Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.     

Induction of CD4(+) and CD8(+) Bordetella pertussis toxin subunit S1 specific T cells by immunization with synthetic peptides.

In this study two synthetic peptides from the Bordetella pertussis toxin subunit S1 were conjugated to human anti-idiotypic antibodies and used as an immunogen in cancer patients to induce immunity. The aims of the present report are to explain why no carrier or adjuvant effect of the conjugated pertussis peptides could be established regarding induction of responses against the anti-idiotype and to explore the type and quality of induced anti-pertussis immune responses. The lack of carrier and adjuvant effect of the peptides might be related to the fact that the anti-idiotypic antibodies by themselves include helper epitopes and that none of the patients had a detectable T cell response against any of the selected peptides before immunization, which might be a requirement for an adjuvant effect. However, three of four immunized patients mounted a humoral as well as cellular response against the pertussis peptides used. The induced T cell immunity was restricted to one of the two peptides in responding patients. Established T cell lines and MHC blocking studies indicated that the T cell epitopes of the two peptides had a different MHC restriction. The type of T cell response induced seemed to govern the humoral response. The only durable antibody response was accompanied by the presence of a CD4(+) T cell response against the same peptide. Immunization with an anti-idiotype conjugated to synthetic peptides might thus induce both a B and a T cell response against the peptides and the type of induced T cells (CD4 or CD8) governs the quality of the humoral response. Moreover, the possibility of boosting or inducing a response against the antigen from which the peptide sequences were deduced also seemed feasible.     

Characterization of T cell responses to the RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis in BALB/c mice

Porphyromonas gingivalis is a Gram-negative bacterium strongly associated with chronic periodontitis, an inflammatory oral disease. A major virulence factor common to all characterized strains of P. gingivalis is the RgpA-Kgp proteinase-adhesin complexes (RgpA-Kgp complexes). In this study, we investigated T cell proliferative and cytokine responses to the RgpA-Kgp complexes and identified T cell epitopes in BALB/c mice utilizing Pepscan methodology. T cell proliferative responses were found to be predominantly directed toward the proteinase catalytic domains. Eleven T cell epitopes were identified using RgpA-Kgp-primed lymph node T cells (IL-4 dominant) and 21 using an RgpA-Kgp-specific T cell line (IFN-gamma dominant), with 5 T cell epitopes, including the immunodominant epitope peptide 22, common to both T cell populations. Peptide 22 ((439)ANYTAHGSETAWADP(453)) from the Kgp proteinase catalytic domain induced a Th2 cytokine response in mice, and peptide 22-primed T cells had a Th2 cytokine profile when stimulated with the RgpA-Kgp complexes. Truncation and alanine scanning of peptide 22 identified the minimum epitope ((442)TAHGSETAWA(451)), and residues His(444), Glu(447), and Trp(450) as critical for T cell proliferation. With a view to vaccine development, peptide 22 was incorporated into a synthetic peptide polymer. Peptide 22 polymer induced strong T cell proliferation and crossreactivity to native RgpA-Kgp complexes. In conclusion, we have identified a major T cell epitope of P. gingivalis and established that antigenicity of the T cell epitope is retained when delivered as a peptide polymer. The strategies employed here may have potential in the development of a synthetic peptide vaccine for P. gingivalis.     

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.     

Identification and characterization of T helper epitopes in the nucleoprotein of influenza A virus

