Immune response of various animals to Akabane disease live virus vaccine

When various animals and routes of inoculation were examined for antibody response to Akabane disease live virus vaccine, the intracerebral (ic) inoculation of mice induced a better antibody response than the subcutaneous (sc) inoculation of calves, guinea pigs, hamsters, mice, or rats. Immunogenicity was compared among lots of this vaccine by performing ic inoculation of mice and sc inoculation of calves and guinea pigs. As a result, there was no distinct significant difference between any two lots of the vaccine, regardless of the animal species used. There was a tendency that the larger the dose of inoculation of the virus, the earlier the production of neutralizing (NT) antibody took place in calves inoculated with the vaccine, and the higher the antibody titer and the rate of taking a turn for positivity for antibody became in these calves. When calves immunized with the vaccine and cows in the field possessing NT antibody were given booster inoculation with the vaccine, the antibody titer showed a significant increase in almost all the calves and cows that exhibited an NT antibody titer of 4 or less at the time of booster inoculation. There were, however, no changes in antibody titer in such calves and cows as presenting an NT antibody titer of 8 or more. Calves and pregnant cows immunized with the vaccine were prevented from viremia and fetal infection when challenged by inoculation with virulent virus.     

Effect of HLA on the cellular immune response to varicella-zoster virus

The effect of HLA on varicella-zoster virus (VZV)-specific lymphocyte transformation (LTF) was studied in 100 normal immune adults and 64 children who were immunized with live attenuated varicella vaccine. In the normal adults, a statistically significant association was observed between low responsiveness and the presence of A2 (p less than 0.025), and also between high responsiveness and the presence of Aw24 (p less than 0.05). A similar but clearer association, i.e. low responsiveness with A2 (p less than 0.005) and high responsiveness with Aw24 (p less than 0.025), was observed in the vaccinated children. In these children, Aw31 was also found to be related to low responsiveness (p less than 0.05). These results suggest that the VZV-specific cellular immune response is in some way influenced by HLA.     

Leishmania major: species specific delayed hypersensitivity reaction induced by exogenous secreted antigen in the guinea pig

The cellular response to Leishmania major (L. major) is usually evaluated in vivo by the delayed-type-hypersensitivity (DTH) test using leishmanin. Leishmanin can give false-positive reactions in areas where there is a background of leishmaniasis. In a previous study, it was shown that a 56 kDa antigen purified from promastigote and culture supernatant of L. major induce strong DTH reactions in sensitized guinea pigs. In this study, the species-specificity of this antigen was further investigated. Three groups of guinea pigs were sensitized with L. major, L. tropica, and L. infantum and both flanks of sensitized animal were injected intradermally with purified 56 kDa antigen or soluble leishmania antigen (SLA). The extent of indurations were measured after 24, 48, and 72 h. In animals which were sensitized with three species of leishmania, only those immunized with L. major showed skin reactions to purified antigen by an increase in skin thickness. Since complex antigen mixtures such as SLA and leishmanin show cross-reactivity and can be non-specific, the result obtained here suggest that 56 kDa antigen may be a useful diagnostic tool for species specific diagnosis in field studies of leishmaniasis.     

Immunization with the immediate-early tegument protein (open reading frame 62) of varicella-zoster virus protects guinea pigs against virus challenge.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.     

乙型脑炎减毒活疫苗(SA_(14)-14-2)在豚鼠体内保护作用的免疫机制的研究

本实验将乙脑减毒活疫苗ＳＡ_（１４）－１４－２株以不同疫苗病毒量（３．８７ＰＦＵ／ｍｌ和５．８７ＰＦＵ／ｍｌ）分别一次免疫豚鼠,观察其对强毒攻击后抑制毒血症和抗体形成的能力。结果显示疫苗（５．８７ＰＦＵ／ｍｌ）免疫组豚鼠攻击前虽然中和抗体阴性或很低,但经攻击感染后不同时间内均未出现病毒血症,对照组豚鼠则于第２,３,４天全部出现病毒血症。表明一次活疫苗免疫后能有效地抑制病毒血症的产生。免疫后３０天虽然免疫组的豚鼠中和抗体很低,但攻击感染后抗体迅速增长。第四天的抗体滴度为１：８～３２,第５天达１：１２８～２５６,第１４天抗体高达１：５１２～１０２４;而对照组抗体则上升很慢,第７天才出现低水平抗体（１：４）。血凝抑制抗体增长的动态与中和抗体近似。表明活疫苗免疫后虽然中和抗体水平不高,但一经感染可迅速产生高滴度抗体达到保护作用。     

Immunization with recombinant varicella-zoster virus expressing herpes simplex virus type 2 glycoprotein D reduces the severity of genital herpes in guinea pigs.

