Use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease.

Monoclonal antibodies HCl and HD1, directed against herpes simplex virus type 1 (HSV-1) glycoproteins gC and gD, respectively, were evaluated for their ability to passively immunize mice against acute virus-induced neurological disease after footpad inoculation with HSV-1 or herpes simplex virus type 2 (HSV-2). Control virus-infected mice receiving a single intraperitoneal injection of normal serum died within 7 to 10 days after the spread of virus from footpad to spinal cord and brain. However, a single intraperitoneal injection of either HCl or HD1 antibody protected mice from neurological illness and death when administered to HSV-1 (strain HTZ)-infected mice at either 2 h before virus challenge or at 24 h after virus inoculation. To determine the in vivo specificity of the antibodies, passive transfer studies were performed with mice infected with the MP strain of HSV-1, a mutant of HSV-1 (mP) which is defective in the production of glycoprotein gC. Whereas HD1 antibody decreased the incidence of neurological illness in MP- and mP-infected mice, HCl antibody, which protected mP-infected animals, failed to protect mice infected with the MP strain. When HD1 antibody was administered to HSV-2 (strain G)-infected mice at either 2 h before virus challenge or at 6 h (but not 24 h) after virus inoculation, 100% of the infected animals receiving HD1 antibody survived. In contrast, 100% of HSV-2 (strain G)-infected animals passively immunized with HCl antibody developed neurological illness and died. These results provide in vivo evidence that the HSV-induced glycoprotein gC expresses type-specific antigenic determinants, whereas glycoprotein gD expresses type-common determinants.     

Experimental keratitis in rabbits by human HSV-1 variants: prevention and treatment

The efficacy of different therapies and vaccine preparations was assessed for treating or preventing herpetic ocular keratitis induced by experimental inoculation in rabbits with two HSV-1 variants that display different pathogenetic potential. Early administration of acyclovir (ACV) promoted fast healing and prevented neurologic involvements: alpha-interferon (alpha-IFN) was less efficient than ACV; combined therapy with both drugs increased the antiviral effects. In an attempt to prevent the disease, rabbits were vaccinated with a slightly pathogenic HSV-1 variant or with a secreted form of an engineered HSV-1 glycoprotein gB (gB-1s) and were subsequently challenged with a highly pathogenic HSV-1 variant. Immunization of rabbits with gB-1s was much more efficient than immunization with live virus in reducing the severity of herpetic keratitis and in preventing CNS disease.     

A comparison of T cell responses to glycoprotein B (gB-1) of herpes simplex virus type 1 and its non-glycosylated precursor protein, pgB-1.

The ability of non-glycosylated precursor glycoprotein B (pgB) to induce T cell responses in herpes simplex virus (HSV) infected mice was compared with fully glycosylated glycoprotein B (gB) and with whole virus. pgB was as effective as gB in priming for virus- and glycoprotein-specific T cells. pgB could also re-stimulate virus or glycoprotein primed cells in vitro as efficiently as gB. In addition, priming with pgB protected mice against a lethal challenge with HSV type 1 (HSV-1) and could induce the early in vivo production of IL-2 and IL-3 in infected mice. In all of these responses, pgB was as effective as gB. Thus, the carbohydrate side chains on gB do not appear to be necessary for T cell recognition of this protein.     

Escherichia coli-derived envelope protein gD but not gC antigens of herpes simplex virus protect mice against a lethal challenge with HSV-1 and HSV-2

Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed. After active immunization of mice with a gD-1 antigen (Leu₅₃-Ala₃₁₂) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2. In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2. In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E. coli failed to induce protective immunity. Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E. coli. These results indicate, that E. coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E. coli.     

