CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.     

Multiepitope Trojan antigen peptide vaccines for the induction of antitumor CTL and Th immune responses

We describe in this study a strategy to produce synthetic vaccines based on a single polypeptide capable of eliciting strong immune responses to a combination CTL and Th epitopes with the purpose of treating malignancies or preventing infectious diseases. This strategy is based on the capacity of Trojan Ags to deliver exogenous Ags into the intracellular compartments, where processing into MHC-binding peptides takes place. Our previous work demonstrated that Trojan Ags containing a CTL epitope localized to intracellular compartments, where MHC class I-binding peptides were generated in a TAP-independent fashion by the action of various exopeptidases and the endopeptidase furin. In this study, we report that Trojan Ags containing several CTL epitopes joined via furin-sensitive linkers generated all of the corresponding MHC class I-binding peptides, which were recognized by CTL. However, Trojan Ags prepared with furin-resistant linkers failed to produce the MHC class I-binding peptides. We also present data indicating that Trojan Ags bearing both CTL and Th epitopes can generate the corresponding MHC class I- and II-binding peptides, which are capable of stimulating T cell responses. Most significantly, in vivo vaccination of mice with a single injection of multiepitope Trojan Ags resulted in strong CTL and Th responses that translated into significant antitumor responses in a model of malignant melanoma. The overall results indicate that Trojan Ags prepared with furin-sensitive linkers are ideal candidates for producing synthetic multiepitope vaccines for the induction of CTL and Th responses that could be used against a variety of diseases, including cancer.     

Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses

CD8(+) T cells are essential for long-term, vaccine-induced resistance against intracellular pathogens. Here we show that natural antibodies, acting in concert with complement, are endogenous adjuvants for the generation of protective CD8(+) T cells after vaccination against visceral leishmaniasis. IL-4 was crucial for the priming of vaccine-specific CD8(+) T cells, and we defined the primary source of IL-4 as a CD11b(+)CD11c(lo) phagocyte. IL-4 secretion was not observed in antibody-deficient mice and could be reconstituted with serum from normal, but not Btk immune-deficient, mice. Similarly, no IL-4 response or CD8(+) T-cell priming was seen in C1qa(-/-) mice. These results identify a new pathway by which immune complex-mediated complement activation can regulate T-cell-mediated immunity. We propose that this function of natural antibodies could be exploited when developing new vaccines for infectious diseases.     

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.     

Vehicles for genetic vaccines against human immunodeficiency virus: induction of T cell-mediated immune responses

Success of a candidate vaccine against human immunodeficiency virus (HIV) depends on the type, site, strength, longevity and specificity of the immune responses it induces. The specificity of a vaccine is determined by the HIV-derived immunogens it employs in its formulation. Central to the other features is a correct and efficient delivery of the immunogens to the relevant cells of the immune system, which leads to orchestrated actions of millions of cells of several types and functions at multiple sites in the body. Thus, for elicitation of cytotoxic T lymphocyte responses, immunogens have to be delivered to the so called 'professional' antigen-presenting cells in a way that leads to a specific activation and expansion of na?ve or precursor T cells, subsequent maturation of effector functions and, importantly, generation of a potent immunological memory. Many aspects of theseprocesses are currently unknown. However, it is very likely that the immunogenicities of genetic vaccines, i.e. vaccines delivering genes coding for immunogens rather than purified possibly adjuvanted proteins or peptides themselves, are in great part determined by the choice of vaccine vehicles and route of administration. In addition, vaccine immunogenicities can be augmented semi-rationally by immunogen engineering and co-delivering immunomodulatory molecules, and empirically by combining different vehicles expressing the same immunogen in heterologous prime-boost protocols. In any case, a successful vaccination strategy against HIV as well as other chronic viral infections has to elicit better immune responses than the natural infections do.     

T-cell responses to the M3 immune evasion protein of murid gammaherpesvirus 68 are partially protective and induced with lytic antigen kinetics

DNA vaccination with the M3 gene, encoding an immune evasion molecule expressed during both the acute lytic and persistent phases of murid gammaherpesvirus 68 infection, yielded a significantly lower titer of virus in the lung than controls. The protection seen was dependent on T cells, and we mapped an epitope recognized by CD8 T cells. The immune response to this epitope follows the same kinetics as lytic cycle antigens, despite the fact that this gene is expressed in both lytic and persistent stages of infection. This has important implications for our understanding of T-cell responses to putative latency-associated gammaherpesvirus proteins and how vaccination may improve control of these viruses.     

