A synthetic peptide induces long-term protection from lethal infection with herpes simplex virus 2.

Immunization against viral pathogens is generally directed toward the induction of virus neutralizing antibody (VNA) and the maintenance of the potential for a second-set (IgG) response. Indeed, an elevated level of specific antibody is considered a reliable clinical indicator that a state of immunity exists in the host. However, in the case of herpes simplex virus (HSV), the presence of circulating VNA does not necessarily correlate with protection. Thus, it has been found that secondary infections occur in individuals even with high neutralizing titers to HSV, suggesting that antibody to the virus may be useless or even deleterious. In consideration of these facts, we were interested in inducing a T cell response to HSV. We had already shown that synthetic peptides corresponding to the NH3-terminal region of the glycoprotein D (gD) molecule of HSV could induce a strong T cell response when injected into mice, but did not, by themselves, confer protection. In this report, we examined the ability of peptides, covalently coupled to palmitic acid and incorporated into liposomes, to induce virus-specific T cell responses that confer protection against a lethal challenge of HSV-2. We have demonstrated that long-term protective immunity is achieved with a single immunization in the absence of neutralizing antibody when antigen is presented in this form. Furthermore, T cells but not serum from such immune mice can adoptively transfer this protection.

Overlapping T cell antigenic sites on a synthetic peptide fragment from herpes simplex virus glycoprotein D, the degenerate MHC restriction elicited, and functional evidence for antigen-Ia interaction.

Analysis of the B10.A T cell response to synthetic peptides representing the NH2-terminal 23 amino acids from the HSV glycoprotein D sequence revealed two antigenic determinants for T cells: one localized between residues 1-16 and the other between residues 8-23. The 1-16 site, which is helical, was recognized in the context of the Ia molecule, whereas the 8-23 site, which is nonhelical, was recognized in the context of the I-E molecule. The I-E-restricted response was found to be highly MHC degenerate in that T cell hybridomas specific for the 8-23 peptide responded to antigen on APCs derived from B10.A, B10.A(5R), and B10.A(9R) mice and showed differences in antigenic fine specificity with APCs of different haplotypes. These data support the idea of antigen-Ia interaction.

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

Role of virus-induced neuropeptides in the brain in the pathogenesis of rabies.

Rabies virus (RABV) infection is characterized by the rapid neuronal spread of RABV into the CNS before a protective immune response is raised. Therefore, a typical feature of RABV infection is the paucity of inflammatory reactions in the brain. Here we examined whether the induction of immunosuppressive neuropeptides, in particular CGRP, may contribute to the ability of RABV to evade immune responses. RABV infection of mice caused a strong induction of calcitonin gene-related peptide (CGRP) in neurons and fibres in the neocortex as well as in the dentate gyrus and CA1 region of the hippocampus although RABV did not infect neurons in which CGRP expression was upregulated. Neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP) expressing neurons also were not infected by RABV. In contrast, somatostatin neurons were infected by RABV. There was evidence for an RABV-induced increase of VIP and somatostatin but not of NPY. To test how CGRP expression is related to TNFalpha-induced enhancement of CNS innate and adaptive immunity during RABV infection, we used recombinant RABVs that contained either an active (SPBN-TNFalpha(+)) or an inactive (SPBN-TNFalpha(-)) TNFalpha gene. As compared to SPBN-TNFalpha(-), infection with SPBN-TNFalpha(+) attenuated the induction of CGRP but simultaneously enhanced induction of the invariant chain of MHC II, microglial activation and T cell infiltration. In conclusion, distinct neuropeptidergic neurons in the brain are remarkably spared from RABV infection suggesting a pivotal role of neuropeptides during CNS virus infection. Given the inhibitory effect of CGRP on antigen presentation, we propose that the strong RABV-induced upregulation of CGRP in the brain may contribute to the mechanism by which RABV escapes immune detection. Targeting the expression of neuropeptides, in particular CGRP, that are induced during RABV infection may open a new avenue for therapeutic intervention in human rabies.

Use of mouse anti-rabies monoclonal antibodies in postexposure treatment of rabies.

