue tropism, and increased viral pathology in the lung, heart, kidney, and nasal turbinate tissues. Infection with recombinant SARS-CoV viruses bearing disruptions in the gene 7 coding region showed no significant change in replication kinetics, tissue tropism, morbidity, or mortality suggesting that the ORF7a (7a) and ORF7b (7b) proteins are not required for virus replication in immunosuppressed hamsters. This modified hamster model may provide a useful tool for SARS-CoV pathogenesis studies, evaluation of antiviral therapy, and analysis of additional SARS-CoV mutants.

Yellow Fever 17-D Vaccine Is Neurotropic and Produces Encephalitis in Immunosuppressed Hamsters

Immunosuppressed (cyclophosphamide) adult golden hamsters inoculated intraperitoneally (i.p.) with wild-type Asibi yellow fever virus (YFV) developed a rapidly fatal illness. Histopathologic and immunohistochemical studies of tissues from these animals showed typical hepatic changes of severe yellow fever (inflammation, hepatocyte necrosis, and steatosis) without brain involvement. In contrast, 50% of immunosuppressed hamsters receiving the YFV-17D-attenuated vaccine developed a slowly progressive encephalitic-type illness. Brain tissue from these latter animals revealed focal neuronal changes, inflammation, and YFV antigen-positive neurons; however, the liver and spleen appeared normal. YFV was isolated from brain cultures of many of these animals. Immunocompetent (non-immunosuppressed) hamsters inoculated with both viruses developed a subclinical infection. Results of this study indicate that wild-type YFV is hepatotropic in immunosuppressed hamsters, whereas the attenuated YFV-17 is primarily neurotropic. These findings support current recommendations against yellow fever vaccination of immunosuppressed/immunocompromised people and suggest that this hamster model might be useful for monitoring the safety of other live-attenuated YFV vaccines.

EFFECTS OF IMMUNOSUPPRESSION ON WEST NILE VIRUS INFECTION IN HAMSTERS

A research study, comparing the pathogenesis of experimental West Nile virus (WNV) infection in immunocompetent and immunosuppressed golden hamsters, is described. Cyclophosphamide was used to immunosuppress the animals. The immunosuppressed hamsters had a prolonged period of viremia, depressed humoral immune response, more extensive and severe pathology, and higher fatality rate than the untreated immunocompetent animals. Histopathological and immunohistochemical studies of tissues from the two groups showed that pathologic changes in the untreated infected animals were confined to the brain and spinal cord, whereas the histopathological changes and WNV antigen distribution in the immunosuppressed animals were much more extensive and diffuse, involving the adrenal, kidney, heart and lung, and brain and spinal cord. Results of this study in the hamster model provide insight into the increased severity of WNV infection observed in immunosuppressed people.

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters: An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10 5−10 6 in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.

Experimental cryptosporidiosis in hamsters

A new laboratory animal model for experimental cryptosporidiosis is described. Adult immunosuppressed hamsters were infected per os with 0.5 x 10(5) and 1 x 10(5) Cryptosporidium oocysts of calf origin. The mean numbers of oocysts shed per gram of feces per day and the patterns of infection are described. The susceptibility to Cryptosporidium infection, the total number of oocysts shed (a thousand times the infective dose), and the ease of handling in laboratory conditions make hamsters a good animal model for cryptosporidiosis.

Host-mediated transformation: metronidazole

To evaluate the possible genotoxic effects of the drug, metronidazole in the fetus, we employed the hamster embryo host-mediated assay. Pregnant golden Syrian hamsters were fed metronidazole at doses ranging from 200 mg/kg to 900 mg/kg on days 11 and 12 of pregnancy. Embryonic cells obtained from the treated animals were studied in vitro for morphologic evidence of transformation. To further assess the significance of the in vitro finding, cells from mass culture were tested for their ability to grow in soft agar. The drug-treated cells and cells previously treated with diethyl nitrosamine (positive controls) showed comparable growth characteristics. To confirm the neoplastic potential of the drug-treated embryonic cells, subcultivated cells from the tenth passage were implanted into nude mice and irradiated immunosuppressed hamsters. Cells from the 300 mg/kg treatment produced fibrosarcoma in nude mice but not in the irradiated hamsters. Cells from no other dose level employed in the study produced tumors in host animals. It is concluded that metronidazole is capable of vertical transmission of potential genotoxic effects to the fetus.

Heterotransplantation and maturation of a chronic myelogenous leukemia cell line (KCL-22) in vivo.

Heterotransplantation was performed to study the maturational capacity of a new Ph1 chromosome-positive chronic myelogenous leukemia cell line (KCL-22). Five million KCL-22 cells of undifferentiated blasts were implanted intraperitoneally into 15 immunosuppressed newborn hamsters. Of the 15 hamsters, 10 that survived developed granulocytic tumors 16-61 days after implantation. The tumor cells were composed of a mixed population of undifferentiated blasts, neutrophils, and eosinophils and had a human female karyotype with double Ph1 chromosomes identical to that found in the KCL-22 cell line used for transplantation. These findings indicate that Ph1 chromosome-positive cells can be induced to mature along the neutrophil-eosinophil lineage after growth in the heterologous hosts.