By using a series of overlapping synthetic peptides that cover more than 95% of the amino acid sequence of nucleoprotein (NP) of influenza A/NT/60/68 virus, five Th cell epitopes in B10.S (H-2s), BALB/c (H-2d), CBA (H-2k), and B6 (H-2b) mice have been identified. The specificity of Th cell recognition of epitopes is largely dependent on the H-2 haplotype of the responding mouse strain. However, two out of the five Th epitopes defined could be recognized by mice of more than one haplotype, implying that the primary sequence of protein antigens could also influence the selection of dominant T cell epitopes by the immune system. Immunization of B10.S mice with peptide 260-283 generated strong Th cell response against type A influenza viruses. In the other three strains of mice tested, priming with helper peptides induced a stronger antipeptide than antiviral T cell response. However, the low responsiveness to virus in these mice could be partially overcome by immunization with a mixture of several helper peptides. The Th epitopes are defined by the ability of the peptides to stimulate class II MHC restricted CD4+ T cells to proliferate and to produce IL-2 in vitro. When compared with the known epitopes on NP recognised by class I restricted CD8+ cytotoxic T cells, it appears that Th and cytotoxic T cell epitopes are nonoverlapping. The AMPHI and Motifs methods were employed to analyze the sequence of NP and predict the potential dominant sites in the molecule. The predictions are compared with the experimental data obtained and the implications discussed.     

Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation.

DO11.10 transgenic mice, expressing an OVA-specific TCR, were used to study pulmonary T cell responses to inhaled Ags. Before OVA inhalation, the activation of lung parenchymal T cells elicited both strong proliferative responses and IL-2 production. However, following Ag inhalation the proliferative responses of the lung T cells, when restimulated in vitro with OVA323-339 peptide or immobilized anti-CD3, were severely attenuated and associated with a decrease in the level of production of IL-2 but not IFN-gamma. Such immune regulation was tissue-specific, because T cell responses in the lymph nodes and spleens were normal. This dramatic aerosol-induced attenuation of parenchymal T cell proliferation was also observed in BALB/c mice immunized with OVA and in BALB/c mice following adoptive transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In mice that had received Th2 cells, the reduced proliferative responses were associated with a decrease in IL-2 expression but augmented IL-4 and IL-5 production. Invariably, the inhibition of proliferation was a consequence of the action of F4/80+ interstitial macrophages and did not involve alveolar macrophages or their products. These observations demonstrate that clonal expansion of T cells in the lung compartment is prevented following the onset of either Th1- or Th2-mediated inflammation. This form of immune regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses.     

Identification of epitopes within the circumsporozoite protein of Plasmodium vivax recognized by murine T lymphocytes.

The murine cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax was characterized using five synthetic peptides, some of which we identified as corresponding to T cell epitopes. The peptides P308-320, P344-355 and P353-364 were immunogenic, inducing a genetically restricted proliferative response, due to the activation of CD4+ T cells. The peptide P308-320 was recognized only by the lymphocytes of B10 (H-2b) mice. The other two peptides were recognized by primed lymphocytes of H-2a and H-2k mice. Of interest was the finding that one of these peptides, P353-364, induced a proliferative response of a large percentage of immune outbred Swiss mice. Our data provide evidence that, at least in mice, there is recognition of multiple T cell epitopes within the major surface antigen of P. vivax sporozoites.     

Mapping of T helper cell epitopes by using peptides spanning the 19-kDa protein of Mycobacterium tuberculosis. Evidence for unique and shared epitopes in the stimulation of antibody and delayed-type hypersensitivity responses.

In vivo and in vitro T cell responses to overlapping 20-mer peptides that span the entire 19-kDa protein of Mycobacterium tuberculosis have been compared in three different strains of mice. Immunization of the mice with peptides and analysis of specific antibody production is an in vivo assay of Th cell activity. Peptides 1-20 and 61-80 elicited strong IgG1 responses in BALB/cJ, C57BL/10J, and B10.BR mice, indicating that these peptides could stimulate Th cells, possibly of a Th2 phenotype. T cells isolated from peptide-immunized mice were challenged in vitro with peptide, and their proliferative responses were analyzed. T cells from these three strains of mice immunized with peptides 1-20, 61-80, and 76-95 also responded to challenge with specific peptide in vitro. In addition, B10.BR mice and BALB/cJ mice showed antibody and T cell proliferative responses to peptides 136-155 and 145-159, respectively. Thus, in vitro proliferating T cells were found to possess specificities for peptide epitopes that were almost identical to those of the antibody-producing cells. Delayed-type hypersensitivity (DTH) responses to these peptides were also examined in the three strains. Interestingly, the T cells responding in the DTH assay had Ag specificities that were quite different from those identified in the antibody and proliferation assays. These results suggested that DTH Th cells form a separate population from antibody Th and proliferative T cells and these populations of cells were differentially activated, in an Ag-specific manner.     

