IL-22-dependent attenuation of T cell-dependent (ConA) hepatitis in herpes virus entry mediator deficiency.

Coinhibitors and costimulators control intrahepatic T cell responses that trigger acute hepatitis. We used the ConA-induced hepatitis model in the mouse to test if the coinhibitor herpes virus entry mediator (HVEM) modulates hepatitis-inducing T cell responses. Compared with ConA-injected, wild-type (wt) C57BL/6 (B6) mice, HVEM-deficient (HVEM(-/-)) B6 mice showed lower serum transaminase levels and lower proinflammatory IFN-gamma, but higher protective IL-22 serum levels and an attenuated liver histopathology. The liver type I invariant NKT cell population that initiates acute hepatitis in this model was reduced in HVEM(-/-) mice but their surface phenotype was similar to that of untreated or ConA-treated wt controls. In response to mitogen injection, liver invariant NKT cells from HVEM(-/-) B6 mice produced in vivo more IL-22 but lower amounts of IFN-gamma and IL-4 than wt controls. Bone marrow chimeras showed that HVEM deficiency of the liver nonparenchymal cell population, but not of the parenchymal cell population, mediated the attenuated course of the dendritic cell- and T cell-dependent ConA hepatitis. IL-22 is produced more efficiently by liver NKT cells from HVEM(-/-) than from wt mice, and its Ab-mediated neutralization of IL-22 aggravated the course of hepatitis in wt and HVEM(-/-) mice. Hence, HVEM expression promotes pathogenic, proinflammatory Th1 responses but down-modulates protective IL-22 responses of T cells in this model of acute hepatitis.

Elimination of immunodominant epitopes from multispecific DNA-based vaccines allows induction of CD8 T cells that have a striking antiviral potential.

Immunodominance limits the TCR diversity of specific antiviral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain Ags (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV) surface (S), core (C), and polymerase (Pol) proteins and/or the OVA Ag as stress protein-capturing fusion proteins. Priming of mono- or multispecific, HLA-A*0201- or K(b)-restricted CD8 T cell responses by these DNA vaccines differed. K(b)/OVA(257-264)- and K(b)/S(190-197)-specific CD8 T cell responses did not allow priming of a K(b)/C(93-100)-specific CD8 T cell response in mice immunized with multidomain vaccines. Tolerance to the S- Ag in transgenic Alb/HBs mice (that express large amounts of transgene-encoded S- Ag in the liver) facilitated priming of subdominant, K(b)/C(93-100)-specific CD8 T cell immunity by multidomain Ags. The "weak" (i.e., easily suppressed) K(b)/C(93-100)-specific CD8 T cell response was efficiently elicited by a HBV core Ag-encoding vector in 1.4HBV-S(mut) tg mice (that harbor a replicating HBV genome that produces HBV surface, core, and precore Ag in the liver). K(b)/C(93-100)-specific CD8 T cells accumulated in the liver of vaccinated 1.4HBV-S(mut) transgenic mice where they suppressed HBV replication. Subdominant epitopes in vaccines can hence prime specific CD8 T cell immunity in a tolerogenic milieu that delivers specific antiviral effects to HBV-expressing hepatocytes.

IL-20 receptor 2 signaling down-regulates antigen-specific T cell responses.

The recently described cytokines IL-19, IL-20, and IL-24 share structural homology with IL-10 and are therefore classified as members of the IL-10 family of cytokines. Although it has long been speculated that signaling by their heterodimeric receptor complexes (IL-20R1/IL-20R2 and IL-22R/IL-20R2) influences immunological processes, the target cells for this group of cytokines are still unclear. By generating a knockout mouse strain deficient for the common IL-20R beta-chain (IL-20R2), we show that IFN-gamma and IL-2 secretion is significantly elevated after stimulation of IL-20R2-/--deficient CD8 and CD4 T cells with Con A or anti-CD3/CD28 in vitro. IL-10 secretion by activated IL-20R2-/- CD4 cells was diminished. Consistent with our in vitro results, significantly more Ag-specific CD8 IFN-gamma+ and CD4 IFN-gamma+ T cells developed to locally applied DNA vaccines in IL-20R2-deficient mice. In a T cell-dependent model of contact hypersensitivity, IL-20R2 knockout mice were more sensitive to the contact allergen trinitro-chloro-benzene. Thus, IL-20R2 signaling directly regulates CD8 and CD4 T cell answers in vitro and in vivo. For the first time, we provide evidence that IL-19, IL-20, and IL-24 are part of a signaling network that normally down-modulates T cell responses in mice.

