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@#Objective To develop a method for determination of activity and viability of Hansenula polymorpha by dual fluorescent staining with carboxyfluorescein diacetate(CFDA)and propidium iodide(PI).Methods The time durations(5,10,20,40,60 and 120 min for CFDA,5,10,15,20 and 25 min for PI)and working solution concentrations(50,100,200and 300 μg for CFDA,2,10,20 and 30 μmol/L for PI)for the dual fluorescent staining were optimized by single factor test. Under the optimal condition,the H.polymorpha samples at theoretical survival rates of 0,25%,50%,75% and 100%were determined,of which the fluorescent intensity was observed under fluorescent microscope,and the gray value was analyzed by ImageJ software. The live and dead cells were counted,based on which the actual survival and death rates were calculated. Meanwhile,the relationships of actual gray value,actual survival rate and actual death rate to the corresponding theoretical values were analyzed. Activity and viability of three batches of cultured H.polymorpha were detected by CFDA-PI dual fluorescence staining.Results The optimal time durations for staining with CFDA and PI were 60 and 5 min,while the optimal working solution concentrations were 200 and 2 μmol/L,respectively. The actual gray value,actual survival rate and actual death rate of H.polymorpha samples at various theoretical survival rates were significantly correlated to the corresponding theoretical values(R~2=0. 998 3～0. 999 2,P < 0. 05). The CVs of activity and viability values in three detections of three batches of H.polymorpha culture were 3. 20%～4. 03% and 1. 10%～2. 27%, respectively.Conclusion The CFDA-PI dual fluorescent staining was successfully developed,which may be used for determination of activity and vitality of H.polymorpha.
ABSTRACT
Objective] To assess the safety and immunogenicity of booster vaccination 10 μg of recombinant Hansenula hepatitis B vaccine in school-age children , and to provide scientific basis for the development of hepatitis B immunization strategies . [ Methods] A total of 2 101 children were selected as the safety study object and were inoculated with three injections of 10 μg Hansenula hepatitis B vaccine for observation of the incidence of abnormal reaction .Among them 404 schoolchildren were observed for immune effects.And comparison was made in the difference of antibody titers and antibody positive rate before and after immunization . [ Results] Of the 2 101 children, 85 cases of adverse reactions occurred and the incidence rate was 4.05%.Of the 85 cases, 84 had slight reactions , one with moderate reactions and none with severe reactions .After vaccination , the positive rate of protective antibodies increased to 100.00%, and antibody titers rose from 2.55 IU/L to 5 051.90 IU/L, the difference being statistically significant (Z =12.51, P =0.00). [Conclusion] Hansenula hepatitis B vaccine used for booster immunization in schoolchildren is of good security and fine immune effect .
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Objective To evaluate the immunogenicity of the recombinant human papillomavirus type 68b (HPV68b) virus-like particles(VLPs)in a mouse model.Methods The L1 protein of HPV type 68b was successful expressed in the Hansenula polymorpha strain (NVSI-68b-1).Processes including purifi-cation and reconstitution were performed to achieve pure HPV 68b VLPs.The purity, morphology and immu-nogenicity of the purified HPV 68 b VLPs were further analyzed .The BALB/c mice were immunized with HPV68b VLPs formulated on aluminum adjuvant .Pseudovirus-neutralizing antibody ( PsV NAb) assay was performed to detect the neutralizing antibodies in serum samples .Results The HPV 68 b L1 VLPs were ob-tained as indicated by the results of SDS-PAGE, Western blot assay , HPLC, electron microscopy and dy-namic light scattering with a high purity of 95%.Transmission electron microscopy and dynamic light scat-tering analysis revealed that the HPV68b L1 VLPs resembled the native virus with an average particle diame-ter of 50 nm.High levels of HPV68b-neutralizing antibodies were detected in serum samples from the mice immunized with HPV68b L1 VLPs.Moreover, a cross-protective efficacy of HPV68b L1 VLPs for HPV68a was observed .Conclusion This study suggested that the recombinant HPV 68 b VLPs expressed in a Han-senula polymorpha strain might be used as a potential candidate for the development of HPV vaccine .