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.     

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.     

A stimulatory Mls-1 superantigen is destroyed by ultraviolet light while other Mtv-7 antigens remain intact. Significance for Mls-1 unresponsiveness.

Accessory cells present Ag together with costimulatory signals as immunogens and without costimulatory signals as tolerogens. Responsiveness and unresponsiveness are thus alternatives of T cell immune reactions to Ag. Superantigens appear to make an exception; being presented by accessory cells capable of providing costimulatory signals, these Ag induce a strong T cell response but leave T cells unresponsive to a secondary challenge (anergy). We show here that T cell anergy is not a mandatory consequence of superantigen-induced activation. Mls-1- BALB/c recipients of DBA/2 spleen cells mount vigorous Mls-1 responses in vivo but their T cells retain the ability to respond to a subsequent Mls-1 challenge in vitro. We tested the possibility that the inability of DBA/2 spleen cells to inactivate Mls-1-reactive BALB/c T cells was the result of excessive costimulatory activity provided by Mls-1+ DBA/2 B cells. Costimulatory accessory cell activity has been reported to be destroyed by UV light. We exposed superantigen-presenting cells to UV radiation and found that they had lost the ability to stimulate an Mls-1 response without, however, gaining the capacity to render Mls-1-specific T cells anergic. Despite their inability to noticeably stimulate Mls-1-reactive T cells, UV-treated Mls-1+ lymphocytes induced an absolute unresponsiveness in Mls-1- recipients to a second challenge with the superantigen. Our data are in agreement with previous evidence, confirmed here, that BALB/c mice establish immunity against Mls-1+ cells, which causes the accelerated rejection of superantigen-bearing lymphocytes. Thus, our data imply that, whereas it takes stimulatory superantigenic Mtv-7 gene products to induce the activation of superantigen-reactive T cells, nonsuperantigenic Mtv-7 gene products may induce an immune response leading to the elimination of Mtv-7+ lymphoid cells.     

Immune response to human immunodeficiency virus. In vivo administration of anti-idiotype induces an anti-gp160 response specific for a synthetic peptide

Anti-idiotypic antibodies (anti-Id) to chimpanzee antibodies directed against a synthetic peptide corresponding to a native epitope associated with gp41 of human immunodeficiency virus (HIV) envelope glycoprotein were produced in rabbits. The peptide was analogous to amino acid sequences 735 to 752 from the human T cell leukemia virus-IIIB isolate of HIV. Characteristics of the anti-Id preparation included: 1) detection of a shared determinant present on a second chimpanzee and one of three rabbit antibody preparations directed against the synthetic peptide, 2) failure to recognize an idiotype (Id) in BALB/c mouse antisera to the peptide, and 3) partial inhibition of the homologous chimpanzee Id preparation from binding either peptide or a recombinant HIV gp160 preparation. Immunization of BALB/c mice with the anti-Id induced an antipeptide response which bound a recombinant gp160 preparation without subsequent peptide or gp160 exposure. The anti-gp160 containing sera from mice immunized with anti-Id were able to inhibit the Id-anti-Id reaction indicating that an Id-positive antibody response was induced. This Id is not normally expressed in the murine anti-gp 160 immune response to the synthetic peptide and suggests that this anti-Id may activate normally silent clones. This study indicates that Id networks may be operational during the immune response to HIV epitopes. Alternatively, anti-Id may be useful in altering the serologic characteristics of an antibody response to HIV and may offer potential for modulating the immune response in this viral infection.     

Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.     

