HSV-1特异转移因子活性成分的化学本质

亲和色谱法分离TF_(HSV-1)特异活性成分,该成分在高效液相层析(包括离子交换层析和反相层析)产生一个紫外吸收峰,簿层层析后有一个荧光胺显色点而无254nm吸收点。在等电点聚焦分析中该成分为一条考马斯亮蓝染色带。凝胶层析测定特异成分的分子量为12,870 dalton。特异成分不含DNA和游离氨基酸,与Orcinol反应阴性,其水解物含16种氨基酸。实验结果表明,TF_(HSV-1)特异成分是12,870 dalton的多肽。     

抗HSV-1淋巴细胞杂交瘤的建立及应用单克隆抗体对HSV分型

应用HSV-1 SM44株感染的BHK-21细胞及提取的感染细胞膜蛋白抗原免疫Balb/c小鼠。以免疫小鼠脾细胞与小鼠骨髓瘤细胞系Sp_2/0融合,经ELISA筛选仅与HSV-1反应的阳性克隆及克隆化,建立了一株产生抗HSV-1型特异性McAb的杂交瘤细胞系(Mad-2)。经ELISA,IF及抗原吸收试验证明,该细胞系的培养上清及制备的小鼠腹水均与HSV-1呈特异性反应,但不与HSV-2反应。ELISA测定McAb Mad-2的腹水效价为10~(-7)Ig亚类鉴定为IgG-1。应用Mad-2和抗HSV型共同性McAb 1A12及抗HSV-2型特异性McAb CH-A9,对43株临床HSV分离株做了ELISA及IF分型。结果表明,28株口唇、眼部感染分离株和1株宫颈炎分离株均与1A12和Mad-2反应,不与CH-A9反应,为HSV-1。余14株富颈炎分离株均与1A12和CH-A9反应,而不与Mad-2反应,为HSV-2。ELISA和IF分型的结果完全一致。本研究提出作者应用的一套抗HSVMcAb有可能作为HSV型别鉴定的标准试剂。     

A群奈瑟氏脑膜炎球菌荚膜多糖-破伤风类毒素结合疫苗的制备及其免疫原性研究

采用溴化氰(CNBr)活化多糖,以无水己二酸二肼(ADH)作为连接剂,1乙基13(3二甲基氨基丙基)碳化二亚胺(EDAC)为偶联剂制备A群奈瑟氏脑膜炎球菌荚膜多糖(GAMP)与破伤风类毒素(TT)的结合物,经皮下免疫NIH小鼠,用ELISA检测小鼠血清中抗GAMP及抗载体蛋白的IgG抗体水平。用补体介导的体外杀菌试验检测血清中GAMP抗体的杀菌活性。结果显示,实验中制备的多糖衍生物和多糖蛋白质结合物都具有GAMP抗原特异活性。结合物免疫小鼠后可诱生比多糖单独免疫更高水平的GAMP血清IgG抗体,并能形成免疫记忆,产生再次应答。结合物免疫小鼠所诱生的血清GAMP抗体较之多糖组具有更强的体外杀菌活性。表明此方法制备的结合物可获得优于多糖的、稳定的特异免疫原性。     

人巨细胞病毒对单纯疱疹病毒1型抗原表达的影响

我们用免疫胶体金色埋前标记技术和免疫荧光技术研究了人胚肺细胞(HEL)内,人巨细胞病毒(HCMV-AD_(169))对单纯疱疹病毒1型(HSV-ⅠSM_(44))抗原表达的影响,旨在探讨在细胞这一微生境内,一病毒对另一病毒可能发生的影响。电镜下计数HSV-1组和HCMV HSV-1组特异性结合金颗粒数得HSV-1组为657个,HCMV HSV-1组的总数为283个。t检验P<0.01,差别非常显著。并且HSV-1组细胞的胞浆中的病毒颗粒,比HCMV HSV-1组明显多。荧光显微镜下:HSV-1组阳性细胞数为689个HCMV HSV-1组只有484个,经poisson分布u检验,P<0.01,差别非常显著。免疫荧光实验还表明:HSV-1组,抗血清在1:320时仍有荧光清晰的阳性细胞,而HCMV HSV-1组,抗血清在1:160时,却无荧光阳性细胞。细胞病变效应(CPE)动态观察显示:HSV-1组8小时即有细胞病变,24小时蔓延整个单层;而HCMV HSV-1组超感染14小时才有细胞病变。24小时约有75％细胞受累。结果表明HCMV对HSV-1的抗原表达有明显的抑制作用。对抑制作用的可能机理及其在分子生态学中的意义,进行了讨论。     

