Induction of Anti-Meningitis Immunity by Synthetic Peptides: II. Immunoactive Synthetic Fragments of the OpaB Protein from Neisseria meningitidis

Mice of various lines were immunized by 11 synthetic peptides that correspond to the sequences of fragments of the OpaB protein from the outer membrane of Neisseria meningitidis involving the known human T-helper epitopes and all the potential mouse T-helper epitopes calculated for the protein. The mice were immunized with the free peptides without their conjugation with a protein carrier. Most of the peptides were found to induce in mice the production of antipeptide antibodies. Mice protection against the experimental infection by a virulent strain of N. meningitidis of the B serotype was studied, and two peptides were shown to exert the most pronounced protective effect.     

Induction of anti-meningitis immunity using the synthetic peptides. I. The immunoreactive synthetic fragments of porin A from Neisseria meningitidis

Fourteen peptides corresponding to sequences of all the exposed and some of the transmembrane protein regions of porin A from the outer membrane of Neisseria meningitidis strain B:15:P1.7,16 were synthesize. Mice of various lines were immunized with the free peptides not conjugated with any protein carrier. It was shown that the majority of the peptides possess immunogenic properties. Two peptides were identified binding to antibodies present in the serum of mice after meningitis. Protective properties of a number of the synthesized peptides were studied, and three peptide sequences inducing mice protection from an experimental infection with N. meningitidis were identified.     

Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein.

The T-helper cell response to the internal proteins of rabies virus was investigated. The rabies virus nucleoprotein was shown to be a major target antigen for T-helper cells that cross-react between rabies and rabies-related viruses. T-helper cells were assayed in vitro by testing virus-induced lymphocytes for lymphokine secretion in response to antigen. Immunodominant T-helper cell epitopes of the viral nucleoprotein were identified in vitro by using synthetic peptides delineated from the amino acid sequence of the nucleoprotein. The response to synthetic peptides were under Ir gene control. Antigenic peptides were tested in vivo for stimulation of rabies virus-specific T-helper cells. Inoculation of mice with peptides bearing immunodominant T-helper cell epitopes resulted in an accelerated and enhanced neutralizing antibody response upon booster immunization with inactivated rabies virus.     

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.     

Induction of the anti-meningitis immunity with synthetic peptides. III. Immunoactive synthetic fragments of NspA protein from Neisseria meningitidis

Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the bacterium Neisseria meningitidis were synthesized in order to create a synthetic vaccine against the meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with the free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

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.     

Synthesis, immunogenicity and antigenic characteristics of the glycoprotein E fragments from 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.     

Production of antibodies to the alpha7-subunit of human acetylcholine nicotinic receptor with the use of immunoactive synthetic peptides

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the alpha7-subunit of human acetylcholine receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four alpha7-subunit regions containing 16-23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR alpha7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR alpha7-subunit and do not react with the analogous domain of the alpha1-subunit of the ray Torpedo californica AChR.     

Cell mediated immune response elicited in mice after immunization with the P64k meningococcal protein: epitope mapping

The P64k protein of Neisseria meningitidis has been reported as an immunological carrier for weak immunogens. This investigation was aimed at characterizing the T-cell response produced in primed mice and at identifying T helper cell epitopes within this molecule. BALB/c mice subcutaneously immunized with the recombinant antigen provided inguinal lymph node cells (LNC) that proliferated in the presence of P64k in a dose-dependent manner. Proliferating cells secreted IL-4 while the concentration of IL-12 remained unaltered in the culture supernatant. By testing a panel of 59 overlapping synthetic peptides spanning the entire sequence of the antigen a T-cell determinant was localized. Prime-boost and lymphoproliferation experiments, conducted with highly purified synthetic peptides, confirmed that the segment including amino acids 470-485 comprises a T-cell epitope within the P64k molecule.     

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.     

Human recognition of T cell epitopes on the Plasmodium vivax circumsporozoite protein.

In order to identify T cell epitopes recognized by human in the Plasmodium vivax circumsporozoite protein, 28 overlapping synthetic peptides spanning the entire circumsporozoite protein were tested for their ability to stimulate proliferation of PBMC from 22 adults living in a malaria-endemic area of the Colombian Pacific Coast and from four individuals who never had a history of malaria infection. In addition, BALB/c mice were immunized with pools of peptides, and their lymph node cells were stimulated in vitro with individual peptides. Four epitopes were recognized by human lymphocytes but not all of them by mice. One of the epitopes was located inside the central repetitive B cell immunodominant domain. Several of the variants of the repeats were recognized by about one-third of the studied individuals. Another T cell epitope was located in the amino terminus and the other two in the carboxyl region. Peptides were recognized by both immune and nonimmune donors. Some of them were frequently recognized suggesting a lack of genetic restriction, whereas some others were recognized by only a few individuals but induced strong proliferation. These epitopes may be of potential value for a malaria subunit vaccine.     

Structural and immunogenicity analysis of chimeric B-cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV-1.

B-cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T-cell lymphotropic virus type 1 (HTLV-1) by computer-aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T-helper epitopes derived either from the tetanus toxoid (amino acids 947-967) or measles virus fusion protein (amino acids 288-302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus-induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

Induction of antimeningitis immunity by the synthetic peptides: I. The immunoactive synthetic fragments of porin A fromNeisseria meningitidis

Fourteen peptides corresponding to sequences of all the exposed and some of the transmembrane protein regions of porin A from the outer membrane ofNeisseria meningitidis strain B:15:P1.7,16 were synthesized. Mice of various lines were immunized with the free peptides not conjugated with any protein carrier. It was shown that the majority of the peptides possess immunogenic properties. Two peptides were identified binding to antibodies present in the serum of mice after meningitis. Protective properties of a number of the synthesized peptides were studied, and three peptide sequences inducing mice protection to an experimental infection withN. meningitidis were identified.     