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Objective To express virus-like particles(VLP) of enterovirus 71 (EV71) in Han-senula polymorpha.Methods The coding sequences of P1 and 3CD genes of EV71 were optimized accord-ing to codon usage bias of Hansenula polymorpha for achieving high expression , and then cloned into the ex-pression vector PMV of Hansenula polymorpha .The recombinant expression vector PMV-P1-3CD was trans-formed into Hansenula polymorpha AU 0501 .The transformants were stably cultured in selective medium (Yeast Nitrogen Base) and screened for strains with positive P1 and 3CD genes by PCR.Then an induced cultivation on the recombinant strains were performed in a medium supplemented with methanol to a final concentration of 1.0%and the expressed products were analyzed by SDS-PAGE and Western blot assays to select high expression strains .The high expression strains were cultured in 30 L fermentor and its fermenta-tion products were analyzed by electronic microscope after purification .Results EV71 recombinant expres-sion strains were successfully constructed .The results of SDS-PAGE showed that the expressed products had obvious VP3, VP1, VP0 protein bands with molecular weights of 26×103, 33×103 and 35×103, respective-ly, which were consistent with the expected molecular weight of the fusion proteins .Western blot demonstra-ted that the expressed products could be specifically recognized by the polyclonal antibody against EV 71-VP1 at 33 ×10 3 , indicating its high immunoreactivity .ELISA confirmed that the expression level of EV 71 fermen-tation products was reached to 200 mg/L.Electronic microscope analysis showed that the VLP of recombi-nant EV71 were 24-30 nm in diameter with normal structure .Conclusion The virus-like particles of human enterovirus 71 are successfully expressed in Hansenula polymorpha , which provides a foundation for the fur-ther development of EV 71 VLP vaccine .
ABSTRACT
Hansenula anomala (H. anomaly) is part of the normal flora in the alimentary tract and throat. It has been reported to be an organism causing opportunistic infections in immunocompromised patients. However, cases of fungal arthritis caused by H. anomala are rare. We encountered a case of H. anomala arthritis in a 70-year-old man who was treated with an empirical antibiotic treatment and surgery under the impression of septic arthritis. However, the patient did not improve after antibiotic therapy and surgery. Consequently, knee joint aspiration was performed again, which identified fungal arthritis caused by H. anomala. It was treated successfully with amphotericin B and fluconazole. When treating arthritis patients with diabetes, it is important to consider the possibility of septic arthritis by H. anomala and provide the appropriate treatment.
Subject(s)
Aged , Humans , Male , Amphotericin B/therapeutic use , Antifungal Agents/therapeutic use , Arthritis, Infectious/diagnosis , Fluconazole/therapeutic use , Knee Joint , Mycoses/diagnosis , PichiaABSTRACT
Se revisaron en forma retrospectiva las histórias clínicas de todos los pacientes en los cuales se aisló Hansenula anómala en los hemocultivos realizados en el Laboratorio de Microbiología del Hospital Privado Centro Médico de Caracas, en el lapso de 2 años y medio, comprendido entre agosto 1994 y febrero 1997. De 57 pacientes se obtuvo 64 hemocultivos positivos, aislándose 64 especies de Candida. Hansenula anómala se obtuvo en 15 hemocultivos de 15 pacientes diferentes, representando un 26,3 por ciento del total de pacientes, un 23,4 por ciento del total de las candidas y un 30,6 por ciento de las variedades no albicans. La Candida albicans se aisló en 15 pacientes (26,3 por ciento del total). De los pacientes que presentaron Hansenula anómala en sus hemocultivos, el 100 por ciento cumplieron con criterios clínicos de severidad. El rango de edad estaba comprendido entre 1 mes y 76 años, con una media de 43, de los cuales 6 eran masculinos y 9 femeninos. De este grupo de pacientes, 12 ingresaron con patología abdominal, siendo intervenidos quirúrgicamente 10 de estos. De los tres pacientes restantes, dos ameritaron cirugía no abdominal. Todos los pacientes recibieron antibióticos de amplio espectro, nutrición parenteral total y le fue colocado catéteres centrales. Nueve pacientes (60 por ciento) tuvieron criterios de sepsis. Los días de hospitalización oscilaron entre 7 y 101 días, con un promedio de 37. Doce pacientes ameritaron ingreso a la Unidad de Terapia Intensiva. Se inició Anfotericina B en 8 pacientes, con buena respuesta en el 100 por ciento. Seis pacientes recibieron como terapia inicial Fluconazol: 2 con respuesta satisfactoria; 1 fallecido no relacionado con la infección por Hansenula anómala y los tres restantes ameritaron cambio a Anfotericina B. Un paciente no recibió tratamiento antifúngico desconociéndose su evolución.
