Moist Heat Disinfection of HBV
| Krugman and co-workers | Kobayashi and co-workers | Lelie and co-workers | ||
|---|---|---|---|---|
| Year of Publication | 1979 | 1984 | 1987 | |
| Initial serum of HBV | ||||
| Strain | MS-2 | JHB001 | NYBC 78-564 | |
| Subtype | ayw | adr | adw | |
| Infectivity (units/mL) | 107.5 | 108 | 107 | |
| Heat-inactivated sample | Inoculum II | Inoculum III | ||
| Dilution of initial serum | 1:10 using sterile distilled water | 1:1000 using phosphate buffer | Inoculum I*** was spiked using HBV serum (NYBC 78-564) so that the final infectivity titer would be 105 CID50/mL. This HBV-spiked solution underwent moist heat disinfection at 103 °C for 90 s. This heat-inactivated solution was ultracentrifuged and was sterile-filtered to produce inoculum II. | Inoculum I*** underwent moist heat disinfection at 103 °C for 90 s under production circumstances and was diluted in PBS so that the protein concentration would be 1 mg/mL. This protein solution was spiked using HBV serum (NYBC 78-564) so that the final infectivity titer would be 105 CID50/mL. This HBV-spiked protein solution underwent moist heat disinfection at 65 °C for 10 h to produce inoculum III. |
| Volume | 20 mL | 1 mL | 50 mL | 50 mL |
| Infectivity titer (CID50/mL) | 106.5 | 105 | 105 | 105 |
| Heat-inactivation method | ||||
| Heating | Electric burner | Liquid paraffin bath at 98 °C | Oil bath | Water bath at 65 °C |
| Cooling | NA | Ice water bath | Ice bath | NA |
| Thermometry | Thermometer | Thermocouple | Data-logger apparatus (Kaye Instruments) | Data-logger apparatus |
| Come-up phase | 43 s | 4 min | 4340 as A0 value | NA |
| Holding time phase | 98 °C for 1 min (A0 value: 3786) | 98 °C for 2 min (A0 value: 7571) | 19762 as A0 value (Temperature range: 103.0 to 104.1 °C) | 65 °C for 10 hr (A0 value: 1138) |
| Come-down phase | 25 min to the room temp | Cooled immediately | 763 as A0 value | NA |
| Estimated A0 value | Conservatively estimated as 3786 | Conservatively estimated as 7571 | 24865 | 1138 |
| Innoculation | ||||
| Inoculated volume | 0.1 mL | 1 mL | 10 mL | 10 mL |
| Route | Intramascularly | Intravenously | Intravenously | |
| Inoculated subjects | 29 susceptible human volunteers | Two susceptible chimpanzees | Two susceptible chimpanzees | |
| Comments | With informed consents conformed to the World Medical Association's Draft Code of Ethics on Human Experimentation (1961) | A male weighing 10.3 kg and a female weighing 8.8 kg from West Africa. Both were negative for HBsAg, anti-HBs and anti-HBc (RIA). | Two wild-caught chimpanzees aged 3–5 yr and negative for HBsAg, anti-HBs and anti-HBc (RIA). The mixture of inoculum II and III was administered to each of the two susceptible chimpanzees. | |
| Results | ||||
| Follow-up period | 8 and a half months | 30 weeks | 28 weeks | |
| Liver blood chemistry | All normal | All normal | All normal | |
| HBsAg (CF or RPHA) | All negative* | NA | NA | |
| HBsAg (RIA) | 26 negatives & 3 positives** | All negative | All negative | |
| anti-HBs (RIA) | NA | All negative | All negative | |
| anti-HBc (RIA) | NA | All negative | All negative | |
| Liver biopsy | NA | All normal | All normal | |
| Comments | *Reported in 1971 and 1973, using the test methods available at the time. **Report in 1979. The stored samples were retested using more sensitive radioimmunoassay method (Ausria II). | Liver blood chemistry tests: total protein, bilirubin, alkaline phosphatase, asparate and alanine aminotransferases, lactic dehydrogenase, γ-glutamine transpeptidase, cholinesterase, leucine aminopeptidase, and triglycerides. HBV markers were tested by radioiimunoassay (Ausria II, Ausab, and Corab, respectively; Abbott Laboratories, North Chicago, IL.) | ***HBV infectivity was eliminated using chemical purification step followed by two steps of moist heat disinfection at 103 °C for 90 s and at 65 °C for 10 h, respectively. Inoculum I, II, and III were prepared under the same circumstances to produce the vaccine. HBV serum (NYBC 78-564) was diluted using 20 L of plasma negative for HBV markers as the starting material. Inoculum I was the ultracentrifuge resuspension in PBS. Elimination of HBV infectivity in inoculum I was confirmed using two chimpanzees. | Liver blood chemistry tests: ALT (alanine aminotransferases) and AST (asparate aminotransferases) detected by kinetic spectrophotometric assay with an Abbott ABA-100 Analyzer using Abbott reagents and reference standards. HBV markers were tested by radioimmunoassay (Ausria II, Ausab, Corab, Abbott Labs). |
NA: not available, CF: complement fixation, RPHA: reverse passive hemaglutination, RIA: radioimmunoassay, HBsAg: hepatitis B antigen, anti-HBs: hepatitis B antibody, anti-HBc: antibody to hepatitisB core antigen. (References 15–22).