Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes

A polypeptide fragment obtained by CNBr cleavage of the hemagglutinin from A/JAPAN/305/57 influenza virus has been purified by using high performance liquid chromatography. The first five N-terminal amino acids as determined by sequential Edman degradations have localized this peptide to the HA2 subunit of the hemagglutinin between residues 103 and 123. This peptide, denoted HA2(103-23), can generate both proliferative and cytolytic responses from spleen cells of BALB/c mice previously immunized with A/JAPAN/305/57. These results demonstrate that a single nonglycosylated fragment of the influenza hemagglutinin as small as 21 amino acid residues is capable of being recognized as an antigenic determinant to generate influenza CTL from primed precursors.     

Immunogenic peptides of influenza virus subtype N1 neuraminidase identify a T-cell determinant used in class II major histocompatibility complex-restricted responses to infectious virus

Six nonoverlapping peptides of the neuraminidase (NA) glycoprotein of influenza virus A/Puerto Rico/8/34 (H1N1) (PR8 virus) were found to be immunogenic for proliferating T cells when injected into BALB/c mice in Freund adjuvant. T cells elicited by peptide immunization could recognize PR8 virus in vitro. However, only one of these peptides, corresponding to residues 79 to 93 of NA (NA 79-93), was able to restimulate T cells of mice immunized with infectious virus. T cells that recognized this peptide were uniformly I-Ed restricted, yet infectious influenza virus was required for responses. NA 79-93-specific T-hybridoma clones raised by immunization either with whole virus or with the synthetic peptide alone each responded to replicative virus and not to UV-inactivated virions. These data suggest that the NA 79-93 T-cell determinant which is commonly presented during an encounter with influenza virus in vivo is processed preferentially from NA synthesized within antigen-presenting cells.     

Single amino acid substitution alters T cell determinant selection during antigen processing of Staphylococcus aureus nuclease.

The effect of amino acid residues outside of T cell determinant regions of Staphylococcus aureus nuclease (Nase) on the activation of T cell hybridomas has been investigated. T cell hybridomas derived from BALB/c mice immunized with Nase were screened against a nested set of overlapping synthetic peptides spanning the entire Nase molecule. Five regions of Nase, encompassing residues 1 to 20, 21 to 40, 61 to 80, 101 to 120, and 112 to 130, were found to be the T cell determinants. Region 61 to 80 is the immunodominant site. Mutants of Nase with a single amino acid substitution outside the defined T cell determinants were tested for their ability to stimulate the T cell hybridomas. The substitution of arginine for glutamic acid at residue 43 markedly reduces the antigenic potency of the protein for I-Ed restricted T cell hybridomas, which recognize Nase peptides comprised of residues 21 to 40 (p21-40) or 112 to 130 (p112-130). In contrast, the stimulatory capacity of this mutant for I-Ad restricted T cell hybridomas remains unchanged. Our results suggest that selective regulation of an immune response may be achieved by appropriately mutagenizing protein Ag.     

Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.     

Immunogenic peptide comprising a mouse hepatitis virus A59 B-cell epitope and an influenza virus T-cell epitope protects against lethal infection.

The coronavirus spike protein S is responsible for important biological activities including virus neutralization by antibody, cell attachment, and cell fusion. Recently, we have elucidated the amino acid sequence of an S determinant common in murine coronaviruses (W. Luytjes, D. Geerts, W. Posthumus, R. Meloen, and W. Spaan, J. Virol. 63:1408-1412, 1989). A monoclonal antibody directed to this determinant (MAb 5B19.2) protected mice against acute fatal infection. In this study, BALB/c mice were immunized with a synthetic peptide of 13 amino acids corresponding to the binding site of MAb 5B19.2, which was either extended with an amino acid sequence of influenza virus hemagglutinin or conjugated to keyhole limpet hemocyanin. Both immunogens induced S-specific antibodies in mice, but only the hemagglutinin-peptide construct protected them against lethal challenge. In contrast to mouse hepatitis virus type 4 (MHV-4), MHV-A59 was not neutralized in vitro by MAb 5B19.2. Neither MHV-A59 nor MHV-4 was neutralized in vitro by antibodies comprising by the synthetic peptides. Our results demonstrated that antibodies elicited with a synthetic peptide comprising a B-cell epitope and a T-helper cell determinant can protect mice against an acute fetal mouse hepatitis virus infection.     

