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Background: This study aimed to evaluate the outcomes of preclinical studies on the safety and immunogenicity of an inactivated COVID-19 vaccine candidate to warrant further clinical evaluation. Method(s): SARS-CoV-2 positive nasopharyngeal swab specimens were confirmed by real-time polymerase chain reaction and next-generation sequencing. The safety and immunogenicity tests of the COVID-19 vaccine were carried out in rats and Rhesus monkeys, and Balb/C mice and Rhesus monkeys, respectively. Result(s): The candidate vaccine was well tolerated and induced promising levels of SARS-CoV-2- specific IgG1, IgG2a, and Granzyme B in Balb/C mice, and anti-SARS-CoV-2 spike IgG and neutralizing antibodies in Rhesus monkeys. Based on cVNT results, the inactivated vaccine in 0.5 and 1 microg/100 microL doses was able to induce a neutralizing effect against the SARS-CoV-2 virus up to a dilution of 1:512 and 1:1000. The protective efficacy of the vaccine candidate was challenged with 2 x108 PFU of live viruses and confirmed by lung CT scan and histopathological evaluations compared to the control group. Repeated intramuscular injection of the candidate vaccine was generally well-tolerated in Rats and Rhesuses. No significant side effects were observed in rats injected with ten full human doses and in the Rhesus monkeys with three full human doses. Conclusion(s): Based on the findings presented in this study, it is recommended that this vaccine be moved into human testing commencing with a phase I clinical trial.Copyright © 2021 Muslim OT et al.
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Reports on antibody titers following CoronaVac administration are still scarce, particularly when it comes to the post-vaccination effectiveness of CoronaVac in the Indonesian population. The purpose of this study is to determine the efficacy of COVID-19 vaccination by comparing the IgG levels against the S1 subunit of SARS-CoV-2 RBD after the first and second vaccinations. The researchers collected venous blood samples from participants after they received the CoronaVac 600 SU/0.5 mL vaccine at two different intervals (14 days and 28 days). Blood was drawn twice (after the first and second vaccinations) and tested for antibodies (positive antibody detection value of 50 AU/mL). Paired data were analyzed by using either the Wilcoxon test (numerical) or the McNemar test (categorical). The median IgG1 levels in the 14-day interval between vaccine doses were 64.40 AU/mL and IgG2 levels were 886.10 AU/mL. Meanwhile, the median IgG1 level was 146.10, and IgG2 level was 688.00.AU/mL in the group with a 28-day interval between vaccine doses. After the first vaccination, 60.00 % of study subjects had positive IgG levels, which increased to 98.57% after the second vaccination. Following the full-dose vaccination, all participants had higher antibody levels, and considered significant. The effect was stronger in the group that received the vaccine at 14-day intervals. CoronaVac has also been shown to increase the prevalence of detectable antibody positivity in study participants.Copyright © 2023 Russian Association of Allergologists and Clinical Immunologists, St. Petersburg Regional Branch (SPb RAACI). All rights reserved.
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Case report Introduction: Toxic epidermal necrolysis (TEN), is an immune-mediated disease characterized by severe mucocutaneous symptoms and is the result of an inflammatory response that leads to keratinocyte necrosis and perivascular lymphocyte infiltration, mostly drug-related. Case report: A 35-year- old male, with a history of recently diagnosed systemic lupus under treatment with prednisone, hydroxychloroquine, mycophenolate and cotrimoxazole forte evolves with persistent proteinuria, it is decided to add losartan, chlorthalidone and atorvastatin. Nevertheless despite immunosuppression, proteinuria and skin involvement persisted, so mycophenolate was suspended and a bolus of cyclophosphamide 1 g was administered. Eight weeks after adjusting treatment, the patient went to the emergency department due to a confluent, pruritic, maculopapular rash with blistering lesions on the trunk, upper limbs, face, and oral mucosa, associated with