Safety of probiotic Escherichia coli strain Nissle 1917 depends on intestinal microbiota and adaptive immunity of the host.

Probiotics are viable microorganisms that are increasingly used for treatment of a variety of diseases. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. Here we examined the role of the intestinal microbiota and the adaptive immune system in preventing translocation of probiotics (e.g., Escherichia coli Nissle). We challenged C57BL/6J mice raised under germfree conditions (GF-raised C57BL/6J mice) and Rag1(-/-) mice raised under germfree conditions (GF-raised Rag1(-/-) mice) and under specific-pathogen-free conditions (SPF-raised Rag1(-/-) mice) with probiotic E. coli strain Nissle 1917, strain Nissle 1917 mutants, the commensal strain E. coli mpk, or Bacteroides vulgatus mpk. Additionally, we reconstituted Rag1(-/-) mice with CD4(+) T cells. E. coli translocation and dissemination and the mortality of mice were assessed. In GF-raised Rag1(-/-) mice, but not in SPF-raised Rag1(-/-) mice or GF-raised C57BL/6J mice, oral challenge with E. coli strain Nissle 1917, but not oral challenge with E. coli mpk, resulted in translocation and dissemination. The mortality rate was significantly higher for E. coli strain Nissle 1917-challenged GF-raised Rag1(-/-) mice (100%; P < 0.001) than for E. coli strain Nissle 1917-challenged SPF-raised Rag1(-/-) mice (0%) and GF-raised C57BL/6J mice (0%). Translocation of and mortality due to strain E. coli Nissle 1917 in GF-raised Rag1(-/-) mice were prevented when mice were reconstituted with T cells prior to strain E. coli Nissle 1917 challenge, but not when mice were reconstituted with T cells after E. coli strain Nissle 1917 challenge. Cocolonization experiments revealed that E. coli mpk could not prevent translocation of strain E. coli Nissle 1917. Moreover, we demonstrated that neither lipopolysaccharide structure nor flagella play a role in E. coli strain Nissle 1917 translocation and dissemination. Our results suggest that if both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain Nissle 1917 may occur and have potentially severe adverse effects. Future work should define the possibly related molecular factors that promote probiotic functions, fitness, and facultative pathogenicity.

Type I IFN-induced, NKT cell-mediated negative control of CD8 T cell priming by dendritic cells.

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8(+) conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d(-/-) mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-gamma, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-gamma but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.

B7-H1 on hepatocytes facilitates priming of specific CD8 T cells but limits the specific recall of primed responses.