Subject(s)
Humans , Male , Adolescent , Adult , Female , Child, Preschool , Child , Middle Aged , Amphotericin B/therapeutic use , Antifungal Agents/therapeutic use , Fluconazole/administration & dosage , Fungemia/complications , Fungemia/pathology , Cross Infection/immunology , Pichia/immunology , Pichia/isolation & purification , Candida/isolation & purification , Infectious Disease Medicine , Medical RecordsABSTRACT
Hansenula anomala is a normal or transient flora of the throat and alimentary tract, and has been reported as an organism causing opportunistic or nosocomial infections in immunocompromised patients, but rarely in immunocompetent hosts. From the review of the literatures, there is no published report on arthritis caused by H. anomala. We experienced a case of H. anomala arthritis in a 59-year old man who had diabetes mellitus and right knee joint swelling with deformity. The findings of magnetic resonance image were consistent with septic arthritis which had bony destruction in distal femur and proximal tibia, abscess formation in joint space, and joint effusion with synovial hypertrophy. A fungus was isolated from a couple of blood cultures and knee joint aspiration fluid, and later identified as H. anomala by yeast biochemical cards. He was successfully treated with extensive debridement and fluconazole for 8 weeks.
Subject(s)
Humans , Middle Aged , Abscess , Arthritis , Arthritis, Infectious , Congenital Abnormalities , Cross Infection , Debridement , Diabetes Mellitus , Femur , Fluconazole , Fungi , Hypertrophy , Immunocompromised Host , Joints , Knee Joint , Pharynx , Pichia , Tibia , YeastsABSTRACT
BACKGROUND/AIMS: The introduction of Hansenula polymorpha for recombinant hepatitis B vaccine production allowed high product yield with plasmid stability and less glycosylation than conventional Saccharomyces cerevisiae system. A Green Cross HG-II vaccine formulated from HBsAg produced by a recombinant strain of the yeast H. polymorpha was evaluated for immunogenicity and safety in an open label triaL METHOFD: A 20 ug dose of Green Cross HG-II vaccine was administered intramuscularly at 0, 1 and 6 months at the deltoid region in 118 healthy adults seronegative for HBV markers. The anti-HBs titers were determined at one month after administration of the third dose of vaccine by radioimmunoassay. RESULTS: The seroconversion rate was 96.8% (90 out of 93), with seroprotective rate of 95.7% (89 out of 93). The geometric mean titers(GMT) of the anti-HBs response was 153.1mIU/ml in seroconverters. An age-dependent effect was observed in the anti-HBs response. But sex-dependent effect was not prominent. Reactogenecity was in incidence and general reactions were short-lasting and a mainly mild in severity. CONCLUSIONS: The results of this study have shown that the Green Cross HG-II vaccine is safe and clinically well tolerated, a nd that it may provide protection against HBV infection.