fever over 38degreeC, that evolved during one week. On admission, the following was confirmed: confluent erythematous macular exanthem associated with multiple flaccid blisters on the chest, upper limbs and neck, Nikolsky's sign (+), keratoconjunctivitis and dryness on the lips. Admission tests included complete blood count with no leukocytosis or eosinophilia, ESR 29 mm/hr, C-RP 19.8 mg/L, no liver profile abnormalities, creatinine 0.8 mg/dl, and urine test with proteinuria 300 mg/dl. Negative infectious study for mycoplasma, herpes 6 virus, cytomegalovirus, Epstein barr virus, hepatitis A, B, C, E and SARS-COV2 virus. Due to severe mucosal skin involvement, TEN/SJS was suspected v/s (TEN)-like Lupus presentation, drugs used prior to admission (chlorthalidone, losartan, atorvastatin) were discontinued, and treatment was started with Hydrocortisone 100 mg every 8 hours IV, Immunoglobulin 2 g/kg daily IV for 4 days, plus skin and mucous membrane care. Patient had a favorable evolution, with resolution of skin and mucosal lesions and no signs of infection. Skin biopsy showed necrotic epidermis, necrotic basal keratinocytes, and sparse lymphocytic inflammatory infiltrate in the papillary dermis, consistent with erythema multiforme/toxic epidermal necrolysis. Conclusion(s): Extensive mucosal involvement is one of the cardinal signs of the presentation of SJS/ETN and given its severity, a high index of suspicion is important with the consequent suspension of suspected drugs and support management for a favorable evolution. In this case the suspected culprit drug was the combination of cyclophosphamide and chlorthalidone, due to reports of increased toxicity of cyclophosphamide in combination with diuretic drugs.
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The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.
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The SARS-CoV-2 virus caused the COVID-19 pandemic is related to the SARS-CoV-1 and MERS coronaviruses, which were resulted in 2003 and 2012 epidemics. Antibodies in patients with COVID-19 emerge 7–14 days after the onset of symptoms and gradually increase. Because the COVID-19 pandemic is still in progress, it is hard to say how long the immunological memory to the SARS-CoV-2 virus may be retained. The aim of this study was to study a ratio between humoral and cellular immunity against the SARS-CoV-2 S protein in COVID-19 convalescents. There were enrolled 60 adults with mild to moderate COVID-19 2 to 12 months prior to the examination. The control group consisted of 15 adults without COVID-19 or unvaccinated. Specific antibodies to the SARS-CoV-2 virus were determined by ELISA with the SARS-CoV-2-IgG-ELISA-BEST kit. To determine the specific IgG and IgA subclasses, the anti-IgG conjugate from the kit was replaced with a conjugate against the IgG subclasses and IgA. Additional incubation with or without denaturing urea solution was used to determine the avidity of antibodies. Peripheral blood mononuclear cells were isolated by gradient centrifugation, incubated with or without coronavirus S antigen for 20 hours, stained by fluorescently labeled antibodies, and the percentage of CD8highCD107a cells was assessed on flow cytometer BD FACSCanto II. In the control group, neither humoral nor cellular immunity against the SARS-CoV-2 S protein was found. In the group of convalescents, the level of IgG antibodies against the SARS-CoV-2 S protein varies greatly not being strictly associated with the disease duration, with 57% and 43% of COVID-19 patients having high vs. low level of humoral response, respectively. A correlation between level of specific IgG and IgA was r = 0.43. The avidity of antibodies increased over time in convalescents comprising 49.9% at 6–12 months afterwards. No virus-specific IgG2 and IgG4 subclasses were detected, and the percentage of IgG1 increased over time comprising 100% 6–12 months after recovery. 50% of the subjects examined had high cellular immunity, no correlations with the level of humoral immunity were found. We identified 4 combinations of humoral and cellular immunity against the SARS-CoV-2 S protein: high humoral and cellular, low humoral and cellular, high humoral and low cellular, and vice versa, low humoral and high cellular immunity.