Altered peptide ligands narrow the repertoire of cellular immune responses by interfering with T-cell priming

Variation in epitopes of infectious pathogens inhibits various effector functions of T lymphocytes through antagonism of the T-cell receptor. However, a more powerful strategy for immune evasion would be to prevent the induction of T-cell responses. We report here mutual 'interference' with the priming of human T-cell responses by a pair of naturally occurring variants of a malaria cytotoxic T-cell epitope. Interference with priming also occurs in vivo for a murine malaria T-cell epitope. Reshaping of the T-cell repertoire by such immune interference during naive T-cell induction may provide a general mechanism for observed patterns of immunodominance and persistence by many polymorphic pathogens.     

Mimotopes of tumor-associated T-cell epitopes for cancer vaccines determined with combinatorial peptide libraries

Cytotoxic T-cells are the most important effector cells in immune responses against tumors. The identification of tumor-associated epitopes for these cells, therefore, has become a key aspect of the development of cancer vaccines. Here, we describe a new approach to the determination of tumor-associated T-cell epitopes which employs combinatorial peptide libraries with singly defined sequence positions in a randomized context. The analysis of the responses of a T-cell clone to these libraries yields the amino acid constituents of the epitope which can be combined to obtain mimotopes that are suitable as vaccine antigens for the induction of tumor-specific responses.     

Differential tissue-specific regulation of antiviral CD8+ T-cell immune responses during chronic viral infection

The hallmarks of the immune response to viral infections are the expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) after they encounter antigen-presenting cells in the lymphoid tissues and their subsequent redistribution to nonlymphoid tissues to deal with the pathogen. Control mechanisms exist within CTL activation pathways to prevent inappropriate CTL responses against disseminating infections with a broad distribution of pathogen in host tissues. This is demonstrated during overwhelming infection with the noncytolytic murine lymphocytic choriomeningitis virus, in which clonal exhaustion (anergy and/or deletion) of CTLs prevents immune-mediated pathology but allows persistence of the virus. The mechanism by which the immune system determines whether or not to mount a full response to such infections is unknown. Here we present data showing that the initial encounter of specific CTLs with infected cells in lymphoid tissues is critical for this decision. Whether the course of the viral infection is acute or persistent for life primarily depends on the degree and kinetics of CTL exhaustion in infected lymphoid tissues. Virus-driven CTL expansion in lymphoid tissues resulted in the migration of large quantities of CTLs to nonlymphoid tissues, where they persisted at stable levels. Surprisingly, although virus-specific CTLs were rapidly clonally exhausted in lymphoid tissues under conditions of chronic infection, a substantial number of them migrated to nonlymphoid tissues, where they retained an effector phenotype for a long time. However, these cells were unable to control the infection and progressively lost their antiviral capacities (cytotoxicity and cytokine secretion) in a hierarchical manner before their eventual physical elimination. These results illustrate the differential tissue-specific regulation of antiviral T-cell responses during chronic infections and may help us to understand the dynamic relationship between antigen and T-cell populations in many persistent infections in humans.     

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.     

Dendritic cells in the induction of protective and nonprotective anticryptococcal cell-mediated immune responses