BACKGROUND & AIMS: The requirement for costimulation of CD8 T-cell priming and restimulation by nonprofessional antigen-presenting cells is unresolved. Here, we investigated whether B7-H1 (CD274, PD-L1) on hepatocytes (HC) is involved in the specific activation of naive CD8 T cells or activated CD8 T blasts. METHODS: Naive or activated CD8 T cells from transgenic OT-I mice were primed/restimulated by peptide-pulsed HC, and their proliferation and effector response were determined. We used blocking monoclonal antibodies against B7-H1 and HC from B7-H1-deficient mice to assign a costimulatory or coinhibitory role to B7-H1 for CD8 T-cell priming/restimulation. RESULTS: Blockade of B7-H1 on HC down modulated interferon (IFN)-gamma production and proliferation of HC-primed CD8 T cells, indicating a costimulatory role for B7-H1 in priming CD8 T cells. In contrast, the PD-1/B7-H1 interaction inhibited proliferation and interferon-gamma release of effector/memory CD8 T blasts specifically restimulated by peptide-pulsed HC. CONCLUSIONS: B7-H1 differentially modulates the different stages of the specific CD8 T-cell response triggered by HC, and, whereas it costimulates priming and cytokine responses of naive CD8 T cells, it coinhibits their specific local recall of effector cytokine responses. The interaction of CD8 T cells with B7-H1(+) HC can thus fine-tune proliferative and effector responses of specific CD8 T cells reacting locally to nonprofessional antigen-presenting cells infected with hepatotropic agents.

Local accumulation and activation of regulatory Foxp3+ CD4 T(R) cells accompanies the appearance of activated CD8 T cells in the liver.

UNLABELLED: Only small populations of nonactivated, nonproliferating Foxp3(+) CD4 regulatory T cell (T(R)) cells are found in the nonparenchymal cell compartment of the mouse liver while liver-draining celiac nodes contain expanded, activated T(R) cell populations (similar to other lymph nodes). Liver Foxp3(+) CD4 T(R) cells suppress activation of T cell responses. Polyclonal, systemic T cell activation in vivo (via anti-CD3 antibody injection) is accompanied by intrahepatic accumulation of T blasts and a rapid but transient intrahepatic increase of activated, proliferating Foxp3(+) CD4 T(R) cells. Following vaccination, the appearance of peripherally primed, specific CD8 T blasts in the liver is preceded by a transient rise of Foxp3(+) CD4 T(R) cells in the liver. The adoptive transfer of immune CD8 T cells into congenic hosts that express the relevant antigen only in the liver leads to the accumulation of specific donor CD8 T cells and of host Foxp3(+) CD4 T(R) cells in the liver. CONCLUSION: Although it contains only a small population of quiescent Foxp3(+) CD4 T(R) cells, the liver can rapidly mobilize and/or recruit this T cell control in response to the intrahepatic appearance of peripherally or locally generated CD8 T blasts.

Innate immune responses in NF-kappaB-repressing factor-deficient mice.

NF-kappaB-repressing factor (NRF) is a transcriptional silencer protein that specifically counteracts the basal activity of several NF-kappaB-dependent promoters by direct binding to specific neighboring DNA sequences. In cell culture experiments, the reduction of NRF mRNA leads to a derepression of beta interferon, interleukin-8, and inducible nitric oxide synthase transcription. The X chromosome-located single-copy NRF gene is ubiquitously expressed and encodes a protein of 690 amino acids. The N-terminal part contains a nuclear localization signal, the DNA-binding domain, and the NF-kappaB-repressing domain, while the C-terminal part is responsible for double-stranded RNA binding and nucleolar localization. To study the function of NRF in a systemic context, transgenic mice lacking the NRF gene were created. Against predictions from in vitro experiments, mice with a deletion of the NRF gene are viable and have a phenotype that is indistinguishable from wild-type mice, even after challenge with different pathogens. The data hint towards an unexpected functional redundancy of NRF.

Recombinant complexes of antigen with stress proteins are potent CD8 T-cell-stimulating immunogens.

Heat shock proteins (Hsp) of the Hsp70/90 families facilitate cellular immune responses to antigenic peptides or proteins bound to them and have therefore been used as vaccine vehicles. We developed an expression system in which chimeric proteins with an Hsp-capturing, viral J domain fused to diverse antigen-encoding sequences form stable complexes with eukaryotic (Hsp70, Hsp73) or bacterial (DnaK) stress proteins and accumulate to high steady-state levels. J domains from different species (viruses/SV40, bacteria/Chlamydia trachomatis or plants/Arabidopsis thaliana) efficiently capture murine or human stress proteins in this system, thus making different J domains available for vaccine production. A novel expression and purification method was developed to produce native Hsp/antigen complexes in transfectants. These purified Hsp/antigen complexes efficiently elicited antigen-specific CD8 T cell responses in mice when delivered as vaccines without adjuvants. In situ complex formation of antigen with Hsp was critical for CD8 T cell priming. Because the described expression system supports the flexible design of multivalent vaccines, it is an attractive strategy to elicit CD8 T cell responses either to recombinant proteins or to selected antigenic domains of these molecules.