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Background: COVID-19 convalescent plasma (CCP) was shown to reduce disease progression if high-titre CCP is administered in early infection. CCP donors have a risk profile like first time donors, especially with respect to window-period viral transmissions. Pathogen reduction (PR) could mitigate that risk, but may have impact on quality and quantity of plasma proteins, including neutralizing antibodies. It has been shown that different IgG subclasses contribute differently to CCP neutralizing activity, raising the question of a potential impact of PR on different IgG subclasses. Aims: Side-by-side comparison of the impact of 3 commercially available PR technologies on total IgG and subclasses quantity and subclass distribution in CCP. Methods: 36 apheresis CCP donations collected with a MCS+ 9000 plasmapheresis device (Haemonetics S.A., Switzerland), or DigiPla 80 (Sichuan Nigale Biomed, China) plasmapheresis device (650-750 ml) were allocated to 3 study groups with 12 units respectively. The impact of amotosalen/UVA (AS)-treatment (INTERCEPT Blood System, Cerus) against Riboflavin UVB (RB) (Mirasol Pathogen Reduction System, Terumo BCT), AS against Methylene Blue (MB) (Theraflex MB, Macopharma) and RB against MB on the quantity and distribution of IgG and subclasses was assessed in a side-by-side comparison study with a nephelometric analyser (BN II System, Siemens Healthcare). PRtreatment was conducted immediately post collection. All samples for analysis were frozen within 6 h post collection and stored at -75°C until testing. All samples were analysed simultaneously with the same device, the same lot of reagents and the same operator. Results: IgG subclass distributions were not significantly changed post PR-treatment with all 3 technologies (p > 0.05). There was also no significant difference in the median loss of concentration for IgG1 and IgG2 between the three technologies (p > 0.05). The median loss (%) of IgG3 (2.1 and 2.6-fold compared to AS and MBtreatment respectively, p < 0.01) and IgG4 (1.6 and 5.9-fold compared to AS and MB-treatment respectively, p < 0.01) post RBtreatment was significantly higher. The median loss (%) of IgG4 post AS-treatment was significantly higher compared to MB-treatment (3.5-fold, p < 0.01). Summary/Conclusions: The 3 commercially available PR systems do not significantly change the IgG subclass distribution, but impact differently IgG3 and IgG4 post-treatment loss. It was reported that IgG1 and IgG3 play an important role in neutralization, which should be considered when planning PR-treatment for CCP. .
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Introduction. On March 5 2020, the first case of COVID-19 was reported and on March 16, order of quarantine was issued which resulted in interruption of academic activities. Objective. Determine the seroprevalence and risk factors associated to SARS-CoV-2 infection in undergraduate students of the school of medicine in a public university. Methods. Descriptive cross-sectional study by means of a serology survey to a representative sample of the population of undergraduate students of a health sciences school using a rapid test to detect antibodies IgG, IGM or both in a total blood sample by digital punction. Results. On August 2021, 24,92% (IC95% 18,2-33,0) of students of health sciences tested positive for antibodies anti SARS-CoV-2, 21,2% for IgG, 2,3% for IGM and 0,8% for both;75,08% (IC95% 66,9-82,0) tested negative antibodies. Conclusions. The seroprevalence in students was not different from the seroprevalence in general population. Therefore, in order to restart presential academic activities this population must be vaccinated and other nonpharmacological requirements should be accomplished for prevention and control of the pandemics such as natural ventilation of classrooms, use of personal protection equipment – masks, hand washing and keeping physical distancing.
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Introduction: After viral or bacterial sepsis, most intensive care unit (ICU) patients enter a state of profound immunosuppression contributing to patients' worsening. Transgene has developed an immunotherapy based on a viral vector encoding human interleukin-7 (hIL-7) to restore both innate and adaptive immune responses. Here, we assessed the capacity of hIL-7 to improve ex vivo T lymphocyte function from septic shock and COVID-19 patients. Methods: Primary human hepatocytes were transduced with MVAhIL- 7-Fc, a recombinant Modified Vaccinia virus Ankara (MVA) encoding the hIL-7 fused to the human IgG2 Fc fragment, or with empty MVA as control. Cell culture supernatants were harvested for further assays. T cells were collected from ICU patients (septic shock = 11, COVID- 19 = 29) and healthy donors (n = 21). STAT5 phosphorylation, cytokine production (ELISpot and intracellular staining) and cell proliferation were assessed upon TCR stimulation with supernatants containing or not hIL-7 produced after MVA transduction or with the counterpart recombinant hIL-7 (rhIL-7). Results: Patients with viral and bacterial sepsis display T lymphocyte alterations compared to healthy donors with a decreased production of cytokines and a decreased proliferation capacity. Supernatant containing hIL-7 induces STAT5 phosphorylation in CD3 lymphocytes of all patients. With 90% of responders, hIL-7 boosts cytokines production (single and double IFN-TNF) and T lymphocytes proliferation capacity at the same level as rhIL-7 in both cohorts whereas empty MVA has no effect. Conclusions: This study indicates that hIL-7-Fc produced after MVA transduction initiates IL-7 signaling through the phosphorylation of STAT5 and restores ex vivo human lymphocyte functions in cells from septic patients with acquired immunosuppression. This proof-of-concept study, along with experimental results in animal models, supports the clinical development of the MVA-hIL-7-Fc in sepsis immunosuppressed patients.