A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus

The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus.     

Modulation of the T cell response to beta-lactoglobulin by conjugation with carboxymethyl dextran

We have previously prepared beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates with water-soluble carbodiimide and achieved reduced immunogenicity of beta-LG. In the present study, to elucidate the mechanism for the reduced immunogenicity of beta-LG, we investigated changes in the T cell response to beta-LG after conjugation with CMDs differing in molecular weight (about 40 and 162 kDa). Lymph node cells from BALB/c, C3H/He, and C57BL/6 mice that had been immunized with beta-LG or the conjugates were stimulated with beta-LG, and the in vivo T cell response was then evaluated by BrdU (5-bromo-2'-deoxyuridine) ELISA as the ex vivo proliferative response. T cells from the conjugate-immunized mice showed a lower proliferative response than those from the beta-LG-immunized mice. T cell epitope scanning, using synthesized peptides, showed that the T cell epitope profiles of the conjugates were similar to those of beta-LG, whereas the proliferative response to each epitope was reduced. These results indicate that the lower in vivo T cell response with the conjugates was not due to induction of conjugate-specific T cells, but due to a decrease in the number of beta-LG-specific T cells. After the lymph node cells from beta-LG-immunized mice had been stimulated with beta-LG or the conjugates, the efficiency of the antigen presentation of the conjugate to beta-LG-specific T cells was evaluated by BrdU ELISA as the in vitro proliferative response. The antigen presentation of beta-LG to the T cells was reduced by conjugation with CMD. In addition, conjugation with CMD enhanced the resistance of beta-LG to cathepsin B and cathepsin D, which suggest that conjugation with CMD inhibited the degradation of beta-LG by proteases in APC and led to suppression of the generation of antigenic peptides including T cell epitopes from beta-LG. It is therefore considered that the suppressive effect on the generation of T cell epitopes reduced the antigen presentation of the conjugates and that this reduction led to a decrease in the number of beta-LG-specific T cells in vivo. As a result, the decreased help to B cells by T cells would have reduced the antibody response to beta-LG. We conclude that suppression of the generation of T cell epitopes by conjugation with CMD is important to the mechanism for the reduced immunogenicity of beta-LG.     

Identification of an HSV-1/HSV-2 cross-reactive T cell determinant.

The results from a number of studies have documented that the HSV glycoprotein gD is an important target for neutralizing antibodies. In contrast, little is known about the Th cell determinants present on HSV that are required for anti HSV gD antibody production. In our study we have immunized BALB/c mice with a recombinant source of HSV-1 gD lacking the carboxyl-terminal 93 amino acids. T cell hybridomas produced from the immunized animals recognized a single antigenic peptide (amino acids 246-261) in the context of I-Ad. The determinant expressed by gD peptide 246-261 was generated and presented by both HSV-1 and HSV-2 infected APC. Fine specificity analysis using truncated synthetic gD peptides revealed that the minimal amino acids recognized by the T hybrids were identical between HSV-1 and HSV-2. In addition, the minimal peptide-I-Ad binding analysis demonstrated that the minimal peptide sequence required for the binding to I-Ad and for T cell recognition contained two prolines. Thus, this important HSV antigenic determinant would not be expected to form an amphipathic alpha-helix and could therefore be missed by algorithms currently used to predict which amino acid sequences would be antigenic based on the propensity to form helices.     