Identification of T-helper epitopes in the VP1 capsid protein of poliovirus.

Poliovirus-specific T lymphocytes were isolated from virus-immunized mice of different H-2 haplotypes. Immunological characterization of this population indicates that the effector population involved in the observed poliovirus-specific proliferative response was that of CD4-positive T-helper cells. Proliferative responses also were induced within these T-lymphocyte populations upon stimulation with either purified VP1 capsid protein or VP1 synthetic peptides. By using these synthetic peptides, several T-helper epitopes were identified. Generally, proliferative responses were observed in three regions of VP1. Two regions spanning VP1 residues 86 to 120 and 201 to 241 were recognized by T lymphocytes from BALB/c (H-2d), C57BL/6 (H-2b), and C3H/HeJ (H-2k) backgrounds. Analyses using synthetic peptides of nonoverlapping sequences indicated that the region spanning residues 201 to 241 may contain several T epitopes and may account for the strong proliferative response observed. In addition, for two of the three haplotypes examined, T epitopes were observed within residues 7 to 24 of VP1. Additional epitopes which appeared to be restricted to specific H-2 backgrounds were identified. T epitopes within VP1 that are common between different strains of mice appeared to lie within previously identified neutralizing antigenic sites in poliovirus.     

Conserved T and B cell epitopes on the M protein of group A streptococci. Induction of bactericidal antibodies.

To identify conserved T and B cell epitopes on the M protein of group A beta-hemolytic streptococci, overlapping synthetic peptides that span the conserved carboxyl-terminal segment of the M-5 protein were constructed and used to immunize a panel of H-2 congenic mice. Proliferative T cell epitopes were identified and, in many cases, mice immunized with these peptides produced high titer antibodies to the same peptides indicating that these proliferative epitopes could also stimulate Th cells. Peptide-specific T cells and antisera were tested for their reactivity with porcine myosin, tropomyosin, human heart myosin synthetic peptides, and extracts of human pericardial and atrial heart tissue. Although there was minimal response of M peptide-specific T cells to any of these Ag, certain M peptide-specific antisera reacted to immunoblotted porcine myosin and to an immunoblotted extract of human atrial heart tissue. However, two conserved peptides, LRRDLDASREAKKQVEKALE and KLTEKEKAELQAKLEAEAKA, stimulated peptide-specific antibodies in B10.BR and B10.D2 mice respectively, which reacted minimally if at all with human atrial heart tissue extract. Furthermore, antisera to the former peptide, in a bactericidal assay involving human monocytes, could mediate killing of streptococci (82% of bacteria). Although this level of killing is less than that produced by antisera to the highly polymorphic type-specific aminoterminus (up to 100% killing), it provides evidence that conserved epitopes can be the targets of bactericidal antibodies. These conserved epitopes may be useful in a vaccine because they also stimulate T cells, thus allowing development of immunologic memory and natural boosting of an immune response after natural exposure.     

Epitopes of group A streptococcal M protein that evoke cross-protective local immune responses.

The present studies were undertaken to identify conserved epitopes of group A streptococcal M proteins that evoke cross-protective mucosal immune responses. Two synthetic peptides copying conserved regions of type 5 M protein, designated SM5(235-264)C and SM5(265-291)C, were covalently linked to carrier molecules and their immunogenicity was tested in laboratory animals. Rabbit antisera against both peptides cross-reacted with multiple serotypes of group A streptococci, indicating that the peptides contained broadly cross-reactive, surface exposed M protein epitopes. Serum antipeptide antibodies adsorbed to the surface of heterologous type 24 streptococci passively protected mice against intranasal challenge infections. Mice that were actively immunized intranasally with each synthetic peptide covalently linked to the B subunit of cholera toxin were protected against colonization and death after intranasal challenge infections with type 24 streptococci in the absence of serum opsonic antibodies. These data confirm and extend previous observations that conserved M protein epitopes evoke cross-protective local immunity and may serve as the basis for broadly cross-protective M protein vaccines.     

Differing T-cell requirements for recombinant retrovirus vaccines.

Friend murine leukemia virus is a retrovirus complex that induces rapid erythroleukemia and immunosuppression in susceptible strains of adult mice. Using this model, we directly examined the T-cell subsets required for a protective retrovirus vaccine. Paradoxically, recovery in mice immunized with a chimeric envelope containing only T-helper (TH) and B-cell epitopes was dependent on CD8+ T cells as well as CD4+ T cells despite the fact that the vaccine contained no CD8+ cytolytic T-lymphocyte (CTL) epitopes. However, the requirement for CD8+ T cells was overcome by inclusion of additional TH and B-cell epitopes in the immunizing protein. These additional epitopes primed for more rapid production of virus-neutralizing antibody which appeared to limit virus spread sufficiently to protect even in the absence of CD8+ T cells. Inclusion of an immunodominant CTL epitope in the vaccine was not sufficient to overcome dependence on CD4+ T cells. These data suggest that TH priming is more critical for retrovirus immunity than CTL priming.     

Production of antibodies to the α7-subunit of human acetylcholine receptor with the use of immunoactive synthetic peptides

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the α7-subunit of human acetylchloline receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four α7-subunit regions containing 16–23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR α7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR α7-subunit and do not react with the analogous domain of the α1-subunit of the ray Torpedo californica AChR.     

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.