扇贝糖胺聚糖对感染HSV-Ⅰ小鼠免疫功能的影响研究

目的:研究扇贝裙边糖胺聚糖(glycosaminoglycan from Scallop Skirt,SS-GAG)对感染单纯疱疹病毒Ⅰ型(herpes simplex virus type,HSV-Ⅰ)小鼠免疫功能的影响。方法:通过在无菌条件下给予小鼠注射扇贝糖胺聚糖SS-GAG,连续11天,并在给药第三天给小鼠腹腔注射HSV-Ⅰ病毒悬液建立小鼠感染模型,用MTT等方法观察SS-GAG对HSV-Ⅰ感染小鼠腹腔巨噬细胞吞噬活性的影响、对脾脏指数、胸腺指数的影响以及对脾淋巴细胞转化能力等免疫指标的影响。结果:与病毒对照组相比,扇贝糖胺聚糖SS-GAG低剂量组、中剂量组、高剂量组均能显著增强HSV-Ⅰ感染小鼠的腹腔巨噬细胞的吞噬活性和HSV-Ⅰ感染小鼠的脾脏指数和胸腺指数(P0.01),并且能促进其脾淋巴细胞转化增殖能力(P0.01)。结论:扇贝糖胺聚糖在体内有一定的抗Ⅰ型单纯疱疹病毒作用。其抗病毒作用可能与增强机体免疫功能有关。     

葡萄球菌蛋白A免疫粘附法检测单纯疤疹病毒抗原

本文用葡萄球菌蛋白A(Staphylococcal Protein A,简称SPA)免疫粘附法快速检出单纯疱疹病毒抗原。试验观察了正常人角膜上皮细胞、兔角膜上皮细胞及来感染的传代细胞(Vero细胞等)对SPA的反应。同时,对单纯疱疹病毒Ⅰ型(HSV-1)实验感染性角膜炎的家兔,用SPA免疫粘附法与病毒分离培养法做了对照观察,并对临床诊断为单纯疱疹病毒性角膜炎的患者做了初步观察。结果表明,SPA免疫粘附法特异、简便,适合基层实验室应用。     

BALB/c小鼠作为单纯疱疹病毒1型感染模型的免疫学分析

在单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)小鼠感染及其相关研究中,临床病理和免疫学指标对其分析具有重要技术意义。本研究观察了HSV-1在不同条件下感染BALB/c小鼠后的多个免疫学指标,包括外周血单核细胞(peripheral blood mononuclear cell,PBMC)群体中树突细胞比例及功能、血清中和抗体水平、PBMC中HSV-1抗原特异性T细胞水平,以及潜伏感染期小鼠神经组织中CD8^＋ T细胞浸润情况。结果显示,HSV-1毒株Mckrae、17＋以角膜及滴鼻途径感染3周龄及6周龄BALB/c小鼠后,小鼠PBMC中树突细胞数量增加,并显示出刺激病毒抗原特异性T细胞增殖的能力。病毒感染后35 d,小鼠PBMC中未检测到白细胞介素4(interleukin 4,IL-4)^＋抗原特异性T细胞,但能检测到低水平的γ干扰素(interferon γ,IFN-γ)^＋抗原特异性T细胞;小鼠血清中未检测到或仅能检测到低水平的中和抗体。HSV-1以皮下及足垫注射途径感染BALB/c小鼠90 d后,足垫感染途径较皮下感染诱导出更高水平的血清中和抗体,PBMC中可检测到IL-4^＋及IFN-γ^＋抗原特异性T细胞,但不同毒株及小鼠周龄之间出现T细胞反应程度差异。组织病理学结果表明,各组小鼠三叉神经组织中均有CD8^＋ T细胞浸润。这些结果提示,不同HSV-1毒株以不同途径感染不同周龄BALB/c小鼠后,均可刺激树突细胞成熟及呈递病毒抗原,但血清中和抗体及PBMC中病毒抗原特异性T细胞水平在不同毒株、感染途径及小鼠周龄之间有差异。     