Dendritic cells (DC) can be divided into three subsets, Langerhans cells, myeloid DC (MDC), and lymphoid DC (LDC), based upon phenotypic and functional differences. We hypothesized that different DC subsets are associated with the development of protective vs nonprotective cell-mediated immune (CMI) responses against the fungal pathogen, Cryptococcus neoformans. To test this, mice were immunized with protective and/or nonprotective immunogens, and DC subsets in draining lymph nodes were assessed by flow cytometry. The protective immunogen (cryptococcal culture filtrate Ag-CFA), in contrast to the nonprotective immunogen (heat-killed cryptococci-CFA), the nonprotective immunogen mixed with the protective immunogen (cryptococcal culture filtrate Ag + heat-killed cryptococci-CFA), or controls, stimulated significant increases in total leukocytes, Langerhans cells, MDC, LDC, and activated CD4+ T cells in draining lymph nodes. The protective immune response resulted in significantly increased levels of anticryptococcal delayed-type hypersensitivity reactivity and activated CD4+ T cells at the delayed-type hypersensitivity reaction site. Draining lymph node LDC:MDC ratios induced by the protective immunogen were significantly lower than the ratios induced by either immunization in which the nonprotective immunogen was present. In contrast, mice given the nonprotective immunogen had LDC:MDC ratios similar to those of naive mice. Our data indicate that lymph node Langerhans cells and MDC are APC needed for induction of the protective response. The predominance of LDC in mice undergoing nonprotective responses suggests that lymph node LDC, like splenic LDC, are negative regulators of CMI responses. In addition to showing DC subsets associated with functional differences, our data suggest that the LDC:MDC balance, which can be modulated by the Ag, determines whether protective or nonprotective anticryptococcal CMI responses develop.     

CpG oligonucleotides are potent adjuvants for the activation of autoreactive encephalitogenic T cells in vivo

The mechanism of action of microbial adjuvants in promoting the differentiation of autoimmune effector cells remains to be elucidated. We demonstrate that CpG-containing oligodeoxynucleotides (ODN) can completely substitute for heat-killed mycobacteria in the priming of encephalitogenic myelin-reactive T cells in vivo. The adjuvanticity of the CpG ODN was secondary to their direct ability to induce IL-12 or to act synergistically with endogenous IL-12 to promote Th1 differentiation and encephalitogenicity. T cells primed in the absence of CpG with Ag and IFA alone appeared to be in a transitional state and had not undergone differentiation along a conventional Th pathway. Unlike Th2 cells, they expressed low levels of the IL-12R beta 2 subunit and retained the ability to differentiate into encephalitogenic effectors when reactivated in vitro under Th1-polarizing conditions. These results support the use of CpG ODN as adjuvants but also suggest that they could potentially trigger autoimmune disease in a susceptible individual.     

Targeted nanotherapy for induction of therapeutic immune responses

Nanotechnology permits the design of therapeutic devices with defined structure and molecular composition. Modular designs employing surface-bound ligands provide specific homing devices for loaded cargo, and biocompatible and biodegradable constructs provide surrogate temporary microenvironments. We first present a case for developing 'smart' modular constructs as immunogenic vaccines to prime immune memory against specific pathogens where current vaccines fail. Second, we argue that nanotherapeutic intervention can harness pivotal molecular pathways recently discovered to regulate lineage development between pathogenic TH17 cells associated with autoimmune disease, versus tolerogenic regulatory T cells (Treg). Underpinned by molecular mechanisms that enable exquisitely specific responses in adaptive immunity, targeted nanodevices designed to stimulate either immune aggression or immune tolerance signify the birth of a new era in therapeutics.     

Skin immune responses to peptide and protein antigen are TLR4 independent

Little is known about the innate immune mechanisms regulating adaptive immune responses elicited through the skin. Tissue injury is postulated to liberate Toll like receptor 4 (TLR4) ligands. In this study, we determined whether TLR4 signaling modulates the response to epidermal injury induced by tape stripping (TS) and whether it alters humoral and cellular immune responses generated through epicutaneous immunization with peptide+cholera toxin (CT). The combined use of cholera toxin and TS with antigen promoted optimal antigen-specific CD4(+) and CD8(+) T cell proliferation in Balb/c and C57BL/6 mice, respectively. TLR4 mutant mice had similar T cell responses to wild type mice. Further, OVA-protein specific IgG, IgG(1), IgG(2a), and IgE titers were similar in wild type and TLR4 mutant mice. Thus, TLR4 signaling was not required for the generation of epicutaneous T cell or antibody mediated immune responses and did not alter the quality of the immune responses elicited.     

Bacterial ghosts are an efficient delivery system for DNA vaccines

Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52-60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for beta-galactosidase under the control of the CMV immediate early gene promoter (pCMVbeta) stimulates more efficient Ag-specific humoral and cellular (CD4(+) and CD8(+)) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVbeta-containing ghosts also resulted in the elicitation of beta-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.     