Fine mapping of T-cell determinants of bovine β-lactoglobin

T-cell recognition sites, i.e. T-cell determinants, of bovine β-lactoglobulin, a major allergen in milk, were analyzed in detail. For this purpose, we prepared primary cultures of lymph node cells from three strains of mice, C57BL/6 (H-2^b), C3H/HeN (H-2^k), and BALB/c (H-2^d), and examined the proliferative response of these cells to a complete set of overlapping 15-mer peptides which covered the entire sequence of β-lactoglobulin by shifting in single amino acid steps. We were able to determine the putative core sequence of each T-cell determinant and estimate its relative importance. In the case of C57BL/6 mice, dominant, subdominant, and minor determinants were identified as residues 122–130, 16–26, and 108–122, respectively, as represented by their core sequences. Each determinant peptide induced the production of interferon-γ, the amount of which showed a correlation with the intensity of the proliferative response induced by each determinant. In the case of C3H/HeN mice, a dominant determinant comprised of residues 140–148 was identified together with three subdominant and two minor determinants. Dominant T-cell determinants recognized in BALB/c mice were identified as residues 67–75, 71–79, and 80–88, and six other regions were identified as subdominant determinants. Comparisons between our results and the determinants predicted from relevant MHC-binding motifs reported to date revealed the inadequacy of the motifs in predicting even the dominant determinants. The information obtained by complete mapping of T-cell determinants as done in this study is expected to be helpful in establishment and evaluation of new prediction methods and also may contribute to the development of a new approach to control immune responses by manipulation of the T-cell determinants of allergens. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Mapping an antibody-binding site and a T-cell-stimulating site on the 1A protein of respiratory syncytial virus.

A synthetic peptide modeled on residues 45 to 60 of the 1A protein of respiratory syncytial (RS) virus [1A(45-60)] was constructed and used for immunization of mice and rabbits. The immunoglobulin G fraction of the resulting rabbit antibody, purified on protein A-Sepharose, immunoprecipitated from RS-infected HEp-2 cells a protein with a molecular size of approximately 9.5 kilodaltons, which corresponds to the previously published molecular size of the 1A protein (Y. T. Huang, P. L. Collins, and G. W. Wertz, Virus Res. 2:157-173, 1985). To investigate the T-cell-inducing properties of 1A(45-60), six strains of mice were immunized and their popliteal lymph node cells were tested for proliferation upon restimulation with peptide in vitro. The lymph node cells of all six strains of mice were responsive to restimulation with 1A(45-60) and showed high- and low-responder strain variation. These peptide-primed lymph node cells also proliferated upon in vitro restimulation with RS virus-infected cells. Correlation of proliferation with interleukin 2 production suggested that the responding lymphocytes were T-helper cells. The antibody-binding and T-cell-stimulating sites of 1A were mapped by constructing a series of overlapping synthetic peptides and testing each for ability to react with antiserum prepared by immunization of BALB/C mice with free peptide 1A(45-60) or for ability to restimulate proliferation in 1A(45-60)-primed lymph node cells of BALB/C mice. Human antibody, obtained during confirmed RS virus infection, was similarly tested with the truncated peptides. Antibody-binding activity was reduced after truncation from the carboxy terminus, and a binding site was mapped to residues 51 through 60, the smallest peptide tested. T-cell-stimulating activity in mice was relatively resistant to truncation from the carboxy terminus and sensitive to truncation from the amino terminus. The smallest region which retained significant T-cell-stimulating activity mapped to residues 46 through 56. However, addition of the naturally occurring Cys at residue 45 and extension of the C terminus to residue 62 resulted in maximum T-cell-stimulating activity of the peptide. These data define both a T-cell epitope and a B-cell epitope of the 1A protein of RS virus and suggest that the carboxy terminus of 1A contains a B-cell epitope, involving residues 51 through 60, which is recognized during natural human infection.     

Identification of a T-cell epitope adjacent to neutralization antigenic site 1 of poliovirus type 1.

Proliferative T-cell responses to poliovirus in various strains of mice have been analyzed by using either killed purified virus or capsid protein VP1 synthetic peptides. Following immunization of mice with inactivated poliovirus type 1 (PV1), a specific proliferative response of their lymph node CD4+ T cells was obtained after in vitro stimulation with purified virus. In mice immunized with PV1, PV2, or PV3, a strong cross-reactivity of the T-cell responses was observed after in vitro stimulation with heterologous viruses. By using various strategies, a dominant T-cell epitope was identified in the amino acid 103 to 115 region of capsid polypeptide VP1, close by the C3 neutralization epitope. The T-cell response to VP1 amino acids 103 to 115 is H-2 restricted: H-2d mice are responders, whereas H-2k and H-2b mice do not respond to this T-cell epitope. Immunization of BALB/c (H-2d) mice with the uncoupled p86-115 peptide, which represents VP1 amino acids 86 to 115 and contains both the T-cell epitope and the C3 neutralization epitope, induced poliovirus-specific B- and T-cell responses. Moreover, these mice developed poliovirus neutralizing antibodies.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG. The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.     