Protective immunity against lethal HSV-1 challenge in mice by nucleic acid-based immunisation with herpes simplex virus type-1 genes specifying glycoproteins gB and gD

DNA-based vaccines were employed to assess protective immunity against herpes simplex virus in experimental infections of hairless (strain SKH1) and BALB/c mice. Mice were vaccinated with plasmids containing the herpes simplex virus type-1 (HSV-1) glycoprotein B (gB) or D (gD) genes under the human cytomegalovirus immediate-early promoter control. Vaccines were injected intramuscularly (i.m.) or intraperitoneally (i.p.) as purified DNA alone or as formulations supplemented with different non-ionic block copolymers. Antibody responses were assessed by immunofluorescence and radio-immunoprecipitation assays. Mice inoculated with either gB or gD plasmid, alone or with non-ionic block copolymers CRL 1029 and CRL 1190, produced high levels of antibodies specific for gB or gD. Three weeks after the last vaccination, mice were challenged with a clinical HSV-1 isolate (ABGK-1) by inoculation of a shaved and subsequently scarified area between the third and fourth lumbar vertebrae. Mice immunised with either gD or gB plasmid alone or mixed with copolymers were protected against lethal HSV-1 challenge when immunisation was performed via the i.m. route. Immunisations given via the i.p. route induced humoral responses in some mice and protected the animals against lethal HSV-1 challenge only when the formulations contained copolymers. The BALB/c mouse model was shown to be as good a model as the hairless mouse model.     

Efficacy of herpes simplex virus type 1 immunization in protecting against acute and latent infection by herpes simplex virus type 2 in mice.

ICR mice were immunized with herpes simplex virus type 1 (HSV-1) and later challenged with HSV-2 by footpad inoculation. Both immunized animals and age-matched, nonimmunized controls were observed for ascending neurological disease and latent infection of spinal ganglia resulting from the HSV-2 challenge. Control animals had a 78% incidence of acute and latent infection compared with a 1.7% incidence in immunized mice. The data show immunity to HSV-1 is protective against both acute and latent infection by HSV-2.     

Persistent expression of CD94/NKG2 receptors by virus-specific CD8 T cells is initiated by TCR-mediated signals

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.     

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

HSV—1 SM44株糖蛋白D基因真核表达载体的构建及其免？…

目的 构建ＨＳＶ－１型ＳＭ４４株糖蛋白Ｄ（ｇＤ）基因的真核表达载体,并用此重组质粒直接免疫小鼠,探讨ＨＳＶ－１ ｇＤ基因作为基因疫苗的可能性。方法 从ＨＳＶ－１基因组中扩增ｇＤ的全编码基因,克隆入载体ｐＵＣ１９中,测序鉴定后转入真核表达载体ｐｃＤＮＡ３．１（＋）。所得重组质粒ｐｃＤ－ＮＡ－ｇＤ以电穿孔法转染ＣＨＯ细胞,并以荧光染色法鉴定表达效果。用ｐｃＤＮＡ－ｇＤ免疫小鼠,ＥＬＩＳＡ法检测基因免疫     

Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.     

Route of infection, systemic host resistance, and integrity of ganglionic axons influence acute and latent herpes simplex virus infection of the superior cervical ganglion.

The character of acute and latent herpes simplex virus (HSV) infection of the superior cervical ganglion (SCG) in mice depended on the route by which the virus reached the ganglion, the level of systemic host resistance, and the integrity of postganglionic nerves. Prevention of ganglionic infection by postganglionic neurectomy carried out before intraocular (i.o.) virus challenge established the importance of the neural route in the development of SCG infection. However, hematogenous virus dissemination also led to SCG infection although with reduced frequency compared to that with i.o. inoculation. Enhanced host systemic antiviral resistance had two divergent effects on ganglionic infection depending on the dose and timing of virus inoculation. Thus, both acute and latent ganglionic infections were concomitantly reduced when resistant C57B1/6 mice were challenged with low doses of virus or when less resistant BALB/c mice were actively immunized 1 week before virus challenge. On the other hand, when resistant mice were challenged with high doses of virus or when either active or passive (antibody) immunization was delayed long enough to assure viral access to the ganglion, intraganglionic viral replication during the acute phase of infection was reduced, but the prevalence of subsequent latent infection was either unaffected or actually enhanced. Postganglionic neurectomy, performed after virus had reached the ganglion, altered the course of SCG infection in a direction opposite that of immunization, augmenting the acute phase of viral replication while reducing latency. In athymic nude mice and mice immunosuppressed with cyclophosphamide, intraganglionic viral replication was prolonged. These results emphasize that host factors both extrinsic and intrinsic to the SCG modify the course of ganglionic infection.     