SARS-CoV-2 Beta变异株和甲型流感病毒H3N2双价重组腺病毒载体疫苗可在小鼠诱导免疫保护

评价一种SARS-CoV-2 Beta变异株和甲型流感病毒H3N2新型重组双价疫苗在小鼠模型中的免疫保护效果。本研究构建了表达SARS-CoV-2南非变异株(B.1.351)棘突蛋白1(S1)和H3N2柬埔寨分离株(A/Cambodia/e0826360/2020)血凝素(HA)的重组双价非复制Ad5载体疫苗,命名为HAdV5-S1-2A-HA,经单针肌肉注射免疫BALB/c雌鼠后,采用ELISA、血凝抑制实验、假病毒中和实验与Elispot实验进行体液与细胞免疫学检测,免后3～6周采用H3N2-X31病毒、H1N1-PR8与SARS-CoV-2(B.1.351)进行攻毒保护实验。HAdV5-S1-2A-HA免疫两周后,低剂量组(1×10⁸vp/只)免疫小鼠后可检出HA特异体液免疫与S1特异的细胞免疫应答;而高剂量组(5×10⁹vp/只)诱导小鼠产生了较强的双抗原(S1,HA)特异的体液和细胞免疫应答,并能完全保护小鼠对H3N2-X31攻击,降低SARS-CoV-2(B.1.351)感染后小鼠肺部病毒载量,延迟H1N1-PR8病毒感染后小鼠死...     

重组CHO细胞乙肝表面抗原诱导小鼠细胞免疫应答的研究

为了研究重组CHO细胞乙肝表面抗原(CHO-rHBsAg)在小鼠中诱导T细胞免疫应答的能力,全面评价疫苗的免疫原性,以CHO-rHBsAg免疫BALB/c小鼠,常规制备小鼠脾脏淋巴细胞并在体外以抗原或特异多肽刺激;采用ELISA法测定抗原特异性T淋巴细胞分泌的细胞因子,乳酸脱氢酶法(LDH)测定抗原特异性细胞毒T淋巴细胞(CTL)活性,酶联斑点法(ELISPOT)测定CTL频数(CTLp),应用流式细胞仪分析T淋巴细胞亚群。结果显示,rHBsAg可在小鼠中诱导Th1及Th2类细胞因子;加铝佐剂的rHBsAg较未加佐剂的抗原可诱导较高水平的IFN-γ、CTL克隆及较高百分比的CD8+T淋巴细胞亚群。重组CHO细胞来源的HBsAg可在BALB/c小鼠中诱导一定程度的细胞免疫应答。     

HSV-1 UL41基因编码蛋白的RNase活性及其调控

单纯疱疹病毒1型(herpes simplex virus type-1,HSV-1)UL41基因编码一种皮层蛋白,该蛋白质具有核糖核酸酶(ribonuclease,RNase)活性,能特异性地降解一些宿主和病毒信使RNA(messenger RNA,m RNA),参与宿主免疫逃逸,与其他蛋白质相互作用调控其RNase活性。该文概述了HSV-1UL41基因编码蛋白的RNase活性及其调控机制,以期为该基因的深入研究提供参考。     

Relationship between tumour resistance and the in vitro and in vivo cytotoxicity elicited by Salmonella antigens in immunized mice

Immunization of mice with the attenuated 11RX strain of Salmonella enteritidis (11RX) induces resistance to intraperitoneal (i.p.) tumour growth. Tumour resistance is much greater and lasts for a longer time following i.p. immunization than following intravenous (i.v.) immunization. This paper extends our previous observations that, after this resistance is lost, it can be recalled by a T cell-mediated reaction to an antigenic extract of the bacteria (11RX antigen) which is not protective in unimmunized mice. The duration of this sensitization to 11RX antigen was determined in mice immunized i.p. or i.v. with live 11RX by challenging them at various times after immunization with 131I (or 125I)-labelled Ehrlich ascites tumour (EAT) cells alone or mixed with 11RX antigen. In vivo killing of EAT cells was assayed by monitoring whole-body retention of radioactivity and this was correlated in the same mice with suppression of tumour growth and survival of the mice. The resistance recalled by 11RX antigen was short-lived and in vivo cytotoxic activity had subsided by 6 days after antigen injection. 11RX antigen also recalled the ability of the peritoneal cells to lyse 51Cr-labelled EAT cells in vitro and a close correlation was found between this activity and the cytotoxicity measured in vivo. The adherence properties of the cytotoxic cells and their inhibition by trypan blue indicated that they were macrophages.     