Initiation of immune responses in brain is promoted by local dendritic cells

The contribution of dendritic cells (DCs) to initiating T cell-mediated immune response in and T cell homing into the CNS has not yet been clarified. In this study we show by confocal microscopy and flow cytometry that cells expressing CD11c, CD205, and MHC class II molecules and containing fluorescently labeled, processed Ag accumulate at the site of intracerebral Ag injection. These cells follow a specific pattern upon migrating out of the brain. To track their pathway out of the CNS, we differentiated DCs from bone marrow of GFP-transgenic mice and injected them directly into brains of naive C57BL/6 mice. We demonstrate that DCs migrate from brain to cervical lymph nodes, a process that can be blocked by fixation or pertussis toxin treatment of the DCs. Injection of OVA-loaded DCs into brain initiates a SIINFEKL (a dominant OVA epitope)-specific T cell response in lymph nodes and spleen, as measured by specific tetramer and LFA-1 activation marker staining. Additionally, a fraction of activated SIINFEKL-specific T cells home to the CNS. Specific T cell homing to the CNS, however, cannot be induced by i.v. injection of OVA-loaded DCs alone. These data suggest that brain-emigrant DCs are sufficient to support activated T cells to home to the tissue of DC origination. Thus, initiation of immune reactivity against CNS Ags involves the migration of APCs from nervous tissue to peripheral lymphoid tissues, similarly to that in other organs.     

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.     

A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.     

Induction of humoral immunity in response to immunization with recombinant Mycobacterium bovis BCG expressing the S1 subunit of Bordetella pertussis toxin

Two recombinant Mycobacterium bovis BCG (rBCG) vaccine strains were developed for the expression of cytoplasmically located S1 subunit of pertussis toxin, with expression driven by the hsp60 promoter of M. bovis (rBCG/pPB10) or the pAN promoter of Mycobacterium paratuberculosis (rBCG/pPB12). Both strains showed stable expression of equivalent levels of recombinant S1 in vitro and induced long-term (up to 8 months) humoral immune responses in BALB/c mice, although these responses differed quantitatively and qualitatively. Specifically, rBCG/pPB12 induced markedly higher levels of IgG1 than did rBCG/pPB10, and mice immunized with the former strain developed specific long-term memory to S1, as indicated by the production of high levels of S1-specific IgG in response to a sublethal challenge with pertussis toxin 15 months after initial immunization. When considered in combination with previous studies, our data encourage further evaluation of rBCG as a potential means of developing a low-cost whooping cough vaccine based on defined antigens.     

CD18 is required for intestinal T cell responses at multiple immune checkpoints

The intestinal immune response to oral Ags involves a complex multistep process. The requirements for optimal intestinal T cell responses in this process are unclear. LFA-1 plays a critical role in peripheral T cell trafficking and activation, however, its role in intestinal immune responses has not been precisely defined. To dissect the role of LFA-1 in intestinal immune responses, we used a system that allows for segregation of T cell migration and activation through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 TCR transgenic mice into wild-type BALB/c mice. We find that wild-type mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate decreases in the numbers of Ag-specific T cells in the intestinal lamina propria after oral Ag administration. We also find that in addition to its role in trafficking to intestinal secondary lymphoid organs, LFA-1 is required for optimal CD4(+) T cell proliferation in vivo upon oral Ag immunization. Furthermore, CD18(-/-) DO11.10 CD4(+) T cells primed in the intestinal secondary lymphoid organs demonstrate defects in up-regulation of the intestinal-specific trafficking molecules, alpha(4)beta(7) and CCR9. Interestingly, the defect in trafficking of CD18(-/-) DO11.10 CD4(+) T cells to the intestinal lamina propria persists even under conditions of equivalent activation and intestinal-tropic differentiation, implicating a role for CD18 in the trafficking of activated T cells into intestinal tissues independent of the earlier defects in the intestinal immune response. This argues for a complex role for CD18 in the early priming checkpoints and ultimately in the trafficking of T cells to the intestinal tissues during an intestinal immune response.     

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ?i.e., 4-16(Val12)?. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ?i.e., 4-12(Val12)?, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ?i.e., 5-17(Val12)? lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.