Regulation of autologous immunity to the mouse 5T4 oncofoetal antigen: implications for immunotherapy

Effective vaccination against tumour-associated antigens (TAA) such as the 5T4 oncofoetal glycoprotein may be limited by the nature of the T cell repertoire and the influence of immunomodulatory factors in particular T regulatory cells (Treg). Here, we identified mouse 5T4-specific T cell epitopes using a 5T4 knock out (5T4KO) mouse and evaluated corresponding wild-type (WT) responses as a model to refine and improve immunogenicity. We have shown that 5T4KO mice vaccinated by replication defective adenovirus encoding mouse 5T4 (Adm5T4) generate potent 5T4-specific IFN-γ CD8 and CD4 T cell responses which mediate significant protection against 5T4 positive tumour challenge. 5T4KO CD8 but not CD4 primed T cells also produced IL-17. By contrast, Adm5T4-immunized WT mice showed no tumour protection consistent with only low avidity CD8 IFN-γ, no IL-17 T cell responses and no detectable CD4 T cell effectors producing IFN-γ or IL-17. Treatment with anti-folate receptor 4 (FR4) antibody significantly reduced the frequency of Tregs in WT mice and enhanced 5T4-specific IFN-γ but reduced IL-10 T cell responses but did not reveal IL-17-producing effectors. This altered balance of effectors by treatment with FR4 antibody after Adm5T4 vaccination provided modest protection against autologous B16m5T4 melanoma challenge. The efficacy of 5T4 and some other TAA vaccines may be limited by the combination of TAA-specific T regs, the deletion and/or alternative differentiation of CD4 T cells as well as the absence of distinct subsets of CD8 T cells.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

The immunodominant CD8 T cell response to the human cytomegalovirus tegument phosphoprotein pp65(495-503) epitope critically depends on CD4 T cell help in vaccinated HLA-A*0201 transgenic mice

Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-A(b+)/A2-HHD-II and HLA-DR1(+)/A2-DR1 mice the HLA-A*0201-restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ(+) CD8 T cell frequencies to the pp65(495-503)/(e6) epitope and low responses to the pp65(320-328)/(e3) and pp65(522-530)/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65(Δ501-503) DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65(487-503) domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.     

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.     

Vaccinia virus-specific CD4+ T cell responses target a set of antigens largely distinct from those targeted by CD8+ T cell responses

Recent studies have defined vaccinia virus (VACV)-specific CD8(+) T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4(+) T cell responses. In this study, we identified 14 I-A(b)-restricted VACV-specific CD4(+) T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for approximately 20% of the total anti-VACV CD4(+) T cell response and are derived from 13 different viral proteins. Surprisingly, none of the CD4(+) T cell epitopes identified was derived from VACV virulence factors. Although early Ags were recognized, late Ags predominated as CD4(+) T cell targets. These results are in contrast to what was previously found in CD8(+) T cells responses, where early Ags, including virulence factors, were prominently recognized. Taken together, these results highlight fundamental differences in immunodominance of CD4(+) and CD8(+) T cell responses to a complex pathogen.     

DNA fusion vaccines induce targeted epitope-specific CTLs against minor histocompatibility antigens from a normal or tolerized repertoire

We have designed DNA fusion vaccines able to induce high levels of epitope-specific CD8(+) T cells, using linked CD4(+) T cell help. Such vaccines can activate effective immunity against tumor Ags. To model performance against minor histocompatibility (H) Ags important in allogeneic hemopoietic stem cell transplantation, responses against the H2D(b)-restricted Uty and Smcy male HY epitopes have been investigated. Vaccination of females induced high levels of tetramer-specific, IFN-gamma-producing CD8(+) T cells against each epitope. Vaccines incorporating a single epitope primed effector CTL able to kill male splenocytes in vitro and in vivo, and HY(Db)Uty-specific vaccination accelerated rejection of syngeneic male skin grafts. Priming against either epitope established long-term memory, expandable by injection of male cells. Expanded CD8(+) T cells remained specific for the priming HY epitope, with responses to the second suppressed. To investigate vaccine performance in a tolerized repertoire, male mice were vaccinated with the fusion constructs. Strikingly, this also generated epitope-specific IFN-gamma-producing CD8(+) T cells with cytotoxic function. However, numbers and avidity were lower than in vaccinated females, and vaccinated males failed to reject CFSE-labeled male splenocytes in vivo. Nevertheless, these findings indicate that DNA fusion vaccines can mobilize CD8(+) T cells against endogenous minor H Ags, even from a profoundly tolerized repertoire. In the transplantation setting, vaccination of donors could prime and expand specific T cells for in vivo transfer. For patients, vaccination could activate a potentially less tolerized repertoire against similar Ags that may be overexpressed by tumor cells, for focused immune attack.     

