Synthesis,Immunogenicity, and Antigenicity of the Glycoprotein E Fragments of the Tick-Borne Encephalitis Virus

Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.     

Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16- to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

Synthetic fragments of the NS1 protein of the tick-borne encephalitis virus exhibiting a protective effect

Potentially immunoactive regions of the NS1 nonstructural protein of the tick-borne encephalitis virus that can stimulate the antibody formation in vivo and protect animals from this disease were chosen on the basis of theoretical calculations. Eleven 16-to 27-aa peptides containing the chosen regions were synthesized. The ability of the free peptides (without any high-molecular-mass carrier) to stimulate the production of antipeptide antibodies in mice of three lines and ensure the formation of protective immunity was studied. Most of these peptides were shown to exhibit the immunogenic activity in a free state. Five fragments that can protect mice from the infection by a lethal dose of tick-borne encephalitis virus were found.     

Study of the antigenic structure of tick-borne encephalitis virus using synthetic peptides]

A number of peptides, fragments of the envelope protein E of the tick-borne encephalitis virus (Sofjin strain), were synthesized. Their binding to the polyclonal antiserum to protein E was studied. Rats were immunized with both the free peptides and their KLH-conjugates, and the resulting antisera were tested for their reactivity toward protein E and for their neutralizing activity toward the virus in cell culture. The only peptide corresponding to the 98-113 sequence of protein E was shown to be bound by the protein E antiserum in EIA. Two-fold immunization of rats with KLH-conjugates of the peptides corresponding to the 98-113, 130-143, and 394-403 sequences of protein E resulted in antipeptide antibodies capable of binding the native protein E, and the antibodies to the 98-113 and 394-403 peptides were capable of neutralizing the virus.     

Results of using the ELISA method in studying tick-borne encephalitis and rabies

The results of using the indirect variant of ELISA for the study of serum samples from humans and white mice for the presence of antibodies to tick-borne encephalitis virus and rabies virus are presented. The high sensitivity and specificity of this method have been confirmed.     

Study of the antigenic structure of the E1 glycoprotein of the Venezuelan equine encephalomyelitis virus using monoclonal antibodies]

The collection of eight rat and mouse hybridomas secreting the high affinity monoclonal antibodies to glycoprotein E1 of the Venezuelan equine encephalomyelitis has been obtained. The antigenic structure of E1 protein has been studied with the use of these antibodies for the strains Trinidad, TC-83 and 230 of the virus. Antigenic map of glycoprotein E1 based on competition radioimmunoanalysis is proposed. Five sites are mapped including eight epitopes binding monoclonal antibodies. Antibodies to sites E1-1, E1-3 and E1-5 are crossreactive in interaction with the virus of Venezuelan equine encephalomyelitis, while antibodies to site E1-5 interact also with the virus of tick-borne encephalitis. Antibodies to site E1-1 possess the protective effect and lack the neutralizing effect in tissue cultures. Antibodies to all sites of E1 protein are devoid of ability to neutralize the Venezuelan equine encephalitis virus.     

A Japanese encephalitis virus peptide present on Johnson grass mosaic virus-like particles induces virus-neutralizing antibodies and protects mice against lethal challenge

Protection against Japanese encephalitis virus (JEV) is antibody dependent, and neutralizing antibodies alone are sufficient to impart protection. Thus, we are aiming to develop a peptide-based vaccine against JEV by identifying JEV peptide sequences that could induce virus-neutralizing antibodies. Previously, we have synthesized large amounts of Johnson grass mosaic virus (JGMV) coat protein (CP) in Escherichia coli and have shown that it autoassembled to form virus-like particles (VLPs). The envelope (E) protein of JEV contains the virus-neutralization epitopes. Four peptides from different locations within JEV E protein were chosen, and these were fused to JGMV CP by recombinant DNA methods. The fusion protein autoassembled to form VLPs that could be purified by sucrose gradient centrifugation. Immunization of mice with the recombinant VLPs containing JEV peptide sequences induced anti-peptide and anti-JEV antibodies. A 27-amino-acid peptide containing amino acids 373 to 399 from JEV E protein, present on JGMV VLPs, induced virus-neutralizing antibodies. Importantly, these antibodies were obtained without the use of an adjuvant. The immunized mice showed significant protection against a lethal JEV challenge.     