The immunogenicity of adenovirus vectors limits the multispecificity of CD8 T-cell responses to vector-encoded transgenic antigens.

We investigated whether the immunogenicity of antigens delivered by recombinant E1-deleted adenovirus (Ad) is impaired by the concomitant priming of specific immunity to protein antigens of the vector. A comparative evaluation of the immunogenicity of hepatitis B surface antigen (HBsAg or S) or ovalbumin (OVA) was carried out in mice injected with either the antigen-encoding Ad vector or a corresponding plasmid DNA vaccine. Recombinant Ad, but not the plasmid DNA vaccine, induced long lasting, specific CD8 T-cell immunity to immunodominant epitopes of the two antigens. In contrast, the HBsAg-encoding pCI/S DNA, but not the Ad/S vaccine, was shown to prime CD8 T-cell responses to subdominant HBsAg epitopes. Ad/S-primed CD8 T-cell responses to immunodominant epitopes of vector-encoded or capsid-delivered Ad proteins apparently suppressed CD8 T-cell priming to subdominant HBsAg epitopes. In B-cell-deficient mice, the established, Ad-specific T-cell immunity induced by vaccination with an irrelevant Ad vector impaired the priming of HBsAg-specific CD8 T-cell responses by Ad/S. It is clear, therefore, that a T-cell immunity specific for Ad proteins (either delivered with the Ad capsid or transcribed from the Ad genome) is efficiently primed by vaccination with Ad vectors, and can limit the immunogenicity (particularly of subdominant epitopes) of Ad vector-encoded transgenic antigens.

Fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies.

Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product-specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.

Tumor suppression by IFN regulatory factor-1 is mediated by transcriptional down-regulation of cyclin D1.

IFNs have been ascribed to mediate antitumor effects. IFN regulatory factor-1 (IRF-1) is a major target gene of IFNs. It inhibits cell proliferation and oncogenic transformation. Here, we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of nontransformed cells by IRF-1. These include cell cycle-regulating genes. An indirect target is cyclin D1. Activation of IRF-1 decreased cyclin D1 expression and cyclin-dependent kinase 4 kinase activity concomitant with change in the levels of hyperphosphorylated retinoblastoma protein. These effects are mediated by inhibition of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway and a transcriptional repression of cyclin D1. As shown by in vitro assays and tumor growth in nude mice, IRF-1-mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Conversely, decrease of cyclin D1 by RNA interference experiments prevents transformation and tumor growth. The data show that cyclin D1 is a key target for IRF-1-mediated tumor-suppressive effects.

Host gene expression in the colon of gnotobiotic interleukin-2-deficient mice colonized with commensal colitogenic or noncolitogenic bacterial strains: common patterns and bacteria strain specific signatures.