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Introduction: Timeliness of diagnosis and treatment of MIS-C has increased amid the COVID-19 pandemic. Methods: A child was admitted to our clinic (male, 14 years old). He was in contact with a COVID-19 patient 17 days before. Upon admission, the patient complained of a rise in body temperature to 40° C, abdominal pain, vomiting, and diarrhea. Hemorrhagic rash on the skin of the upper and lower extremities, hyperemia of the mucous membrane of the lips and tongue, arterial hypotension were found. Hospitalized at ICU. In laboratory tests: WBC 3.42 × 109/ l, RBC 4 × 1012/ l, HB 111 g/l, HTC 31, PLT 31 × 109/ l, CRP 283 mg/l, PCT 6.66, D-dimer 9.2, LDG 194 U/l, ferritin 989 mcg/l, ALT 54 U/l, GGT 79 IU/l, albumin 32 g/l;proteinuria 0.75 g\l, hematuria. Diagnosis: MIS-C associated with COVID-19. Results: Prescribed: Meropenem 20 mg/kg/d, methylprednisolone 2 mg/kg/d. After 8 h-septic shock. 0.3 μg/kg/min norepinephrine was started. ECG-a violation of repolarization with ST elevation up to 0.3 mm. Echocardiography-a decrease in the left ventricular ejection fraction to 47%, pericardial effusion. Ultrasound examination of the abdominal cavity: hepatosplenomegaly. Dobutamine 3 μg/kg/min was added to the therapy. An increase in PCT up to 19.8 was found. IV IgG 2 g/kg was added to the therapy. On the 3rd day of therapy, regression of all symptoms was obtained. On the 8th day, the child was transferred from the ICU to the pediatric department. On the 12th day he was discharged home. Conclusions: Thus, the timely diagnosis of MIS-C associated with COVID-19 and the appointment of intensive therapy with the inclusion of methylprednisolone and IV IgG allows achieving a positive result in the shortest possible time. Consent to Publish: Written informed consent was obtained from the next of kin.
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SARS-CoV-2 still remains to be the cause of the Covid-19 pandemic, with more than 200 million infections and over 4.5 million deaths worldwide. With the onset of this pandemic, vaccine development efforts began on an unprecedented scale in the world, and it was seen that some vaccines received global application permissions in a very short period of time. For now, it has been proven that current COVID-19 vaccines are highly effective in protecting against serious illness and death. However, there remains much to learn about the immune response of vaccines and the duration of protection, and how we can further optimize vaccines against new variants. As of today, some inactivated vaccines (eg. Sinovac, Sinopharm, BharatBiotech), mRNA vaccines (eg.Pfizer&Bionthec, Moderna), Non-Replicating Viral Vector ones (eg. Oxford, Astra- Zeneca, Sputnik V) and recombinant protein vaccines (eg.Novovax) have been approved for immediate use, but determining the longterm effectiveness of vaccination with existing vaccines and moving towards adolescent and pediatric applications, as well as the reports on some serious side effects seen especially with mRNA and vector vaccines, remain important issues. In addition, the difficulties of poor countries' access to vaccines, and serious vaccine insecurity or rejection in almost all countries constitute major obstacles against the termination of the pandemic. The extension of the time required for the formation of herd immunity leads to the emergence of new variants. The rapid spread of these variants leading to a significant decrease in the effectiveness of the existing vaccines brings about a need for the modification of the current vaccines. During this period, we developed a different vaccine candidate and two vaccine formulations that can be easily adapted to emerging variants. In our study, a fragment protein (P1;MW: 33 kDa) containing the Receptor-Binding Domain (RBD) in the spike S1 region, a fragment protein in the S2 region (P2;MW: 17.6) and nucleocapsid protein (N;MW: 46 kDa) were expressed in Escherichia coli, and subsequently the recombinant proteins were purified. It was determined that each of these three protein antigens reacted strongly with recovered Covid-19 patient sera. The combination of these three proteins was adsorbed with one adjuvant or two adjuvants led to the development of two formulations. In mouse immunization studies, these vaccine candidates elicited very high titers of anti-P1 IgG and IgG2a, anti-P2 IgG and IgG2a, and anti-N IgG and IgG2a. In the live virus neutralization assays, high virus neutralizing antibody levels were observed, and by obtaining specific interferon-gamma (INF-gamma) from immunized mouse splenocytes, we proved that a good cellular immunity was achieved too. These findings, overall, validated high immunogenicity of the P1 and P2 proteins of the S region and the N protein to order to develop an effective vaccine candidate against SARS CoV-2 infection. In addition, a variant recombinant protein was designed by our group and prepared in the same way. This protein that contains the most important point mutations of the known variants is being intended to be incorporated into our candidate vaccine. Our studies are being conducted in this direction and we aim at starting Phase I trials as soon as possible. In conclusion, such alterations/adaptations in vaccine formulations are highly important to optimize current vaccines. In order to reduce the worldwide effects of SARS-CoV-2 variants, it is necessary to develop new generation COVID-19 vaccines besides an urgent elimination of the vaccine inequality all over the world.