A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response.

A solid-phase 8-mer random combinatorial peptide library was used to generate a panel of mimotopes of an epitope recognized by a monoclonal antibody to the F protein of measles virus (MV). An inhibition immunoassay was used to show that these peptides were bound by the monoclonal antibody with different affinities. BALB/c mice were coimmunized with the individual mimotopes and a T-helper epitope peptide (from MV fusion protein), and the reactivity of the induced anti-mimotope antibodies with the corresponding peptides and with MV was determined. The affinities of the antibodies with the homologous peptides ranged from 8.9 x 10(5) to 4.4 x 10(7) liters/mol. However, only one of the anti-mimotope antibodies cross-reacted with MV in an enzyme-linked immunosorbent assay and inhibited MV plaque formation. Coimmunization of mice with this mimotope and the T-helper epitope peptide induced an antibody response which conferred protection against fatal encephalitis induced following challenge with MV and with the structurally related canine distemper virus. These results indicate that peptide libraries can be used to identify mimotopes of conformational epitopes and that appropriate immunization with these mimotopes can induce protective antibody responses.     

Naked DNA vaccines expressing the prM and E genes of Russian spring summer encephalitis virus and Central European encephalitis virus protect mice from homologous and heterologous challenge.

Naked DNA vaccines expressing the prM and E genes of two tick-borne flaviviruses, Russian spring summer encephalitis (RSSE) virus and Central European encephalitis (CEE) virus were evaluated in mice. The vaccines were administered by particle bombardment of DNA-coated gold beads by Accell gene gun inoculation. Two immunizations of 0.5 to 1 microg of RSSE or CEE constructs/dose, delivered at 4-week intervals, elicited cross-reactive antibodies detectable by enzyme-linked immunosorbent assay and high-titer neutralizing antibodies to CEE virus. Cross-challenge experiments demonstrated that either vaccine induced protective immunity to homologous or heterologous RSSE or CEE virus challenge. The absence of antibody titer increases after challenge and the presence of antibodies to E and prM, but not NS1, both before and after challenge suggest that the vaccines prevented productive replication of the challenge virus. One vaccination with 0.5 microg of CEE virus DNA provided protective immunity for at least 2 months, and two vaccinations protected mice from challenge with CEE virus for at least 6 months.     

A comparison of the immune response induced by DNA or by an inactivated vaccine against tick-borne encephalitis

BALB/c mice were immunized with recombinant plasmid DNA pSVK3-ENS1 and pcDNAI-NS3 containing, respectively, genes E-NS1 and NS3 of tick-borne encephalitis (TBE) virus. Antibodies to TBE virus proteins were detected in the blood sera of the immunized animals by the method of the enzyme immunoassay. Though the titers of virus-specific antibodies in the sera of mice immunized with protein vaccines exceeded those registered after immunization with DNA vaccines, essential protective immunity was observed after the use of both vaccines.     

Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype

Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.     

Neutralizing Antibodies Protect against Lethal Flavivirus Challenge but Allow for the Development of Active Humoral Immunity to a Nonstructural Virus Protein

Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.     

Biological properties of strains of tick-borne encephalitis virus isolated in the natural foci of the eastern part of the Russian plain

Strains of tick-borne encephalitis virus isolated in the natural foci of infection in the eastern part of the Russian Plain (the Kirov region) were examined for their biological properties. The strains examined were 69 strains isolated from ticks Ixodes persulcatus, 62 strains obtained from patients with the clinically manifest form of tick-borne encephalitis and 56 strains isolated from the blood of patients with the inapparent form of infection. Comparative studies on laboratory animals (albino mice, golden hamsters, suckling guinea pigs and other mammals) as well as comparative serologic studies provided evidence which suggested that all virus isolates from the Kirov region were antigenically identical with the strain "Sofin" isolated in the Far East and represent thus a single causative agent of the tick-borne encephalitis virus infection. This strain of virus is supposed to exist in two variants, in dependence on ecological conditions: one of these variants is the eastern variant (strain Sofin and strains from the Kirov region) and the other one is the western variant of tick-borne encephalitis virus.     

A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.     

Identification of an immunodominant neutralizing and protective epitope from measles virus fusion protein by using human sera from acute infection.

