AT-2灭活HIV-1病毒及纯化回收鉴定

目的制备具备完整空间构型且纯度在90%以上的灭活HIV-1病毒,用于HIV疫苗研究。方法应用化学制剂2,2’-dithiodipyridine（aldrithiol-2;AT-2）灭活HIV-1病毒,对灭活的病毒超速离心浓缩并洗涤去除灭活用的化学制剂AT-2。采用分子筛技术去除灭活病毒中残存的杂质蛋白质。结果 250μmol/L AT-2与HIV-137℃作用1 h可以彻底灭活病毒的感染性,同时保留病毒的免疫原性。灭活的病毒纯化后检测不到AT-2的残留,检测牛血清蛋白残余量低于50 ng/mL。结论灭活的HIV-1病毒经过纯化后纯度达到95.6%,可以满足作为HIV疫苗研究的免疫刺激剂的使用要求。     

Replication-defective mutants of herpes simplex virus (HSV) induce cellular immunity and protect against lethal HSV infection.

Live viruses and live virus vaccines induce cellular immunity more readily than do inactivated viruses or purified proteins, but the mechanism by which this process occurs is unknown. A trivial explanation would relate to the ability of live viruses to spread and infect more cells than can inactivated virus. We have used live but replication-defective mutants to investigate this question. Our studies indicate that the immune responses of mice to live virus differ greatly from the responses to inactivated virus even when the virus does not complete a replicative cycle. Further, these studies indicate that herpes simplex virus-specific T-cell responses can be generated by infection with replication-defective mutant viruses. These data indicate that the magnitude of the cellular immunity to herpes simplex virus may be proportional to the number or quantity of different viral gene products expressed by an immunizing virus.     

Viricidal effects of Lactobacillus and yeast fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40 degrees C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30 degrees C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20 degrees C but was inactivated more rapidly at 30 and 40 degrees C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20 degrees C and day 6 at 30 degrees C. The porcine picornavirus was inactivated by day 7 at 30 degrees C but survived the entire test period at 20 degrees C. Frog virus 3 was inactivated by day 3 at 20 degrees C and day 2 at 30 degrees C.     

Intranasal immunization with inactivated influenza virus enhances immune responses to coadministered simian-human immunodeficiency virus-like particle antigens

Intranasal immunization with inactivated influenza virus vaccine can provide protective immunity, whereas many other antigens are less effective when used for mucosal immunization. To determine whether influenza virus could enhance immune responses to an antigen coadministered to a mucosal surface, we studied the intranasal immunization of mice with a mixture of simian-human immunodeficiency virus (SHIV) virus-like particles (VLPs) and inactivated influenza virus. Compared to mice immunized with SHIV VLPs alone, mice coimmunized with SHIV VLPs and inactivated influenza virus showed significant increases in serum immunoglobulin G (IgG) and mucosal IgA antibodies specific to the human immunodeficiency virus envelope protein, neutralizing activities, numbers of gamma interferon- and interleukin 4-secreting lymphocytes, and cytotoxic-T-lymphocyte activities. The levels of enhancement of immune response by coimmunization with inactivated influenza virus were equivalent to those induced by inclusion of immunostimulatory CpG oligodeoxynucleotides (CpG DNA). We also observed that SHIV VLPs bind to influenza virus virions, forming mixed aggregates. These results indicate that inactivated influenza virus can play a role as a mucosal adjuvant to coadministered antigens.     

Inactivation and removal of influenza A virus H1N1 during the manufacture of plasma derivatives

Although transmission of pandemic influenza A virus H1N1 2009 is still occurring globally, little has been reported about how this outbreak has affected the safety of plasma derivatives. To evaluate the safety of plasma derivatives, dedicated virus clearance processes used during their production were investigated for their effectiveness in eliminating this virus of recent concern. In this study, influenza A virus H1N1 strain A/NWS/33 (H1N1) was chosen as a surrogate. H1N1 was completely inactivated by fraction IV fractionation as well as pasteurization during the manufacture of albumin. H1N1 was also effectively removed into the precipitate by fraction III fractionation and completely inactivated by low pH incubation as well as pasteurization during the manufacture of intravenous immunoglobulin. H1N1 was completely inactivated within 1 min of solvent/detergent treatment using 0.3% tri (n-butyl) phosphate and 1.0% Triton X-100 and also completely inactivated within 10 min of dry-heat treatment at 98 °C during the manufacture of factor VIII. H1N1 was completely removed by virus filtration process using Viresolve NFP filter and also completely inactivated by pasteurization during the manufacture of anti-thrombin III. These results indicate that all the virus clearance processes commonly used have sufficient H1N1 reducing capacity to achieve a high margin of safety.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Viricidal Effects of Lactobacillus and Yeast Fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40°C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30°C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20°C but was inactivated more rapidly at 30 and 40°C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20°C and day 6 at 30°C. The porcine picornavirus was inactivated by day 7 at 30°C but survived the entire test period at 20°C. Frog virus 3 was inactivated by day 3 at 20°C and day 2 at 30°C.     