Neonatal administration of IL-12 enhances the protective efficacy of antiviral vaccines

Neonates are highly susceptible to infectious agents and are known to display polarized expression of Th2-like cytokines and Abs. This neonatal immune bias has important implications for the development of vaccine strategies, particularly against viral infections. We now report that coadministration of IL-12 and an influenza subunit vaccine at birth enhances the protective efficacy of antiviral vaccination. Immunization and treatment with IL-12 within 24 h of birth resulted in elevated expression of IFN-gamma, IL-10, and IL-15 mRNA in the spleens of newborn mice compared with animals exposed to vaccine only. In addition, these animals showed dramatic increases in IFN-gamma-, IL-2-, and IL-4-secreting cells, and in IgG2a Ab levels upon adult challenge compared with mice primed with vaccine alone. Most importantly, animals vaccinated and simultaneously treated with IL-12 at birth displayed enhanced survival after lethal challenge with infectious influenza virus as adults compared with infected animals that had been primed with vaccine alone. This augmented protection required B cells and could be transferred to naive mice by immune serum. Collectively, these results provide evidence that administration of IL-12 to neonates induces a Th1-like response in newborns and elicits protective antiviral immune memory.     

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.     

Induction of specific immune responses by polycation-based vaccines

The s.c injection of tumor Ag-derived, MHC class I-binding peptides together with cationic poly-amino acids (e.g., poly-L-arginine; pR) has been shown to protect animals against a challenge with tumor cells expressing the respective peptide(s). Given our only restricted knowledge about immunogenic tumor-associated peptides, we sought to determine whether this pR-based vaccination protocol would also induce protective cancer immunity if large proteins were used instead of peptide epitopes. We found that the intracutaneous administration of the model Ag beta-galactosidase (beta-gal) together with pR (referred to as pR-based protein vaccine; pR-PV) was significantly more potent in protecting mice against the growth of beta-gal-expressing RENCA cells than the protein alone. Coadministration of pR enhanced both the beta-gal-induced specific humoral and CD8 response. The protective effect required CD8(+), but neither CD4(+) T lymphocytes nor beta-gal-specific Abs. beta-Gal priming of protective CD8(+) T lymphocytes was found to be CD4(+) T cell-independent, to take place within the draining lymph nodes, and to be accomplished by day 5 after vaccination. Ablation of the injection sites as early as 1.5 h after pR-PV administration still led to protection in a large proportion of the animals, indicating that certain protein Ags administered intradermally in the context of polycations are quickly transported to the draining nodes, where they induce molecular and cellular events resulting in the helper-independent priming and expansion of Tc1 cells. However, optimal protection required the prolonged presence of the injection site, suggesting that pR-PV injection facilitates the formation of a cutaneous depot of Ag-charged cells capable of migration and T cell activation.     

Aged mice develop protective antitumor immune responses with appropriate costimulation

There is a clear decrease in CD8(+) T cell effector function with aging, a loss once thought to be intrinsic to the CD8(+) T cells. Recent studies suggest, however, that this decline may be a consequence of altered stimulatory signals within the aged lymphoid microenvironment. In this study, we compared the immune responses of young and old mice against the BM-185 pre-B cell lymphoma expressing enhanced GFP (EGFP) as a surrogate tumor Ag. Young animals develop protective immune responses when immunized with BM-185-EGFP, but aged mice do not and ultimately succumb to the tumor. However, expression of CD80 (B7.1) on the BM-185-EGFP (BM-185-EGFP-CD80) results in rejection of the tumor by both young and old animals. Additionally, injection of BM-185-EGFP-CD80 cells in young mice promotes the development of long-lasting memory responses capable of rejecting BM-185 wild-type tumors. Aged animals similarly injected did not develop antitumor memory responses. Interestingly, old animals immunized with the BM-185-EGFP-CD80 cells plus injections of the agonist anti-OX40 mAb did develop long-lasting memory responses capable of rejecting the BM-185 wild-type tumors with the same vigor as the young animals. We show that old mice have the capacity to develop strong antitumor responses and protective memory responses as long as they are provided with efficient costimulation. These results have important implications for the development of vaccination strategies in the elderly, indicating that the aged T cell repertoire can be exploited for the induction of tumor immunity.