Immunogenicity of herpes simplex virus type 1 glycoproteins expressed in vaccinia virus recombinants

Vaccinia virus recombinants expressing glycoproteins B (vgB11), D (VgD52), E (gE/7.5 and gE/4B), G (gG-vac), H (gH-vac), and I (gI-vac) of HSV-1 were used to compare the protective response to these individual glycoproteins in the mouse. Glycoprotein D induced the best neutralizing antibody titers and the most increased rates of HSV clearance from the ear as well as good protection from the establishment of latent HSV infections in the sensory ganglia. Glycoprotein B also induced good neutralizing antibody titers and as great a protection from the establishment of latency as gD although the rate of virus clearance from the ear was not as great as after immunization with gD. Glycoprotein E induced weak neutralizing antibody but gG, gH, and gI did not show a neutralizing antibody response. At higher challenge doses of virus (10(6) PFU HSV-1 in the ear), gE induced a protective response by increasing the rate of virus clearance and reducing the acute infection of ganglia as compared to negative control immunized mice. However there was no protection from the establishment of latent infections after immunization with gE. No protective response was seen to gG, gH, or gl.     

Immunization with DNA vaccine expressing herpes simplex virus type 1 gD and IL-21 protects against mouse herpes keratitis

The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 ?g/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-? and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-? and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.     

Immunogenicity of purified glycoprotein gB of herpes simplex virus

Summary The efficacy of a herpes simplex virus (HSV) component vaccine consisting of viral glycoprotein gB was examined in a mouse system. Immunization of mice with HSV type 1 (HSV-1) gB emulsified in Freund's complete adjuvant or with HSV-1 gB adsorbed to aluminum gel was fully protective against subsequent challenge with HSV-1 or HSV type 2. Latent infection in the trigeminal ganglion was also prevented by immunization with gB.With 6 Figures     

Chronic progressive deficits in neuron size, density and number in the trigeminal ganglia of mice latently infected with herpes simplex virus

Numerous epidemiological studies have proposed a link between herpes simplex virus (HSV) infection and several common chronic neuropsychiatric and neurodegenerative diseases. Experimental HSV infection of mice can lead to chronic behavioral and neurological deficits and chronic pain. While neuron injury and loss are well-documented consequences of the acute phase of infection, the pathologic consequences of latent HSV infection are poorly understood. To determine whether latent HSV infection can cause neuronal injury in mice, trigeminal ganglia (TG) derived from adult BALB/c mice 1, 12 and 31 weeks after corneal HSV type 1 (HSV-1) inoculation were analyzed for evidence of productive or latent HSV-1 infection, inflammation and changes in neuron size, density and number. We found that latent HSV-1 infection between 12 and 31 weeks after corneal virus inoculation was associated with inflammation and progressive deficits in mean neuron diameter, neuronal nucleus diameter, neuron density and neuron number in the TG relative to mock-infected controls. The extent of neuronal injury during latent infection correlated with the extent of inflammation. These studies demonstrate that latent HSV infection is associated with progressive neuronal pathology and may lead to a better understanding of the role of HSV infections in chronic neurological diseases.     