Whole inactivated SIV vaccine grown on human cells fails to protect against homologous SIV grown on simian cells

Several groups have reported protection against experimental SIV infection in macaques immunized with a whole inactivated virus vaccine. The aim of the current study was to investigate whether five macaques vaccinated with whole inactivated SIV and previously shown to be protected against challenge with two divergent strains of SIV grown on human cells could resist challenge with a subsequent homologous SIV grown on macaque cells. We show here that this same vaccine did not protect when the challenge virus was grown on primary cells of monkey origin.     

Adjuvant effects of dimethyl dioctadecyl ammonium bromide, complete Freund's adjuvant and aluminium hydroxide on neutralizing antibody, antibody-isotype and delayed-type hypersensitivity responses to Semliki Forest virus in mice.

Outbred mice were inoculated subcutaneously with inactivated Semliki Forest virus (SFV) in saline and combinations of the virus with complete Freund's adjuvant (CFA) aluminium hydroxide (Al) and dimethyl dioctadecyl ammonium bromide (DDA). The immune response was evaluated for delayed-type hypersensitivity, for total ELISA antibodies and antibody-isotypes and for neutralizing antibodies. DDA was the most efficient adjuvant in inducing DTH, CFA the second and Al induced a DTH response that was only slightly higher (statistically not significant) than that induced by the inactivated virus without adjuvants. All adjuvants enhanced the production of ELISA antibodies to similar levels. However, the levels of neutralizing antibodies induced were low in mice which were inoculated with the inactivated SFV alone or mixtures of the virus with Al. DDA induced high levels of neutralizing antibodies and CFA induced intermediate levels. The pattern of antibody-isotypes induced by DDA and CFA was different from the pattern induced by the inactivated virus or by the virus mixed with Al: DDA and CFA induced low amounts of IgG1 antibodies and relatively higher amounts of IgG2a and IgG2b antibodies while the inactivated virus and the mixture of the virus with Al induced higher proportions of IgG1 antibodies. In sera from convalescent mice the majority of antibody activity resided in the IgG2a and IgG2b immunoglobulin subclasses, while IgG1 antibodies were undetectable.     

Preparation of a purified, inactivated Japanese encephalitis (JE) virus vaccine in Vero cells

An attenuated Japanese encephalitis (JE) virus SA14-14-2 (PDK) was adapted to Vero cells, a continuous cell line that has been licensed for human vaccine production, by serial passages. The resulting virus was purified by tangential flow ultrafiltration followed by sucrose density gradient ultracentrifugation, giving 2.3 mg purified virus per liter of culture supernatant. Treatment with 0.05% formalin for 4 days at 22 °C completely inactivated viral infectivity while preserving its antigenicity. The purified, inactivated JE virus was formulated with alum hydroxide and administered to mice by intraperitoneal route. In terms of its ability to induce anti-JE neutralizing antibody and to protect the immunized animal against neurovirulent virus challenge, the purified, inactivated JE virus formulated with alum was equivalent to the exiting commercial mouse brain-derived vaccine (JE-VAX, Aventis Pasteur Inc.).     

Critical Arginine Residue for Maintaining the Bacteriophage Tail Structure

The addition of 0.2 m l-arginine to various T-even bacteriophage preparations inactivated the virus preparations irreversibly. The virus particles were even more sensitive to added d-arginine and l-homoarginine than to l-arginine but were unaffected by arginine analogues with either an altered carboxyl group or guanidyl group. Treatment of phage T2H with 2,3-butanedione, a reagent which specifically reacts with the guanidyl portion of arginine residues, resulted in the apparent in-activation of most of the virus particles. However, after incubation of the treated particles at pH 7.5 at 37 C for 1 hr in the absence of butanedione, the original virus titer almost completely returned. The reactivation was completely inhibited by the presence of 0.2 m d-arginine. It appeared that the virus protein coat was sufficiently plastic so that the initial conformational change resulting from the alteration of an arginine residue (to possibly an ornithine residue) was at least partially reversible and that the virus tail proteins then refolded to produce a stable and active virus particle. These reactivated virus particles were not sensitive to inactivation by d-arginine but could now be rapidly inactivated by l-ornithine. Virus particles inactivated by arginine have altered tail structures. They have contracted tail sheaths still attached to tail plates and still contain tail cores. These properties of virus particles indicate that there is a free carboxyl group and a guanidyl group spatially equivalent to an arginine residue on one component of the virus tail which bind reversibly by means of polar linkages to another tail component. These bonds maintain the integrity of the virus tail. Added arginine appears to compete with this endogenous viral arginine for the binding sites and then to favor an irreversible conformational change.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine.

