日本第53届实验动物学会年会介绍

[目的]建立SIVp27杂交瘤细胞株,并对其分泌的SIVp27单克隆抗体进行初步鉴定。[方法]使用基因重组的SIVp27蛋白免疫BALB/c小鼠,采用杂交瘤技术使用半固体培养基法建立杂交瘤细胞株,制备单克隆抗体。通过染色体核型对杂交瘤细胞株进行鉴定;采用Westernblot、免疫荧光法、酶联免疫吸附法确定单克隆抗体的交叉反应性、相对亲和力、抗原识别表位、免疫球蛋白的类型和亚类,对单克隆抗体进行鉴定。[结果]获得四株可稳定分泌SIVp27单克隆抗体的杂交瘤细胞,1C3、2B6为IgG1类,2E12为IgG2b类,3G3为IgG2a类。四株单抗均能识别SIV的p27蛋白,与逆转录病毒SRV、STLV无交叉反应,2B6、2E12与HIVp24有交叉反应。免疫荧光法检测腹水效价为1:10240~40960。1C3、2B6、2E12、3G3染色体平均数分别为103、97、96、101。2E12与3G3识别不同的抗原表位。[结论]成功地制备出四株SIVp27单克隆抗体,均具有良好的特异性和亲和力,为进一步建立免疫分析方法,进行SIV/SAIDS及其艾滋病相关研究,奠定了基础。     

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli beta-galactosidase induced durable beta-gal-specific IgG and CD8(+) T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.     

Variable region 4 of SIV envelope correlates with rapid disease progression in morphine-exposed macaques infected with SIV/SHIV

We analyzed the association between the evolution of the V3-V5 regions of env and disease progression in our SIV/SHIV macaque model of morphine dependence and AIDS. Previous studies revealed two distinct disease patterns--fast progression and normal progression. To determine the effect of the two distinct patterns of disease in the evolution of SIV/17E-Fr envelope, we analyzed env sequences from three morphine-dependent macaques that developed accelerated AIDS and three morphine-dependent macaques that developed AIDS at a slower rate and compared them to control macaques. Morphine-dependent animals exhibited a higher percentage of diversity in both plasma and CSF compartments within V4 when compared to controls. Divergence from the inoculum was significantly greater in the morphine group as compared to controls in CSF but not in plasma. We also found a direct correlation in V4 evolution and rapid disease progression. These results indicate that morphine dependence plays a role in the pathogenesis of SIV/SHIV infection and env evolution.     

A genetically engineered live-attenuated simian-human immunodeficiency virus that co-expresses the RANTES gene improves the magnitude of cellular immunity in rhesus macaques

Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4⁺ Th cell-proliferative response and by inducing an antigen-specific IFN-γ ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4⁺ Th responses and IFN-γ ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4⁺ T cell responses.     

Influence of Naturally Occurring Simian Foamy Viruses (SFVs) on SIV Disease Progression in the Rhesus Macaque (Macaca mulatta) Model

We have investigated the influence of naturally occurring simian foamy viruses (SFVs) on simian immunodeficiency virus (SIV) infection and disease in Indian rhesus macaques. Animals were divided into two groups based upon presence or absence of SFV; in each group, eight monkeys were injected with SIV_mac239 virus obtained from a molecular clone and four were injected with medium. Blood was collected every two weeks for evaluation of SIV infection based upon T cell-subsets, plasma viral load, development and persistence of virus-specific antibodies, and clinical changes by physical examination and hematology. Comparative analysis of SFV+/SIV+ and SFV−/SIV+ monkey groups indicated statistically significant differences in the plasma viral load between 6–28 weeks, particularly after reaching plateau at 20–28 weeks, in the CD4⁺ and CD8⁺ T-cell numbers over the entire study period (2–43 weeks), and in the survival rates evaluated at 49 weeks. There was an increase in the plasma viral load, a decreasing trend in the CD4⁺ T cells, and a greater number of animal deaths in the SFV+/SIV+ group. The results, although based upon a small number of animals, indicated that pre-existing SFV infection can influence SIV infection and disease outcome in the rhesus macaque model. The study highlights consideration of the SFV status in evaluating results from SIV pathogenesis and vaccine challenge studies in monkeys and indicates the potential use of the SFV/SIV monkey model to study the dynamics of SFV and HIV-1 dual infections, recently reported in humans.     