Epitope mapping of HIV-specific CD8+ T cell responses by multiple immunological readouts reveals distinct specificities defined by function

The limited success of HIV vaccine candidates to date highlights our need to better characterize protective cell-mediated immunity (CMI). While HIV-specific CD8(+) T cell responses have been defined largely by measuring gamma interferon (IFN-γ), these responses are not always protective, and it is unclear whether the same epitopes would predominate if other functional parameters were examined. Here, we assessed the epitope specificity of HIV-specific CD8(+) T cell responses by multiparametric flow cytometry, measuring five CD8(+) T cell functions (IFN-γ, macrophage inflammatory protein 1β [MIP-1β], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], and proliferative capacity) in 24 chronically HIV-infected individuals. Sixty-nine epitope-specific responses to 50 epitopes within p24 were measured. Surprisingly, most epitope-specific responses were IFN-γ negative (50/69 responses). Many responses had polyfunctional (33%) and proliferative (19%) components. An inverse association between IL-2 and proliferation responses was also observed, contrary to what was described previously. We confirm that long-term nonprogressors (LTNP) have more polyfunctional responses and also have higher-magnitude and broader p24-specific proliferation and higher levels of IL-2 and TNF-α production than do progressing controls. Together, these data suggest that the specificity of CD8(+) T cell responses differs depending on the immunological readout, with a 3.5-fold increase in breadth detected by including multiple parameters. Furthermore, the identification of epitopes that elicit polyfunctional responses reinforces the need for the comprehensive evaluation of HIV vaccine candidates, and these epitopes may represent novel targets for CMI-based vaccines.     

Differences between T cell epitopes recognized after immunization and after infection

Evidence suggests that cellular immune responses play a crucial role in the control of HIV and SIV replication in infected individuals. Several vaccine strategies have therefore targeted these CD8(+) and CD4(+) responses. Whether vaccination induces the same repertoire of responses seen after infection is, however, a key unanswered question in HIV vaccine development. We therefore compared the epitope specificity induced by vaccination to that present postchallenge in the peripheral blood. Intracellular cytokine staining of PBMC stimulated with overlapping 15/20-mer peptides spanning the proteins of SIV were measured after DNA/modified vaccinia Ankara vaccination of eight rhesus macaques. Lymphocytes from 8 animals recognized a total of 39 CD8 epitopes and 41 CD4 epitopes encoded by the vaccine. T cell responses were again monitored after challenge with SIVmac239 to investigate the evolution of these responses. Only 57% of all CD8(+) T cell responses and 19% of all CD4(+) T cell responses present after vaccination were recalled after infection as measured in the peripheral blood. Interestingly, 29 new CD8 epitopes and 5 new CD4 epitopes were recognized by PBMC in the acute phase. These new epitopes were not detected after vaccination, and only some of them were maintained in the chronic phase (33% of CD8 and no CD4 responses). Additionally, 24 new CD8 epitopes and 7 new CD4 epitopes were recognized by PBMC in the chronic phase of infection. The repertoire of the immune response detected in the peripheral blood after immunization substantially differed from the immune response detected in the peripheral blood after infection.     

Therapeutic effect of a T helper cell supported CTL response induced by a survivin peptide vaccine against murine cerebral glioma

Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides with predicted binding to mouse H2-K^b and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-K^b-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were screened by flow cytometry for binding of dimeric H2-K^b:Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN_57–64 and SVN_82–89) generated specific CD8+ T cells. We chose to focus on the SVN_57–64 peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN_53–67), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell help. We tested the SVN_53–67 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a significant prolongation of survival. The SVN_53–67 vaccine was significantly more effective than the SVN_57–64 core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated cytokine support within a single peptide.     

Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.     

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.     

Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain

Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.     

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.     

A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+) T cells are important for the generation and maintenance of functional CD8(+) cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4(+) T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+)/CD8(+) T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+) and CD8(+) T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4(+) T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4(+) T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+) T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+) T cells and antibody responses- elicited by other HIV immunogens.     