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.     

Preparation of FMD type A87/IRN inactivated vaccine by gamma irradiation and the immune response on guinea pig

FMD is one of the most economically damaging diseases that affect livestock animals. In this study FMD Virus type A87/IRN was multiplied on BHK21 cells. The virus was titrated by TCID50 method, it was 10^7.5/ml. The FMD virus samples were inactivated by gamma ray from 60Co source at −20°C. Safety test was done by IBRS2 monolayer cell culture method, also antigenicity of irradiated and un-irradiated virus samples were studied by Complement Fixation Test. The Dose/Survival curve for irradiated FMD Virus was drawn, the optimum dose range for inactivation of FMDV type A87/IRN and unaltered antigenicity was obtained 40–44 kGy. The inactivated virus samples by irradiation and ethyleneimine (EI) were formulated respectively as vaccine with Al(OH)3 gel and other substances. The vaccines were inoculated to Guinea pigs and the results of Serum Neutralization Test for the normal vaccine and radio-vaccine showed protective titer after 8 months. The potency test of the inactivated vaccines was done, PD50 Value of the vaccines were calculated 7.06 and 5.6 for inactivated vaccine by EI and gamma irradiation respectively.     

Effects of Temperature and pH on Survival of Free Nuclear Polyhedrosis Virus of Autographa californica

The effects of temperature and low pH on replication and survival of nonoccluded Autographa californica nuclear polyhedrosis virus were investigated. No virus replication or formation of polynuclear inclusion bodies occurred at 37°C. The virus was immediately inactivated upon exposure to pH 2.0 and was inactivated within 1 h at pH 4.0. The virus titer slowly declined, a 3-orders of magnitude reduction in virus titer, at pH 5.0 during a 4-h exposure. Virus survival at pH 6.0 was equal to that of the control in cell culture medium 199 MK (pH 7.12).     

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.     

Leucocyte migration inhibition test as an index of immunological response to measles virus II. Immunogenic activity of measles virus.

Experiments discussed in this paper aimed at obtaining, using Cercopithecus aethiops monkeys information on immunogenic activity of measles virus as well as kinetic and persistence of immunological reaction at active and inactivated virus.     

A dot-immunobinding assay on nitrocellulose with psoralen inactivated Herpesvirus simiae (B virus)

An enzyme-immunoassay performed with Herpesvirus simiae (B virus) and H. simplex antigens inactivated with a psoralen derivative and long-wavelength ultraviolet light irradiation is described. Although B virus is a known human pathogen requiring extreme care in its handling, the use of inactivated antigens in the test allows its performance without biosafety containment. The test utilizes nitrocellulose sheets dotted with antigen for the assay of antibody against B virus in nonhuman primate sera. Antigen-antibody complexes are detected visually as red dots by the use of enzyme-conjugated antiglobulin second antibody and a substrate that produces an insoluble product. The test is more rapid, sensitive and specific than the serum neutralization test it is intended to replace. Of 150 macaque monkey sera tested, 83 were negative by the enzyme and neutralization tests, 56 were positive by both tests and 11 were positive by enzyme-assay but negative by neutralization. Positive sera reacted with both simian and human viral antigens in the enzyme assay but with greater intensity against B virus. Absorption with H. simplex removes reactivity with this virus without reducing the B virus response.     

Adjuvant effect ofBacillus firmus in intranasal immunization of guinea pigs with inactivated type B influenza virus

Intranasal immunization of guinea pigs with inactivated type B influenza virus plus inactivated Bacillus firmus as an adjuvant compared to the virus alone yields higher titers of serum hemagglutination-inhibiting antibodies and virus-neutralizing antibodies. This phenomenon could be useful in standard serology, especially in the preparation of immune sera against highly pathogenic strains for in vitro diagnosis.     

Hog Cholera Antibodies in Pigs Vaccinated with an Aujeszky-Vaccine Based on Antigen Produced in IB-RS-2 Cells

The cell line IB-RS-2 is confirmed to be persistently infected with hog cholera virus. Vaccination of pigs against Aujeszky’s Disease with an inactivated vaccine based on antigen produced on this cell line induced neutralizing antibodies against hog cholera virus.