Immunization of mice and hamsters with a cocktail of mouse MAbs specific for rabies virus nucleocapsid protein and glycoprotein protected animals not only when challenged with a lethal dose of rabies virus after immunization, but also in post-exposure situations. Hamsters treated with the MAb cocktail 3 h after virus inoculation were completely protected from lethal rabies virus infection, and 80% of the animals survived when the MAb cocktail was given 36 h after virus challenge. The potential usefulness of this MAb cocktail for the postexposure treatment of human rabies is discussed.

The phenotype of the RABV glycoprotein determines cellular and global virus load in the brain and is decisive for the pace of the disease.

The Rabies lyssavirus glycoprotein (RABV-G) is largely responsible for the neuroinvasiveness of the virus and the induction of antiviral immune responses. To study the effects of RABV-G we compared the G of the attenuated RABV variant SPBN with that of the pathogenic DOG4 strain. Infection via the olfactory route caused 100% mortality in mice with both virus variants. Of note, with the attenuated SPBN, progression of the disease was accelerated, microglia response less pronounced and IL-6 expression higher than in the presence of RABV-G from the pathogenic DOG4. However, while virus spread was less extensive, viral gene expression in individual neurons was actually higher in SPBN-infected brains without causing apoptosis of infected neurons. These differences between the two variants were not observed in infected neuronal cultures indicating that the effects of RABV-G on virus spread and viral gene expression depend on factors only present in the intact brain.

Pathogenesis of rabies.

Rabies is a central nervous system (CNS) disease that is almost invariably fatal. The causative agent is rabies virus (RV), a negative-stranded RNA virus of the rhabdovirus family. RV pathogenesis, like that of other viruses, is a multigenic trait. Recent findings indicate that in addition to the RV G protein viral elements that regulate gene expression, especially expression of the L gene, are also likely to play a role in RV pathogenesis. In vivo, RV infects almost exclusively neurons, and neuroinvasiveness is the major defining characteristic of a classical RV infection. A key factor in the neuroinvasion of RV is transsynaptic neuronal spread. While the ability of RV to spread from the post-synaptic site to the pre-synaptic site is mediated by the RV G protein, the RV P protein might be an important determinant of retrograde transport of the virus within axons. Although the mechanism(s) by which an RV infection cause(s) a lethal neurological disease are still not well understood, the most significant factor underlying the lethal outcome of an RV infection appears to be the neuronal dysfunction due to drastically inhibited synthesis of proteins required in maintaining neuronal functions.

Antibody-mediated clearance of viruses from the mammalian central nervous system.

The novel role of antibody in clearing virus from the central nervous system without the help of other immune effectors is an important phenomenon that has only recently been documented. Possible routes for antibodies across the blood-brain barrier and how they work in the CNS are discussed here.

New developments in the pre- and post-exposure treatment of rabies.

Two approaches have been made to develop the alleged "second generation rabies vaccines". One utilizes recombinant DNA, expressing the G protein gene in a heterotypic host, the vaccinia virus, and has proven efficacious in protecting animals from rabies infection. The second approach posits that only a portion of the native viral antigen may be required to induce protective response. To define regions of the subunit molecule recognized by different immune effectors, epitopes on the G protein and nucleoprotein were identified which are recognized by B and T cells. These epitopes were delineated using chemically or enzymatically cleaved fragments of the native antigens or overlapping synthetic peptides covering the entire antigenic amino acid sequence. Protein fragments or synthetic peptides evincing antigenic activity were tested for ability to induce virus-specific immune responses in vivo. Immunization with synthetic peptides carrying epitopes for B and T cells conferred solid protection against lethal rabies virus infection.

Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain.