BACKGROUND: Specific pathogen-free (SPF), but not germfree (GF), interleukin (IL)-2-deficient (IL-2-/-) mice develop inflammatory bowel disease (IBD) at 10 to 15 weeks of age. Gnotobiotic IL-2-/- mice monocolonized with E. coli mpk develop IBD at 25 to 33 weeks of age but not B. vulgatus mpk, E. coli Nissle 1917, or mice cocolonized with both E. coli mpk and B. vulgatus. METHODS: To determine genes regulated by these commensal bacteria, host gene expression in the colon of 8-week-old IL-2-/- mice was compared by using microarrays and semiquantitative reverse-transcription polymerase chain reaction. Colonization with E. coli mpk/B. vulgatus or SPF microbiota altered the gene expression profile more profoundly than monocolonization with either B. vulgatus, E. coli mpk or E. coli Nissle indicating that the complexity of the gene expression pattern is influenced by the diversity of the microbiota. RESULTS: A small but distinct group of genes could be defined which might be associated with colitis development. Thus, 8 week old E. coli mpk IL-2-/- mice rone to colitis compared to E. coli Nissle, B. vulgatus and E. coli mpk/B. vulgatus IL-2-/- mice displayed a lower expression of the anti-inflammatory RegIII family genes such as RegIII[gamma] and pancreatitis associated protein (PAP) and peroxisome proliferator-activated receptor-[gamma] regulated genes such as adipsin and adiponectin. CONCLUSION: The increased expression of these genes in B. vulgatus colonized mice might be associated with prevention of E. coli mpk triggered colitis in E. coli mpkM/B. vulgatus IL-2-/- mice.

Different sources of "help" facilitate the antibody response to hepatitis D virus delta antigen.

Repeated injections of hepatitis D antigen (HDAg) delivered either as a recombinant protein, or expressed from a DNA vaccine elicited no (or only very low) antibody responses in inbred mouse strains. Codelivery of oligonucleotides (ODN) with immune-stimulating sequences (ISS) with the protein antigen, or ISS in DNA vaccines (encoding HDAg) did not overcome the low intrinsic immunogenicity of this small viral antigen for B cells. In contrast, codelivery of immunogenic, heterologous proteins (either mixed to recombinant HDAg as recombinant proteins, or fused to HDAg sequences as chimeric antigens expressed from DNA vaccines) provided specific, CD4+ T cell-dependent "help" that supported efficient priming of antibody responses to HDAg. Chimeric proteins in which selected HDAg fragments were fused in frame with immunogenic, heterologous protein fragments produced by DNA vaccines allowed the mapping of antibody-binding HDAg domains of the viral antigen. The described approach thus facilitates induction of serum antibody responses against native viral antigens with low immunogenicity for B cells.

Complexes of DNA vaccines with cationic, antigenic peptides are potent, polyvalent CD8(+) T-cell-stimulating immunogens.

A priority in current vaccine research is the development of multivalent vaccines that support the efficient priming of long-lasting CD8(+) T-cell immunity. We developed a novel vaccination strategy that used synthetic, cationic (positively charged), and antigenic peptides complexed to negatively charged nucleic acids: antigenic, major histocompatibility complex-class I-binding epitopes fused with a cationic sequence derived from the HIV tat protein (tat50-57: KKRRQRRR) were mixed with nucleic acids (e.g., CpG-containing oligonucleotides) to quantitatively form peptide/nucleic acid complexes. The injection of these complexes efficiently primed long-lasting, specific CD8(+) T-cell immunity of high magnitude. This chapter describes a novel strategy to codeliver complexes of cationic/antigenic peptides bound to antigen-encoding plasmid DNA vaccines in a way that enhances the immunogenicity of both components for T cells.

A stress protein-facilitated antigen expression system for plasmid DNA vaccines.

In DNA vaccination, an exciting new immunization technique with potential applications in clinical medicine, expression plasmid DNA containing antigen-encoding sequences cloned under heterologous promoter control are delivered by techniques that lead in vivo to antigen expression in transfected cells. DNA vaccination efficiently primes both humoral and cellular immune responses. We developed a novel expression system for DNA vaccines in which a fusion protein with a small, N-terminal, viral DnaJ-like sequence (J domain) is translated in frame with C-terminal antigen-encoding sequences. The J domain stable bind to constitutively expressed, cytosolic stress protein hsp73 and triggers intracellular accumulation of antigen/hsp73 complexes. The system supports enhanced expression of chimeric antigens of >800 residues in length in immunogenic form. A unique advantage of the system is that even unstable or toxic proteins (or protein domains) can be expressed. We describe the design of DNA vaccines expressing antigens with a stress protein-capturing domain and characterize the immunogenicity of the antigens produced by this expression system.