Polyclonal sera obtained from African children with acute measles were used to screen a panel of 15-mer overlapping peptides representing the sequence of measles virus (MV) fusion (F) protein. An immunodominant antigenic region from the F protein (p32; amino acids 388 to 402) was found to represent an amino acid sequence within the highly conserved cysteine-rich domain of the F protein of paramyxoviruses. Epitope mapping of this peptide indicated that the complete 15-amino-acid sequence was necessary for high-affinity interaction with anti-MV antibodies. Immunization of two strains of mice with the p32 peptide indicated that it was immunogenic and could induce antipeptide antibodies which cross-reacted with and neutralized MV infectivity in vitro. Moreover, passive transfer of antipeptide antibodies conferred significant protection against fatal rodent-adapted MV-induced encephalitis in susceptible mice. These results indicate that this epitope represents a candidate for inclusion in a future peptide vaccine for measles.     

Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions affixed to tissue culture plates. The monoclonal antibody had a greater ability to compete for virus binding, which suggested that differences in neutralizing titers may relate to the relative affinities of these antisera for the peptide conformation in the native structure.     

Synthetic peptide designs based on immunoactive fragments of the VP1 protein of the foot-and-mouth disease virus strain A22

Peptide constructs consisting of 44-53 aa were synthesized on the basis of sequences 135-159, 170-190 and 197-213 of VP1 from the foot-and-mouth disease A22 strain. Immunogenic and protective properties of the peptide constructs were studied in guinea pigs and mice of three lines. The constructs were shown to induce higher levels of antibodies and exhibit higher protective effects than the separate peptides. The most active among the peptides studied was the construct involving the VP1 fragments 135-160 and 170-190: it protected pigs from the experimental infection by the foot-and-mouth disease virus.     

Priming for rotavirus neutralizing antibodies by a VP4 protein-derived synthetic peptide.

In the rotavirus SA11 surface protein VP4, the trypsin cleavage sites associated with the enhancement of infectivity are flanked by two amino acid regions that are highly conserved among different rotaviruses. We have tested the ability of synthetic peptides that mimic these two regions to induce and prime for a rotavirus neutralizing antibody response in mice. After the peptide immunization schedule, both peptides induced peptide antibodies, but neither was able to induce virus antibodies, as measured by an enzyme-linked immunosorbent assay or a neutralization assay. However, when the peptide-inoculated mice were subsequently injected with intact SA11 virus, a rapid and high neutralizing antibody response was observed in mice that had previously received the peptide comprising amino acids 220 to 233 of the VP4 protein. This neutralizing activity was serotype specific; however, this peptide was also able to efficiently prime the immune system of mice for a neutralizing antibody response to the heterotypic rotavirus ST3 when the ST3 virus was used for the secondary inoculation.     

Expression of the NS1 gene of tick-borne encephalitis virus in gram-negative bacteria from the mouse nasopharynx

Bacteria were isolated from the nasopharynx of BALB/c mice and electroporated with pUR290(NS1)2 containing two copies of tick-borne encephalitis virus (TBEV) strain Sofjin NS1 under the control of the lac promoter. The plasmid persisted in transformants for at least ten passages. The NS1 gene expression was detected in Gram-negative enterobacteria via immunoblotting with monoclonal antibodies against TBEV nonstructural glycoprotein NS1. Recombinant NS1 was detected in bacterial cells and in the culture medium. Intranasal immunization with recombinant bacteria activated production of antibodies against NS1 in serum of BALB/c mice. The humoral immune response to NS1 failed to protect immunized mice from a TBEV challenge.     

Localization of the antigenic segment of the tick-borne encephalitis virus envelope protein using monoclonal antibodies]

The largest cyanogen bromide fragment (GP-14,5; coordinates 78-176) of E protein belonging to the envelope of the tick-borne encephalitis (TBE) virus (Far Eastern subtype, strain Sofjin) interacted with five out of twelve E-specific monoclonal antibodies (MAbs). Having compared; efficiencies of some MAbs binding to the antigens of TBE viruses of Far Eastern and West European subtypes and primary structures of analogous peptides of these viruses, we suggested the epitopes of these MAbs to be located in the vicinity of 89 and/or 116-th amino acid residues of E protein. Effect of denaturing agents and reduction followed by carboxymethylation on the protein E antigenic properties was studied.