Varicella-zoster virus (VZV) is an attractive candidate for a live-virus vector for the delivery of foreign antigens. The Oka vaccine strain of VZV is safe and effective in humans, and recombinant Oka VZV (ROka) can be generated by transfecting cells with a set of overlapping cosmid DNAs. By this method, the herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) gene was inserted into an intergenic site in the unique short region of the Oka VZV genome. Expression of gD2 in cells infected with the recombinant Oka strain VZV (ROka-gD2) was confirmed by antibody staining of fixed cells and by immunoblot analysis. Immune electron microscopy demonstrated the presence of gD2 in the envelope of ROka-gD2 virions. The ability of ROka-gD2 to protect guinea pigs against HSV-2 challenge was assessed by inoculating animals with three doses of uninfected human fibroblasts, fibroblasts infected with ROka VZV, or fibroblasts infected with ROka-gD2. Neutralizing antibodies specific for HSV-2 developed in animals immunized with ROka-gD2. Forty days after the third inoculation, animals were challenged intravaginally with HSV-2. Inoculation of guinea pigs with ROka-gD2 significantly reduced the severity of primary HSV-2 infection (P < 0.001). These experiments demonstrate that the Oka strain of VZV can be used as a live virus vector to protect animals from disease with a heterologous virus.     

Development and characterization of a live varicella vaccine (Oka strain)

A live varicella vaccine (Oka strain) was developed by serial passage of the Oka strain isolated in our laboratory, in human embryonic lung cells (11 times at 34 C) and guinea pig embryo cells (12 times at 37 C). It is slightly temperature sensitive at 39 C and shows a higher ratio of infectivity in guinea pig embryo cells to infectivity in human embryo cells than wild-type strains. The DNA digest with Hpa I enzyme of the Oka strain contained one unique fragment (K), although its mobility differed only slightly from that of the corresponding fragment of wild-type strains. Studies with clinical varicella zoster virus (VZV) isolates from vaccinees indicated that tests on the ratio of infectivity in guinea pig embryo fibroblasts (GPEF) to that in human embryo fibroblasts (HuEF) and the profile of the DNA digest with Hpa I are useful for differentiation of the vaccine strain from wild-type strains. The vaccine virus showed stable immunogenicity during at least 15 further repeated passages in human diploid cells, a character which seems helpful for production of a large quantity of vaccine virus for practical use.     

Varicella vaccination: evidence for frequent reactivation of the vaccine strain in healthy children

Wild-type varicella zoster virus (VZV) causes chickenpox, a common childhood illness characterized by fever and a vesicular rash and rare serious complications. Wild-type VZV persists in a latent form in the sensory ganglia, and can re-activate to cause herpes zoster. More than 10 million American children have received the live attenuated Oka strain VZV vaccine (OkaVZV) since its licensure in 1995. Pre-licensure clinical studies showed that mean serum anti-VZV levels among vaccinees continued to increase with time after vaccination. This was attributed to immunologic boosting caused by exposure to wild-type VZV in the community. Here, we examine the alternative, that large-scale asymptomatic reactivation of OkaVZV might occur in vaccinees. We analyzed serum antibody levels and infection rates for 4 years of follow-up in 4,631 children immunized with OkaVZV. Anti-VZV titers decreased over time in high-responder subjects, but rose in vaccinees with low titers. Among subjects with low anti-VZV titers, the frequency of clinical infection and immunological boosting substantially exceeded the 13%-per-year rate of exposure to wild-type varicella. These findings indicate that OkaVZV persisted in vivo and reactivated as serum antibody titers decreased after vaccination. This has salient consequences for individuals immunized with OkaVZV.     

Novel polyvalent live vaccine against varicella–zoster and mumps virus infections

The varicella–zoster virus (VZV) Oka vaccine strain (vOka) is a highly immunogenic and safe live vaccine that has long been used worldwide. Because its genome is large, making it suitable for inserting foreign genes, vOka is considered a candidate vector for novel polyvalent vaccines. Previously, a recombinant vOka, rvOka‐HN, that expresses mumps virus (MuV) hemagglutinin‐neuraminidase (HN) was generated by the present team. rvOka‐HN induces production of neutralizing antibodies against MuV in guinea pigs. MuV also expresses fusion (F) protein, which is important for inducing neutralizing antibodies, in its viral envelope. To induce a more robust immune response against MuV than that obtained with rvOka‐HN, here an rvOka expressing both HN and F (rvOka‐HN‐F) was generated. However, co‐expression of HN and F caused the infected cells to form syncytia, which reduced virus titers. To reduce the amount of cell fusion, an rvOka expressing HN and a mutant F, F(S195Y) were generated. Almost no syncytia formed among the rvOka‐HN‐F(S195Y)‐infected cells and the growth of rvOka‐HN‐F(S195Y) was similar to that of the original vOka clone. Moreover, replacement of serine 195 with tyrosine had no effect on the immunogenicity of F in mice and guinea pigs. Although obvious augmentation of neutralizing antibody production was not observed after adding F protein to vOka‐HN, the anti‐F antibodies did have neutralizing activity. These data suggest that F protein contributes to induction of immune protection against MuV. Therefore this recombinant virus is a promising candidate vaccine for polyvalent protection against both VZV and MuV.     