An Epidermoid Carcinoma Cell Line Derived From Hamster 7,12- Dimethylbenz[a]anthracene-Induced Buccal Pouch Tumors2

w cell line, HCPC I, was established from an epidermoid carcinoma of the Syrian golden hamster cheek pouch. The carcinoma was induced by applications three times a week of a 0.5% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in heavy mineral oil. The HCPC I cell line has been maintained through 60 passages over 15 months. Electron microscopy of the cell line revealed tonofilaments and desmosomes, thereby confirming its epithelial nature. Presence of keratin in the HCPC I cells was demonstrated by both histochemical and indirect immunofluorescence studies. The population-doubling time of HCPC I, estimated from the exponential growth phase of the growth curve established for the cell line, was 12 hours. The plating efficiency of the cell line was estimated at 35%. When an inoculum size of HCPC I, numbering 2.4 X 10(7) cells, was transplanted to the cheek pouch of an immunosuppressed inbred hamster, a papillary tumor, measuring 5 X 1 mm and microscopically diagnosed as a well-differentiated epidermoid carcinoma, developed 1 week post transplantation. Further successful transplantation was achieved with 1.8 X 10(7) HCPC I cells inoculated into the cheek pouch of a nonimmunosuppressed inbred hamster. A raised plaquelike mass, microscopically diagnosed as invasive epidermoid carcinoma, developed in the hamster cheek pouch 5 weeks post transplantation. Furthermore, 5 nonimmunosuppressed hamsters that received 1 X 10(6) HCPC I cells on their cheek pouches developed tumors microscopically diagnosed as anaplastic carcinomas 14 weeks post transplantation. The largest of these tumors measured 2 cm in diameter.

Oncogenity of BK virus for immunosuppressed hamsters.

Tumors were induced by BK virus (BKV) inoculated intravenously in 3-week-old Syrian golden hamsters immunosuppressed with anti-lymphocyte serum or methylprednisolone acetate alone or in association with gamma-radiation (60Co). The induced neoplasms were ependymoma, carcinoma of pancreatic islets, lymphoma, osteosarcoma, undifferentiated sarcoma, kidney and renal pelvis carcinoma, pheochromocytoma and hemangiosarcoma. High levels of insulin and glucagon and altered concentrations of glucose were detected in blood of animals with tumors of pancreatic islets. No antibodies to BKV tumor antigen (TAg) and low levels of hemagglutination-inhibition antibodies to BKV viral coat protein Ag were detected in hamster sera. BKV TAg was found in tumors by complement fixation. Blot hybridization analysis of tumor DNA showed the presence of both free and integrated BKV genomes in tumor cells. BKV DNA inoculated intravenously and subcutaneously in immunosuppressed or immunocompetent hamsters was not oncogenic, whereas it was weakly oncogenic when inoculated intracerebrally.

Characteristics of a brain lymphoma cell line derived from primary intracranial lymphoma.

A brain lymphoma cell line, designated TK, was established from biopsy material of a patient with primary intracranial lymphoma. The TK cell line grew in suspension with clumps of cells and consisted of primitive lymphoid cells. TK cells and original lymphoma cells from which the cell line was derived bore surface immunoglobulin and lacked Epstein-Barr virus-determined nuclear antigen. Transplantation of the TK cells into immunosuppressed newborn hamsters gave rise to invasive growth of tumors with metastasis to the brain and eyes. These findings provide definitive evidence for a B-cell origin of the brain lymphoma in this case.

Enhanced intrauterine transmission of herpes simplex virus infection in immunosuppressed hamsters.

Herpes simplex virus (HSV) infection during pregnancy in man may result in abortion, fetal death, perinatal infection and malformations (1–6). The source of infection in the newborns is obscure; it is unknown whether they are infected in utero transplacentally or their infection is acquired during delivery through the birth canal. There is some evidence suggesting vertical transmission of HSV infection in the mother (7–9). In animal experiments, vertical transmission of HSV infection has been demonstrated in rabbit, mouse, hamster and cat (10–14). We have documented elsewhere (15) that type 1 and 2 HSV caused placental lesions and vertical transmission of the infection in the mother into the fetus when the virus was inoculated intravenously (IV) at the middle and late stage of pregnancy.

Chemotherapy against human melanoma in the hamster cheek pouch.

Human malignant melanoma was transplanted to the cheek pouch of immunosuppressed (corticosteroid-treated) hamsters. Significant differences were observed in tumor growth constants for animals treated with methotrexate, DTIC, BCNU, and vincristine (P < 0.05) compared to controls. However, no appreciable effect was demonstrated for Adriamycin, 5-FU, and cytoxan. In addition, single doses as high as 400 mg/kg body weight of methotrexate were no more effective than other agents now used against this neoplasm.

Cell cycle kinetics of uninfected and feline leukemia virus-infected canine lymphoma cell lines: effects of methotrexate treatment.

The cell cycle kinetics of uninfected and feline leukemia virus-infected canine lymphoma cell lines were determined by autoradiography (PLM method) as follows: DT-5: generation time (TC), 15.2 h; pre-synthetic gap phase (TG1), 3.2 h; DNA-synthetic phase (TS), 8.2 h; post-synthetic gap ph se (TG2), 3.3 h; visible mitotic phase (TM), 0.5 h. 11028: TC, 13.6 h; TG1, 1.9 h; TS, 7.7 h; TG2, 3.4 h; TM, 0.6 h. 11028+FeLV (11028 productively infected with feline leukemia virus): TC, 11.2 h; TG1, 0.2 h; TS, 8.3 h; TG2, 2.1 h; TM, 0.6 h. Exposure of the lymphoma cell lines to methotrexate (MTX) in vitro produces dose-related increases in cellular volume, associated with reductions in cellular proliferation. The relative sensitivities of these cell lines to MTX, measured by the ID50 MTX concentrations for DT-5, 11028, and 11028+FeLV are 118 nM, 122 nM, and 28 nM respectively. The cell kinetic effects of the ID50 MTX concentrations added to cultures of lymphoma cells pulse-labeled with tritiated thymidine are an approximately 2-h prolongation of TC, attributable to a lengthening of TS, with other cell cycle phases not significantly altered. These cell lines are highly tumorigenic when transplanted into the cheek pouches of immunosuppressed hamsters, with inocula of 10(4) cells producing rapidly growing, well vascularized tumors.