Protective T-cell-based immunity induced in neonatal mice by a single replicative cycle of herpes simplex virus

Newborns are very susceptible to infections because their immune systems are not fully developed and react to antigen exposure preferentially with unresponsiveness. UV-inactivated herpes simplex virus type 1 (HSV-1) represents such an antigen and does not induce an immune response in neonates. In contrast, protective T cells were primed in newborn mice by a single replicative cycle of DISC HSV-1 given once within 24 h of birth. Each of the HSV-1-primed CD4(+) or CD8(+) T cells induced in wild-type or interferon-deficient mice conferred resistance to naive animals exposed to a lethal virus challenge. Inactivated HSV-1, injected at variable doses up to 10(4) times that of DISC HSV-1, was ineffective in inducing any detectable immune responses in neonates. Thus, the capacity of HSV-1 to replicate once, but not the number of virus particles per se, was decisive in inducing protective T-cell-associated immunity in newborn mice.     

Blocking and unblocking of cell-mediated anti-tumor immunity in mice, as detected by the leucocyte adherence inhibition test

Further observations have been made using the in vitro leucocyte adherence inhibition (LAI) test previously described. The test has been slightly modified and shown to give highly reproducible results in detecting cell-mediated immunity in peritoneal cells of mice exposed to tumors, and in demonstrating factors which modify this immunity.Two different methylcholanthrene-induced tumors stimulated specific reactivity in their hosts as shown by the leucocyte adherence inhibition test employing soluble tumor extracts as antigens. When serum from tumor-bearing mice was added to the mixture, reactivity was specifically blocked. Serum from mice after tumor removal lacked the ability to block the leucocyte adherence inhibition reaction, but specifically reversed the effect of blocking serum (unblocking).Thus three important phenomena of tumor immunity, previously discovered by tedious and complex techniques, are now detectable by a brief, simple procedure.     

HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein

Autophagy is postulated to play a role in antiviral innate immunity. However, it is unknown whether viral evasion of autophagy is important in disease pathogenesis. Here we show that the herpes simplex virus type 1 (HSV-1)-encoded neurovirulence protein ICP34.5 binds to the mammalian autophagy protein Beclin 1 and inhibits its autophagy function. A mutant HSV-1 virus lacking the Beclin 1-binding domain of ICP34.5 fails to inhibit autophagy in neurons and demonstrates impaired ability to cause lethal encephalitis in mice. The neurovirulence of this Beclin 1-binding mutant virus is restored in pkr(-/-) mice. Thus, ICP34.5-mediated antagonism of the autophagy function of Beclin 1 is essential for viral neurovirulence, and the antiviral signaling molecule PKR lies genetically upstream of Beclin 1 in host defense against HSV-1. Our findings suggest that autophagy inhibition is a novel molecular mechanism by which viruses evade innate immunity and cause fatal disease.     

Protection of mice against herpes simplex virus infection by a Lactobacillus casei preparation (LC 9018) in combination with inactivated viral antigen

Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection.     

Interleukin-12- and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type 1

Interferon (IFN) type I (alpha/beta IFN [IFN-alpha/beta]) is very important in directly controlling herpes simplex virus type I (HSV-1) replication as well as in guiding and upregulating specific immunity against this virus. By contrast, the roles of IFN type II (IFN-gamma) and antibodies in the defense against HSV-1 are not clear. Mice without a functional IFN system and no mature B and T cells (AGR mice) did not survive HSV-1 infection in the presence or absence of neutralizing antibodies to the virus. Mice without a functional IFN type I system and with no mature B and T cells (AR129 mice) were unable to control infection with as little as 10 PFU of HSV-1 strain F. By contrast, in the presence of passively administered neutralizing murine antibodies to HSV-1, some AR129 mice survived infection with up to 10(4) PFU of HSV-1. This acute immune response was dependent on the presence of interleukin-12 (IL-12) p75. Interestingly, some virus-infected mice stayed healthy for several months, at which time antibody to HSV-1 was no longer detectable. Treatment of these virus-exposed mice with dexamethasone led to death in approximately 40% of the mice. HSV-1 was found in brains of mice that did not survive dexamethasone treatment, whereas HSV-1 was absent in those that survived the treatment. We conclude that in the presence of passively administered HSV-1-specific antibodies, the IL-12-induced IFN-gamma-dependent innate immune response is able to control low doses of virus infection. Surprisingly, in a significant proportion of these mice, HSV-1 appears to persist in the absence of antibodies and specific immunity.     