The cytotoxic T-lymphocyte (CTL) response to rabies virus glycoprotein has been studied. A primary in vivo CTL response was obtained following inoculation of A/J mice with 10 micrograms of glycoprotein, but only when in the form of reconstituted glycoprotein-lipid vesicles. These glycoprotein-lipid vesicles were prepared with lipids from BHK-21 cells, and did not incorporate mouse major histocompatibility complex (MHC) antigens. Secondary in vitro stimulation of rabies virus-specific CTL was obtained with inactivated virus and with larger quantities of glycoprotein. This response, but not that induced by rabies virus-infected stimulator cells, was dependent on the presence of radiation-resistant accessory cells which could not be replaced with T-cell growth factors. An analysis of the molecular requirements for stimulation of CTL by glycoprotein revealed that cleavage by cyanogen bromide (CNBr) or limited tryptic digestion actually enhanced stimulation of CTL. In contrast, reduction and alkylation destroyed activity. Following separation of CNBr or tryptic fragments by gel electrophoresis or high-pressure liquid chromatography (HPLC) (under nonreducing conditions), a nominal determinant of glycoprotein was identified. One CNBr peptide (residues 103-178) and one peak of tryptic peptides were found to stimulate rabies virus-specific CTL. The tryptic peak was further analyzed by Edman degradation-sequencing, and found to consist of three peptides with amino terminals at residues 130, 251 and 279. This evidence suggests that a nominal determinant of glycoprotein responsible for stimulating rabies virus-specific CTL is located between residues 130-178 of the glycoprotein, and incorporates a single disulfide loop (159-169) which is necessary for biologic activity.

Comparison of the ribonucleoproteins of different rabies virus serotypes by radioimmunoassay.

Radioimmunoassay (RIA) provides a sensitive serological procedure for detecting rabies virus ribonucleoprotein (RNP) as well as its specific antibodies, RIA was carried out using highly purified RNPs labelled by the chloramine-T method. This paper describes optimal conditions for iodination of RNP with high specific activity. The optimal concentrations of 125I, RNP, chloramine-T, and reducing agent as well as the effect of pH on the reaction were investigated. RIA proved to be extremely sensitive for detection of homologous antibodies. In competition experiments the part-relationship of the group-specific RNPs of the three rabies virus serotypes (HEP, MOK and LBV) was confirmed.

High level expression of a human rabies virus-neutralizing monoclonal antibody by a rhabdovirus-based vector.

Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or consistent in their biological activity. An ideal reagent would consist of a panel of human monoclonal antibodies. Such antibodies are now available, their only drawback being the cost of production. Using recombinant technology, we constructed a rabies virus-based vector which expresses high levels (approximately 60 pg/cell) of rabies virus-neutralizing human monoclonal antibody. The vector is a modified vaccine strain of rabies virus in which the rabies virus glycoprotein has been replaced with a chimeric vesicular stomatitis virus glycoprotein, and both heavy and light chain genes encoding a human monoclonal antibody have been inserted. This recombinant virus can infect a variety of mammalian cell lines and is non-cytolytic, allowing the use of cell culture technology routinely employed to produce rabies vaccines.

The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure.

To provide a more defined and safer replacement for the human rabies immune globulin (HRIG) from pooled serum which is currently used for treatment of exposure to rabies virus we have developed a series of human rabies virus-specific monoclonal antibodies. Mouse-human heterohybrid myeloma cells producing rabies virus-specific human monoclonal antibodies were prepared using B cells obtained from volunteers recently-immunized with a commercial rabies virus vaccine (HDCV). Cell lines producing antibody which neutralized the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus strain in vitro were cloned and the resulting monoclonal antibodies characterized for isotype, specificity against a variety of rabies virus isolates, and neutralization capacity. The ability of the monoclonal antibodies to neutralize a variety of rabies virus strains in vitro correlated with their binding specificity for these viruses in an enzyme-linked immunoadsorbant assay (ELISA). A number of these antibodies have proven suitable for the formulation of a prophylactic human monoclonal antibody-based reagent which would provide significant advantages to the HRIG in having defined, reproducible specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

Localization and immunological characterization of antigenic domains of the rabies virus internal N and NS proteins.