Cell-mediated immunity following experimental vaccinations with Candida albicans ribosomes

The aim of the present study was to ascertain whether cell-mediated immunity (CMI) is induced in animals by vaccination with Candida albicans ribosomes. Delayed type hypersensitivity (DTH) was detected in vivo in ribosome-vaccinated mice and guinea pigs by the footpad swelling and skin tests, respectively. The observed DTH was similar to that induced by live C. albicans organisms. A lymphocyte transformation assay was used for in vitro detection of CMI. The tritiated thymidine incorporation assays revealed that spleen lymphocytes from mice immunized with C. albicans ribosomes were stimulated by the ribosomal antigen. The findings establish that C. albicans ribosomes are able to induce CMI in experimental animals.     

Inactivated Eastern Equine Encephalomyelitis Vaccines Prepared in Monolayer and Concentrated Suspension Chick Embryo Cultures

Eastern equine encephalomyelitis vaccines were prepared with virus propagated in stationary monolayer cultures and in concentrated suspension cultures of primary chick embryo cells. Virus pools for vaccine preparation were inactivated by three different methods: 0.05% formalin, 41 C heat, and 0.16% beta-propiolactone. Heat-and beta-propiolactone-inactivated vaccines maintained high hemagglutinating titers in the fluid state for at least 10 months, whereas formalin-inactivated vaccines lost their hemagglutinating activity within a few hours after treatment. The hemagglutinin of beta-propiolactone-inactivated virus particles was more dense than the hemagglutinin of the parent virus particles, as determined by sucrose density gradient centrifugation. The increase in density may be due to the degradation or removal of the lipid from the virus envelope. When administered to guinea pigs, all three vaccines stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibodies and afforded protection against a live virus challenge. Test results showed that vaccines prepared with virus propagated in concentrated suspension cultures were more immunogenic and stimulated greater serologic responses in guinea pigs than vaccines derived from monolayer-propagated virus. The beta-propiolactone-inactivated vaccine was most protective, the heat-inactivated (41 C) vaccine next, and the formalin-inactivated vaccine least potent.     

T lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunization with live attenuated varicella vaccine

Varicella-zoster virus (VZV) specific cytotoxicity was investigated during acute primary VZV infection, in naturally immune subjects and after vaccination with the live attenuated varicella vaccine by using T cell cultures (TCC) generated by stimulating PBMC with VZV Ag and autologous VZV-superinfected lymphoblastoid cell lines as targets. Lysis of VZV-infected lymphoblastoid cell lines was observed by TCC from acutely infected subjects, naturally immune subjects, and recipients of the varicella vaccine. VZV glycoprotein I induced cytotoxic T cells but killing was less efficient than killing by TCC stimulated with VZV Ag. The TCC were primarily CD4+ (mean 86.6%) T lymphocytes with 15.2% of the cells coexpressing Leu-19. TCC were predominantly restricted by HLA class II as demonstrated by lack of any blocking using class I mAb and blocking of 15 to 71% by L243, a mAb to class II. Unrestricted killing as measured by killing of K562 cells occurred in all TCC but was minimally greater than that observed against uninfected autologous targets. Phenotypes of PBMC during acute infection had an initial increase in CD4+ cells and an overall decrease in the percentage of circulating Leu-11+ (CD16). No enhanced K562 killing was demonstrated in PBMC from subjects with acute infection compared to subjects without infection. CD4+ CTL may function as an important primary host response in acute varicella. Immunization with live attenuated varicella vaccine induced VZV-specific, memory CTL responses comparable to those of naturally immune subjects. The demonstration of their persistence long after primary VZV infection may indicate a role for CTL in restriction of viral replication during episodes of VZV reactivation from latency.     

Tropism of varicella-zoster virus for human CD4+ and CD8+ T lymphocytes and epidermal cells in SCID-hu mice.