Synthesis of the gB and gC glycoproteins of herpes simplex virus type 1 in the absence of viral DNA synthesis

Using an inhibitor of DNA synthesis as well as a temperature-sensitive (ts), DNA negative mutant of herpes simplex virus type 1 (HSV-1), we have examined the role of viral DNA synthesis on the expression of the virus-specific glycoproteins. Analysis by immunoblotting employing monospecific antisera revealed that only gC glycoprotein synthesis was completely inhibited in the absence of viral DNA synthesis. In contrast, the gB glycoprotein was detectable in significant, albeit somewhat reduced, amounts. These data suggest that gC is a ‘true-late’ protein of HSV-1 while gB is not. In addition, temperature shift-up experiments with the ts mutant (tsA1) suggest that some viral gene product(s) is needed continuously to achieve wild-type levels of gC synthesis.     

A thymidine kinase deficient HSV-2 strain causes acute keratitis and establishes trigeminal ganglionic latency,but poorly reactivates in vivo

The incidence of herpetic keratitis following in-tranasal or direct ocular infection with thymidine kinase-negative (TK^?) strains of herpes simplex virus (HSV)-2 has not been well studied, and the role of the TK gene in the establishment of latency and virus reactivation is controversial. To determine whether a TK^? strain of HSV-2 could establish trigeminal ganglionic latency and be reactivated in vivo to produce recurrent keratitis or nervous system infection, an animal model of acute and recurrent infection was utilized. Rabbits were infected by the intranasal or ocular routes, and latency was reactivated by immuno-suppression. Virus shedding in nasal and ocular secretions was monitored, and the eyes were examined for the presence of corneal epithelial lesions during acute and reactivated infections. Central nervous system (CNS) and trigeminal ganglionic tissues were assayed by histologic, virologic, and in situ hybridization techniques. All rabbits intranasally infected shed virus in both ocular and nasal secretions, whereas only 30% of rabbits infected in the eyes shed virus in nasal secretions. Virus was recovered from co-cultivation cultures, but not from cell-free ho-mogenates, of trigeminal ganglionic and CNS tissues from animals inoculated by both routes. The incidence of keratitis was much greater after direct ocular inoculation, although both routes of inoculation produced CNS and ganglionic inflammatory lesions. Keratitis healed in 92% of the animals infected by the ocular route by 26 days post infection. Of rabbits initially infected in the eyes and then subjected to drug-induced reactivation, only 30% shed virus, which was limited to a 24 hour period; there was no reappearance of epithelial keratitis, no animal became blind, and none died. In contrast, latently infected control rabbits uniformly reactivated. These studies show that this TK^? HSV-2 strain (i) replicates in the eye, (ii) is neuroinvasive but non-neurovirulent following intranasal and direct ocular infection; (iii) sheds in the eye more frequently and for longer periods after ocular than after intranasal inoculation; (iv) induces epithelial keratitis that usually heals spontaneously; (v) establishes latency in trigeminal ganglionic neurons, but no other ganglionic cells; and, (vi) reactivates in a small proportion of animals, but does not produce recurrent ocular lesions following drug-induced immunosuppres-sion. Thus, the TK gene appears directly involved in HSV latency and reactivation in vivo. © 1994 Wiley-Liss, Inc.     

Functional cross-reactivity between the glycoprotein B of herpes simplex virus type 1 and Epstein-Barr virus

A monoclonal antibody (T157) directed against gB-1, the glycoprotein B (gB) of herpes simplex virus-1 (HSV-1) shows positive indirect immunoflourescent staining with an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line B95-8. SDS PAGE and Western blot analysis show that B95-8 cells contain a 110,000 MW protein that co-migrates with the 110,000-115,000 MW gB-1. The gB-1 homologue of EBV (gB-EBV), immunopurified using a T157 affinity column, cross-stimulates HSV-1 immune T cells to proliferate in vitro. Mice immunized by a single subcutaneous injection of 30 microg gB-EBV in saline developed significant protection against HSV-1 challenge infection. Therefore gB-EBV can be considered a potential candidate vaccine and as an antigen to examine the cell-mediated immune response mounted by the host to limit virus spread during productive infection. The significance of a better understanding of the immune response to this and other EBV proteins of productive infection as an alternative to limit tumour growth by preventing virus spread is discussed.