To locate epitopes on internal antigens of rabies virus, purified N and NS proteins of the nucleocapsid were cleaved at methionine, tryptophan or glutamic acid residues, transferred to nitrocellulose and immunostained using monoclonal antibodies (MAbs) specific for N and NS proteins, respectively. Five MAb-positive fragments of N protein and one fragment of NS protein were located after NH2-terminal amino acid sequence analysis within the deduced amino acid sequences of N and NS proteins. Antigenic analysis of synthetic overlapping peptides corresponding to the amino acid sequences of these fragments localized two major antigenic sites of N protein and one antigenic site of NS protein. Like the N- and NS-specific MAbs, anti-peptide antisera produced against the different synthetic antigens either reacted in a type-common fashion with all rabies virus strains, or in a type-specific manner with a restricted number of strains. The synthetic peptides corresponding to the three antigenic regions of the N and NS proteins also stimulated proliferation of human T lymphocytes derived from vaccinees who received inactivated rabies virus vaccine. This suggested that the antigenic regions of N and NS proteins are recognized by both B and T cells.

Rabies virus cross-reactive murine T cell clones: analysis of helper and delayed-type hypersensitivity function.

Three T cell clones derived from rabies virus-immunized BALB/c mice were analysed for specificity and function. The clones proved to be broadly cross-reactive by responding to different rabies virus isolates (PM, ERA, CVS, HEP) and other representatives of the genus Lyssavirus, like the Duvenhage-6 (DUV6) and Mokola (MOK) viruses. The clones detected three different epitopes: an epitope expressed on the matrix protein (M) shared by PM, HEP, MOK and DUV6 viruses (clone AA8), an epitope expressed on the M-protein shared by PM, ERA, CVS, HEP and MOK viruses (clone 35A) and finally an epitope expressed on the glycoprotein (G-protein) shared by PM, ERA, CVS, HEP and MOK viruses (clone BG2). Antigen recognition of all clones proved to be MHC-restricted and they all displayed the CD4+ CD8- phenotype. Intravenous inoculation of the T cells in syngeneic mice, which had been injected intracutaneously in the ear with HEP virus, resulted in a localized DTH reaction characteristic for TH1 cells. In vitro, the clones were able to provide help to rabies virus-primed B cells, resulting in the production of virus-specific antibodies directed against all the four structural proteins of rabies virus. Further analysis of this antibody response revealed that part of it was directed against antigenic determinants of the G-protein which induce virus neutralizing antibody.

Rhodopsin and retinoblastoma. A monoclonal antibody histopathologic study.

Rhodopsin was identified in formaldehyde-fixed, paraffin-embedded human fetal retina, and in five retinoblastomas using monoclonal antibody (MAb) MAb-E. The binding pattern corresponding to rhodopsin immunoreactivity was then compared with S-antigen using another monoclonal antibody, MAbA9-C6. Rhodopsin and S-antigen were first observed in the 18-week-old human fetal eye, at a stage preceding photoreceptor differentiation. In adult eyes containing normal photoreceptor cells, rhodopsin immunoreactivity was restricted to the rod outer segments, whereas S-antigen immunoreactivity was localized to the entire photoreceptor cell. In retinoblastomas both monoclonal antibodies bound to the same area of the tumor; however, different and distinct staining patterns associated with each monoclonal antibody were recognized. In four cases, an intense well-circumscribed "halo" pattern, characteristic of cell-surface binding, was associated with rhodopsin, whereas the binding pattern associated with S-antigen was intense, well localized, and cytoplasmic in all cases. Our results show that some well-differentiated retinoblastomas express both rhodopsin and S-antigen, and as such express proteins that participate in the initial events in the phototransduction of vision.

Expression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyelitis.

In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE became evident 5 days following cell transfer. C1q positive cells, as identified by immunohistology, were preferentially localized in grey and white matter of the hippocampus and basolateral cortex. The C1q positive cells resembled microglial cells in morphology. The correlation of C1q expression with the development of neurological disease as well as the dramatic increase of C1q within brain regions with inflammatory lesions suggest that local biosynthesis of C1q may play a role in the pathogenesis of Borna virus induced and autoimmune encephalomyelitis.

Detection of Borna disease virus-reactive antibodies from patients with affective disorders by western immunoblot technique.