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Introduction: Introduction: Euglycemic diabetic ketoacidosis (EuDKA) is a rare but increasingly reported serious adverse effect of SGLT2 inhibitors. There is not much published literature on the incidence of EuDKA and the factors associated with it. Though SGLT2 inhibitors were introduced as glucose lowering agents, recent trials have demonstrated their favourable cardiovascular outcome in heart failure and ability to retard progression of proteinuric kidney disease, including in non-diabetics. Hence use of this class of drugs is anticipated to increase exponentially, given the combined high global burden of diabetes, coronary artery disease and chronic kidney disease. As we embark to use the SGLT2 inhibitors in different clinical scenarios, it becomes imperative to report their adverse effects encountered in uncommon clinical conditions as well. Methods: Case history: A 42-year-old gentleman with history of type 2 diabetes mellitus for 15 years and coronary artery disease, presented with difficulty in climbing stairs and walking for 5 days with progressive difficulty in getting up from bed. He did not have past history of covid infection and had been immunised with 1 dose of covid vaccine. On examination, patient had normal hemodynamics. There was flaccid quadriparesis with areflexia and truncal muscle weakness. Nerve conduction study confirmed acute demyelinating polyradiculoneuropathy. His baseline laboratory investigations revealed normal renal parameters but metabolic acidosis was noted at the time of admission. Patient was started on iv immunoglobulin 2mg/kg and the motor weakness improved from grade 2/5 to 4/5. However, the high anion gap metabolic acidosis worsened over the next 4 days and patient developed acidotic breathing. His sugars were within normal limits and the patient was on metformin, glimeperide, vildagliptin, voglibose and dapagliflozin. As blood lactate levels were normal with urine acetone positivity, euglycemic diabetic ketoacidosis secondary to SGLT2 inhibitor was suspected and all the oral hypoglycemic agents were stopped. He was started on hydration and insulin infusion. After 48 hours of stopping dapagliflozin, acidosis resolved completely and the patient was reintroduced back on the other 4 class drugs. At follow-up, there was no recurrence of acidosis and patient was able to walk with support and physiotherapy. [Formula presented] Results: Discussion: SGLT2 inhibitors cause glycosuria and directly induce glucagon release from pancreas. Combined with insulin deficiency, this results in lipolysis, fatty acid oxidation and ketogenesis. They also cause increased renal reabsorption of ketone bodies. The precipitating factors for EuDKA identified so far include abrupt reduction in insulin dosage, reduced oral intake, infections, surgery, excess alcohol use, volume depletion, type 1 diabetes and heavy physical exercise. This is the first reported case of SGLT2 inhibitor-induced EuDKA in a patient with Guillain-Barre syndrome. As symptoms of dehydration may not be significant due to lack of hyperglycemia in EuDKA, there may be a delay in the diagnosis of this complication. Conclusions: Conclusion: The possibility of EuDKA to be kept in mind while evaluating metabolic acidosis in a diabetic patient on SGLT2 inhibitors. Temporarily withholding the SGLT2 inhibitors during an intercurrent illness will prevent the occurrence of the above serious adverse effect. No conflict of interest