Safety, immunogenicity, and efficacy of an alphavirus replicon-based swine influenza virus hemagglutinin vaccine

A single-cycle, propagation-defective replicon particle (RP) vaccine expressing a swine influenza virus hemagglutinin (HA) gene was constructed and evaluated in several different animal studies. Studies done in both the intended host (pigs) and non-host (mice) species demonstrated that the RP vaccine is not shed or spread by vaccinated animals to comingled cohorts, nor does it revert to virulence following vaccination. In addition, vaccinated pigs develop both specific humoral and IFN-γ immune responses, and young pigs are protected against homologous influenza virus challenge.     

Heteroclitic peptides enhance human immunodeficiency virus-specific CD8 T cell responses

The inability of human immunodeficiency virus (HIV)-specific CD8⁺ T cells to durably control HIV replication due to HIV escape mutations and CD8⁺ T cell dysfunction is a key factor in disease progression. A few HIV-infected individuals termed elite controllers (EC) maintain polyfunctional HIV-specific CD8⁺ T cells, minimal HIV replication and normal CD4⁺ T lymphocyte numbers. Thus, therapeutic intervention to sustain or restore CD8⁺ T cell responses similar to those persisting in EC could relieve terminal dependence on antiretrovirals. Vaccination with HIV peptides is one approach to achieve this and our objective in this study was to determine whether certain HIV peptide variants display antigenic superiority over the reference peptides normally included in vaccines. Eight peptide sets were generated, each with a reference peptide and six variants harboring conservative or semi-conservative amino acid substitutions at positions predicted to affect T cell receptor interactions without affecting human class I histocompatibililty-linked antigen (HLA) binding. Recognition across peptide sets was tested with >80 HIV-infected individuals bearing the appropriate HLA alleles. While reference peptides were often the most antigenic, cross-reactivity with variants was common and in many cases, peptide variants were superior at stimulating interferon-γ production or selectively enhanced interleukin-2 production. Although such heteroclitic activity was not generalized for all individuals bearing the HLA class I allele involved, these data suggest that heteroclitic peptide variants could improve the efficacy of therapeutic peptide vaccines in HIV infection.     

The inside out of lentiviral vectors

Lentiviruses induce a wide variety of pathologies in different animal species. A common feature of the replicative cycle of these viruses is their ability to target non-dividing cells, a property that constitutes an extremely attractive asset in gene therapy. In this review, we shall describe the main basic aspects of the virology of lentiviruses that were exploited to obtain efficient gene transfer vectors. In addition, we shall discuss some of the hurdles that oppose the efficient genetic modification mediated by lentiviral vectors and the strategies that are being developed to circumvent them.     

中国恒河猴SIVmac239艾滋病模型亚急性期的实验观察

目的:观察SIVmac239中国恒河猴艾滋病模型亚急性期内T淋巴细胞亚群、病毒载量指标变化情况,探讨中国恒河猴艾滋病模型亚急性期病毒学相关指标变化特征.方法:采用SlVmac239病毒株静脉接种健康中国恒河猴复制中国恒河猴艾滋病动物模型,观察猴免疫缺陷病毒(simian immunodeficiency virus,SIV)感染后动物模型亚急性期内T淋巴细胞亚群指标变化、病毒载量变化情况.同时记录动物模型临床症状情况.结果:健康恒河猴接种病毒株后3个月体重及BMI指数比较稳定,各时间点差异不显著,在感染第10 ～14天病毒载量达到高峰(6.72±0.44)个1g,此后整体表现为水平波动下降趋势.T淋巴细胞亚群在感染后出现显著改变,CD3,CD4,CD8计数和百分比有不同程度的变化;CD4/CD8比值感染后2周内明显下降,一般在病毒载量高峰时表现为最低比值为(0.86±0.29),随后比值缓慢波动上升.感染3个月内共有3只感染猴临床症状明显,其中1只出现腹泻、2只出现摄食下降等临床症状.结论:中国恒河猴艾滋病动物模型与国外印度恒河猴艾滋病动物模型亚急性期相似,研究内容为中国恒河猴应用于艾滋病模型研究的标准化建立补充了更详尽的亚急性期变化内容.