Efficient lung recruitment of respiratory syncytial virus-specific Th1 cells induced by recombinant bacillus Calmette-Guérin promotes virus clearance and protects from infection

Infection by the respiratory syncytial virus (RSV) can cause extensive inflammation and lung damage in susceptible hosts due to a Th2-biased immune response. Such a deleterious inflammatory response can be enhanced by immunization with formalin- or UV-inactivated RSV, as well as with vaccinia virus expressing the RSV-G protein. Recently, we have shown that vaccination with rBCG-expressing RSV Ags can prevent the disease in the mouse. To further understand the immunological mechanisms responsible for protection against RSV, we have characterized the T cell populations contributing to virus clearance in mice immunized with this BCG-based vaccine. We found that both CD4(+) and CD8(+) T cells were recruited significantly earlier to the lungs of infected mice that were previously vaccinated. Furthermore, we observed that simultaneous adoptive transfer of CD8(+) and CD4(+) RSV-specific T cells from vaccinated mice was required to confer protection against virus infection in naive recipients. In addition, CD4(+) T cells induced by vaccination released IFN-γ after RSV challenge, indicating that protection is mediated by a Th1 immune response. These data suggest that vaccination with rBCG-expressing RSV Ags can induce a specific effector/memory Th1 immune response consisting on CD4(+) and CD8(+) T cells, both necessary for a fully protective response against RSV. These results support the notion that an effective induction of Th1 T cell immunity against RSV during childhood could counteract the unbalanced Th2-like immune response triggered by the natural RSV infection.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Induction of protective CD4+ T cell-mediated immunity by a Leishmania peptide delivered in recombinant influenza viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity.     

Enhanced priming of multispecific,murine CD8+ T cell responses by DNA vaccines expressing stress protein-binding polytope peptides

A polytope DNA vaccine (pCI/pt10) was used that encodes within a 106-residue sequence 10-well characterized epitopes binding MHC class I molecules encoded by the K, D, or L locus (of H-2(d), H-2(b), and H-2(k) haplotype mice). The pCI/pt10 DNA vaccine efficiently primed all four K(b)/D(b)-restricted CD8(+) T cell responses in H-2(b) mice, but was deficient in stimulating most CD8(+) T cell responses in H-2(d) mice. Comparing CD8(+) T cell responses elicited with the pCI/pt10 DNA vaccine in L(d+) BALB/c and L(d-) BALB/c(dm2) (dm2) mice revealed that L(d)-restricted CD8(+) T cell responses down-regulated copriming of CD8(+) T cell responses to other epitopes regardless of their restriction or epitope specificity. Although the pt10 vaccine could thus efficiently co prime multispecific CD8(+) T cell responses, this priming was impaired by copriming L(d)-restricted CD8(+) T cell responses. When the pt10 sequence was fused to a 77-residue DnaJ-homologous, heat shock protein 73-binding domain (to generate a 183-residue cT(77)-pt10 fusion protein), expression and immunogenicity (for CD8(+) T cells) of the chimeric Ag were greatly enhanced. Furthermore, priming of multispecific CD8(+) T cell responses was readily elicited even under conditions in which the suppressive, L(d)-dependent immunodominance operated. The expression of polytope vaccines as chimeric peptides that endogenously capture stress proteins during in situ production thus facilitates copriming of CD8(+) T cell populations with a diverse repertoire.     

Nondominant CD8 T cells are active players in the vaccine-induced antitumor immune response

We previously reported that CD8(+) T cells are directed predominantly toward the immunodominant Her-2/neu (neu) epitope RNEU(420-429) in nontolerized FVB/N but not tolerized HER-2/neu (neu-N) mice. In this study, we screened overlapping peptides of the entire neu protein and identified six new epitopes recognized by vaccine-induced neu-N-derived T cells. Evaluation of individual nondominant responses by tetramer staining and IFN-γ secretion demonstrate that this repertoire is peripherally tolerized. To address the role that the complete CD8(+) T cell repertoire plays in vaccine-induced antitumor immunity, we created a whole-cell vaccine-expressing neu cDNA that has been mutated at the RNEU(420-429) anchor residue, thereby abrogating activation of immunodominant epitope responses. Studies comparing the mutated and nonmutated vaccines indicate that nondominant CD8(+) T cells can induce antitumor immunity when combined with regulatory T cell-depleting agents in both neu-N and FVB/N mice. Collectively, these studies demonstrate that the neu-directed T cell repertoire is not intrinsically incapable of eradicating tumors. Rather, they are suppressed by mechanisms of peripheral tolerance. Thus, these studies provide new insights into the function of the complete T cell repertoire directed toward a clinically relevant tumor Ag in tumor-bearing hosts.     

Immunologic hierarchy,class II MHC promiscuity,and epitope spreading of a melanoma helper peptide vaccine

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4⁺ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8⁺ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4⁺ and CD8⁺ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4⁺ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3_281–295 (49 %) and tyrosinase_386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8⁺ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4⁺ and CD8⁺ T cell responses against melanoma antigens. CD4⁺ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8⁺ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.