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.     

Glycoprotein G of herpes simplex virus 2 as a novel vaccine antigen for immunity to genital and neurological disease

The envelope glycoproteins of herpes simplex virus 1 (HSV-1) and HSV-2, with the exception of glycoprotein G, elicit cross-reactive B- and T-cell responses. Human vaccine trials, using the cross-reactive glycoproteins B and D, have shown no protection against genital HSV-2 infection or disease. In this study, the mature form of glycoprotein G (mgG-2) of HSV-2 was used for immunization of mice, either alone or in combination with adjuvant CpG, followed by an intravaginal challenge with a lethal dose of a fully virulent HSV-2 strain. Mice immunized with mgG-2 plus CpG showed low disease scores and a significantly higher survival rate (73%) than mice immunized with mgG-2 alone (20%) or controls (0%). Accordingly, limited numbers of infectious HSV-2 particles were detected in the spinal cord of mice immunized with mgG-2 plus CpG. The observed protection was associated with a gamma interferon (IFN-γ) response by splenic CD4(+) T cells upon antigen restimulation in vitro and in vaginal washes 1 day postinfection. The majority of sera collected from mice immunized with mgG-2 plus CpG showed macrophage-mediated antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis, while no neutralization activity was observed. In conclusion, we have shown that immunization with the type-specific mgG-2 protein in combination with CpG could elicit protective immunity against an otherwise lethal vaginal HSV-2 challenge. The mgG-2 protein may therefore constitute a promising HSV-2 vaccine antigen to be considered for future human trials.     

Growth inhibition of a B cell leukemia: evidence implicating an anti-idiotype immune response for protective tumor immunity

BCL1, a spontaneous surface IgM (mu lambda)-positive (sIgM+) B cell leukemia of BALB/c (Igha) origin rarely grows in the Ig heavy chain (Igh) congenic mouse C.B-20 (Ighb) but is highly metastatic and lethal in the host strain of origin. Previous studies indicated that BCL1 tumor immunity in C.B-20 mice was associated with a T cell-mediated immune response against H-40, a minor histocompatibility (H) antigen controlled by a gene linked to the Igh locus. However, we observed that BCL1 leukemia grew progressively in BAB-14 (Igha/b) mice, a strain capable of generating an anti-H-40 immune response. This suggested that anti-H-40 immunity was insufficient for protection and implied that an Igh-V (variable) region gene product was also important for BCL1 growth inhibition. We therefore evaluated the role of two possible Igh-V region-linked gene products in BCL1 growth inhibition; namely, an Igh-V region-linked minor H antigen or alternatively the BCL1 IgM idiotype (Id). We could find no evidence for an Igh-V region-linked minor H antigen because immunosuppressed (500 R) CB-20 mice reconstituted with C.B-20 anti-BAB-14 splenocytes were susceptible to BCL1 growth, whereas recipients reconstituted with C.B-20 anti-BALB/c splenocytes were resistant to BCL1 challenge. In contrast, C.B-20 mice immunized against purified BCL1 IgM protein could adoptively confer BCL1 tumor immunity. C.B-20 mice immunized against other BALB/c IgM myeloma proteins containing either lambda or kappa light chains failed to protect C.B-20 mice suggesting that recognition of a unique determinant (Id) and not an allotype was crucial for tumor immunity. The BCL1 mu-chain appeared to make the major contribution to the idiotypic determinant because a hybridoma product composed of BCL1 mu-chains and BALB/c kappa-chains still elicited BCL1 immunity. Adoptive transfer of C.B-20 anti-BCL1 Id splenocytes into irradiated recipients that prevented an anti-H-40 response due to H-40 tissue expression failed to adoptively confer BCL1 immunity. Thus, these data suggest that BCL1 growth inhibition requires a T cell-mediated response against both H-40 and the BCL1 Id; these responses must be elicited concurrently in the tumor-bearing host to achieve protective BCL1 immunity.