Somatic mutations in the variable region of the lambda 2 chain of M315 suppression of antibodies to the idiotype of M315

BALB/c mice immunized with purified BALB/c myeloma protein M315 (alpha, lambda 2) produce anti-idiotypic antibody directed predominantly to a combinational (VH-315 + VL-315) determinant(s) of the M315 paratope (Sirisinha and Eisen, 1971; Tungkanak and Sirisinha, 1976). We examined whether the unique B cell response is influenced by pretreatment of mice with fragments or chains derived from M315 before immunization with M315. Intravenous (i.v.) injection of the Fv-315 fragment (VH-315 + VL-315) into normal BALB/c mice seven days before immunization with M315 resulted in marked suppression of anti-M315 idiotype antibodies. Studies on the structural requirement for suppression indicated that VL-315, but not VH-315, is involved. Structural comparison with a defined lambda 2 light (L) chain suggested that three contiguous amino acid residues in the third hypervariable loop of the variable (V) domain of the L chain of M315 are important for down-regulation of production of antibodies to the M315 idiotype.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Immunodominant T cell determinants of aquaporin-4, the autoantigen associated with neuromyelitis optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models.     

Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen

Streptococcus mutans is known to be a major causative organism of human dental caries. A surface protein Ag with a molecular mass of 190 kDa of S. mutans (PAc) is receiving attention as an anticaries vaccine. We have recently determined the complete nucleotide sequence of the gene for PAc. In this study, four peptides were synthesized on the basis of amino acid sequence of PAc. Among these peptides, PAc(301-319) corresponding to the alanine-rich repeating amino acid region was the most strongly bound by polyclonal murine anti-rPAc antibodies. The peptide partially inhibited the binding of polyclonal anti-rPAc antibodies to rPAc. The peptide induced the proliferation of T cells from BALB/c mice immunized with rPAc. Subcutaneous immunization with PAc(301-319) or rPAc emulsified in CFA/IFA induced high serum IgG responses to rPAc and PAc(301-319). In addition, serum IgG responses to a surface protein Ag with a molecular mass of 210 kDa of Streptococcus sobrinus were elicited in mice immunized by s.c. injection with PAc(301-319) or rPAc. Intranasal immunization with PAc(301-319) coupled to cholera toxin B subunit (CTB) or with rPAc and free CTB induced high serum IgG responses to rPAc. The immunization with PAc(301-319) coupled to CTB or rPAc and free CTB suppressed the colonization of murine teeth by S. mutans. These results suggest that intranasal immunization with the peptide or rPAc may be effective for the prevention of dental caries.     

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.     

Identification of a minimal T cell epitope recognized by antinucleosome Th cells in the C-terminal region of histone H4

Autoreactive T cells responding to systemic autoantigens have been characterized in patients and mice with autoimmune diseases and in healthy individuals. Using peptides covering the whole sequence of histone H4, we characterized several epitopes recognized by lymph node Th cells from nonsystemic lupus erythematosus-prone mice immunized with the same peptides, the H4 protein, or nucleosomes. Multiple T epitopes were identified after immunizing H-2d BALB/c mice with H4 peptides. They spanned residues 28-42, 30-47, 66-83, 72-89, and 85-102. Within the region 85-102, a minimal CD4+ T epitope containing residues 88-99 was characterized. Although Abs to peptide 88-99 recognized H4, this peptide does not contain a dominant B cell epitope recognized by anti-H4 Abs raised in BALB/c mice or Abs from NZB/NZW H-2d/z lupus mice. Th cells primed in vivo with H4 responded to H4, but not to peptide 88-99. However, this peptide was able to stimulate the proliferation and IL-2 secretion of Th cells generated after immunization with nucleosomes. H488-99 thus represents a cryptic epitope with regard to H4 and a supradominant epitope presented by nucleosome, a supramolecular complex that plays a key role in lupus. This study shows that in the normal repertoire of naive BALB/c mice, autoreactive Th cells specific for histones are not deleted. The reactivity of these Th cells seems to be relatively restricted and resembles that of Th clones generated from SNF1 ((SWR x NZB)F1; I-Ad/q) lupus mice described earlier.