To investigate the cell tropism and pathogenicity of varicella-zoster virus (VZV) strains, we analyzed VZV replication by using SCID-hu mice that carry human fetal thymus/liver implants under the kidney capsule or as subcutaneous fetal skin implants. MRC-5 cells infected with wild-type VZV or the Oka strain, used in the live attenuated varicella vaccine, were injected into the implants. The implants were surgically removed 2, 7, 14, and 21 days postinfection. The VZV titer from infected thymus/liver implants peaked on day 7 for the wild-type strain and on day 14 for the Oka strain. Histological analysis showed necrotic areas characterized by thymocyte depletion and fibrosis. VZV protein synthesis was detectable by immunohistochemical staining in the necrotic areas and in distant regions that did not show cytopathic changes, and VZV DNA was detected by in situ hybridization in the same distribution. Fluorescence-activated cell sorting analysis of thymocytes harvested at day 7 postinfection showed that VZV proteins were expressed in CD4+, CD8+, and CD4+ CD8+ T cells; VZV was cultured from each T-cell subpopulation. The Oka strain had tropism for human cell types similar to that of wild-type VZV. T lymphocytes released infectious VZV, which is a novel and important observation about the replication of this otherwise highly cell associated virus. VZV-infected skin implants exhibited microscopic epidermal lesions that were indistinguishable histologically from the characteristic lesions of varicella. These experiments demonstrate a unique tropism of VZV for human T lymphocytes, explaining its capacity to cause viremia in natural disease, and demonstrate the value of the SCID-hu model for studies of VZV pathogenesis.     

Live varicella vaccine: prevention of nosocomial infection and protection of high risk infants from varicella infection

For prevention of nosocomial infection, 25 infants including high risk patients received an emergency injection of live varicella vaccine. Three patients developed a rash within 5 days after vaccination, but their symptoms were mild. The other 22 showed no clinical symptoms and gave an immune response. Twenty-two patients receiving immunosuppressive therapy were vaccinated and 20 of them showed a positive response in the varicella skin test. Of 14 vaccinated patients with malignancies, 2 giving a positive skin test, later showed clinical varicella, but their symptoms were not severe. One case with ALL was immunized safely under very poor conditions during the first induction therapy. No complications were observed in any patients.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.     

Molecular and therapeutic aspects of varicella-zoster virus infection

Varicella-zoster virus (VZV) is a highly species-specific member of the Herpesviridae family. The virus exhibits multiple cell tropisms, infecting peripheral blood mononuclear cells and skin cells before establishing latency in sensory neurons. Such tropisms are essential both for primary infection, which manifests itself as chickenpox (varicella), and subsequent reactivation to cause herpes zoster (shingles). The highly cell-associated nature of the virus, coupled with its narrow host range, has resulted in the lack of an animal model that mimics its diseases in humans, thereby greatly hindering the study of events in VZV pathogenesis. Despite this, extensive studies both in vitro and in vivo in small-animal models have provided a fascinating insight into molecular events that govern VZV diseases. In addition, VZV has become the first human herpes virus for which a live attenuated vaccine has been developed.     

HCV多表位抗原基因重组减毒口服活菌苗的研究

把丙型肝炎病毒(hepatitis C virus,HCV)复合多表位抗原基因PCX与β半乳糖苷酶基因(GZ)融合后,构建重组减毒鼠伤寒沙门氏菌口服活菌苗SL3261(pWR/PCX),免疫小鼠及家兔后,于第6周始可检测到低水平的抗GZPCX IgG(1∶200),至3月时最高滴度分别达1∶800及1∶25 600,均显著高于宿主菌SL3261组及空白对照组。在免疫小鼠的肠道灌洗液中可检测到抗GZPCX sIgA\.GZPCX抗原可促进免疫小鼠及家兔淋巴细胞增殖,诱发明显的迟发性超敏反应(DTH)。口服免疫后小鼠体重出现一过性下降,但未见其它明显的毒性作用,安全性较好。本研究从新的角度探讨了HCV复合多表位重组口服活菌苗的可行性,为HCV疫苗的研究提供新的实验依据。     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Age related differences in cellular immune response to vaccine induced rubella infection.

Employing the techniques of in-vitro lymphocyte transformation (LTF) and using Putnam strain of rubella virus as the antigen, the development of rubella specific cellular immune response was studied in different age groups of rubella seronegative normal subjects at various intervals after subcutaneous administration of HPV=77/DE5 live attenuated rubella vaccine. The rubella specific lymphocyte response in children ranging in age from two to twelve years was characterized by significant LTF activity at two months, followed by a gradual decrease. The response in adult subjects 18 to 35 years of age showed a slight delay initially in the appearance and the maximum LTF activity appeared to be 3--4 fold lower (P less than 0.01) than observed in the children. No difference was observed in the maximum antibody titers to rubella virus between these two groups of subjects. These observations suggest that the age related differences in the lymphoproliferative responses might be associated with adverse effects which are known to occur more frequently in adolescent and older patients than in childhood population after vaccine induced rubella infection.