Borna disease (BD) virus is a partially characterized neurotropic agent with a predilection for neurons and astrocytes in the limbic system and cerebrum of infected hosts. Although it usually causes a fatal encephalitis, some laboratory animals which have been experimentally inoculated can develop a persistent non-fatal infection characterized by a neuro-behavioral syndrome akin to human manic-depression. Using immunofluorescent techniques, we previously observed BD virus-specific antibodies in the sera of 4.5% of affectively ill patients, with the highest titers present in bipolar patients. More recently, we have developed a sensitive Western blot assay for the detection of anti-BD virus antibodies to a 38/40 kDa and 24 kDa protein in human serum. In the present study, we screened 138 affectively ill patients and 117 healthy controls and observed a significantly great proportion of patients with antibodies to the 38/40 kDa protein (P < 0.0001), the 24 kDa protein (P < 0.05) and both the 38/40 kDa and 24 kDa proteins (P < 0.025). These data extend prior reports on the presence of BD virus-specific antibodies in psychiatric patients, and suggest that a BD virus-like agent may be associated with affective illness in humans.

S-antigen: identification of the MAbA9-C6 monoclonal antibody binding site and the uveitopathogenic sites.

The location of the monoclonal antibody, MAbA9-C6, binding site and two uveitopathogenic sites in S-antigen have been determined. Using cyanogen bromide, S-antigen was cleaved into nine peptides, designated C1 to C9. MAbA9-C6 bound selectively to one large peptide designated C5, consisting of 122 amino acids. Six peptides (20 to 22 amino acids in length) designated 2,3,K,L,N and M, corresponding to the entire length of peptide C5, were synthesized chemically. In a radioimmunoassay and a dot-binding immunoassay, MAbA9-C6 bound selectively to one of the six peptides, peptide 3, indicating that this region of peptide C5 contains the MAbA9-C6 binding site. Twelve smaller peptides (ten amino acids in length), corresponding to the amino acid sequence of peptide 3, were synthesized and used in a competitive inhibition binding assay. These studies localized the MAbA9-C6 binding site to a small region within peptide 3. In addition, peptide K and peptide M were highly pathogenic for the induction of experimental auto-immune uveitis (EAU). Clinical and histological evidence of a severe uveo-retinitis, indistinguishable from that seen with native S-antigen, was documented in Lewis rats immunized with the synthetic peptides (50 micrograms), 11-12 days following immunization. Our results show that the MAbA9-C6 binding site and the two uveitopathogenic sites lie in close proximity to each other within the region of S-antigen corresponding to peptide C5. Furthermore, microcomputer analysis of the average hydrophilicity/hydrophobicity values of the amino acid sequence corresponding to peptide C5 shows that the MAbA9-C6 binding site and one uveitopathogenic site (peptide K) lie on the adjacent peaks. The significance of these findings and their relationship to the role of S-antigen in the pathogenesis of EAU and the phototransduction of vision is discussed.

S-antigen: characterization of a pathogenic epitope which mediates experimental autoimmune uveitis and pinealitis in Lewis rats.

S-antigen (48K protein) is a photoreceptor cell protein highly pathogenic for the induction of experimental autoimmune uveitis (EAU) and intimately involved in the visual process. EAU is characterized, in part, as a T-cell mediated autoimmune disease which results in a severe inflammation of the uveal tract, and pineal gland. In order to determine specific sites in S-antigen responsible for its pathogenicity we synthesized twenty-three peptides, corresponding to the entire 404 amino acid sequence, and tested each peptide for its ability to induce EAU in Lewis rats. One peptide, peptide M (18 amino acids in length), was found to be highly pathogenic and consistently induced an EAU that was identical to the disease caused by native S-antigen. Clinically, the disease that developed in the eye was characterized by iris and pericorneal hyperemia, followed by inflammatory exudates in the anterior and vitreous chambers. Histopathologically a severe inflammatory response was observed which resulted in the complete destruction of the photoreceptor cell layer of the retina. In order to more fully characterize this pathogenic site, 14 additional smaller peptides (eight to eighteen amino acids in length) corresponding to the left and right portions of peptide M were synthesized. Of these peptides, peptide M16L, M15L, and M12L induced EAU, further localizing this pathogenic site to a small well-characterized region of S-antigen consisting of twelve amino acids. In addition, animals with ocular inflammatory disease had an associated pinealitis characterized by a lymphocytic infiltration of the subcapsular and central area of the pineal gland. The significance of these findings and the relationship of S-antigen in the pathogenesis of EAU and other autoimmune diseases is discussed.