Priming polyvalent immunity by DNA vaccines expressing chimeric antigens with a stress protein-capturing, viral J-domain.

The N-terminal domain of large tumor antigens (T-Ag) of polyomaviruses forms a DnaJ-like structure with a conserved J domain that associates with constitutively expressed stress protein heat shock protein (hsp)73. Mutant (but not wild-type) SV40 T-Ag show stable, ATP-dependent binding to the stress protein hsp73 when expressed in cells from different vertebrate tissues. Intracellular T/hsp73 complexes accumulate to high steady-state levels. From this observation, we designed a vector system that supports stable expression of a large variety of hsp73-capturing, chimeric antigens containing an N-terminal, T-Ag-derived domain, and different C-terminal antigenic domains from unrelated antigens. Most antigenic domains tested could be stably expressed only in eukaryotic cells as fusion protein/hsp73 complexes. The N-terminal 77 residues representing the J domain of T-Ag were required for stable hsp73 binding and efficient expression of chimeric antigens. Hsp73-bound chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular cytolytic T lymphocytes (CTL) responses. The described system supports efficient expression of chimeric, polyvalent antigens and their codelivery with hsp73 as a "natural adjuvant" for enhanced immunogenicity for T and B cells.

The hepatic immune system.

The liver regulates T-cell homeostasis, induces T-cell tolerance, and supports intrahepatic T-cell responses against hepatotropic pathogens. Many data from clinical and preclinical systems provide supportive evidence for these diverse roles of the liver in modulating peripheral (systemic, mucosal, and intrahepatic) T-cell immunity. Little information is available on the cellular and molecular mechanisms that mediate the dual role of the liver in tolerizing T-cell responses and in supporting intrahepatic priming of T-cell responses. Understanding these immunoregulatory effects in the liver may offer insight into clinically relevant immunopathologies of this organ.

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

Cytokine-facilitated priming of CD8+ T cell responses by DNA vaccination.

Immune responses elicited by plasmid DNA vaccination can be enhanced and modulated by codelivery of cytokine-encoding plasmids. We studied whether priming of cytotoxic T lymphocyte (CTL) responses against hepatitis B surface antigen (HBsAg) by DNA vaccines injected either intramusculary or intradermally with the gene gun is enhanced by codelivery of cytokine-encoding plasmids. From a panel of tested cytokine plasmids only mouse IFNbeta, IL-15, and GM-CSF encoding plasmids showed an effect. Intradermal gene gun vaccination with 1 micro g plasmid DNA encoding intracellular HBsAg (large LS) showed enhanced CTL priming when IFNbeta, IL15, or GM-CSF encoding plasmids were codelivered; this was not observed when a DNA vaccine encoding secreted HBsAg (small S) was injected. Intramuscular injection of low (5 micro g) doses of a DNA vaccine encoding large HBsAg did not prime CTL when delivered without cytokines, with IFNbeta or IL15-encoding plasmids. However, codelivery with GM-CSF encoding plasmid DNA primed potent, specific CTL immunity detected either in a cytotoxic assay or by determining the frequency of L(d)-restricted CD8(+) T cells specifically inducible to IFNgamma production. The codelivery of GM-CSF encoding plasmids with the DNA vaccine furthermore enhanced CTL priming to a subdominant, D(d)-restricted epitope of HBsAg. The adjuvant effect of cytokine-encoding plasmids on CTL priming by DNA vaccines is thus complex and depends on: (a) the type of cytokine (or combination of cytokines) codelivered, (b) the type (intracellular vs. secreted) and dose (1-50 micro g) of the DNA vaccine, (c) the method of DNA vaccine delivery ("naked" vs. particle-coated DNA), and (d) the (intramuscular vs. intradermal) route of delivery of the DNA vaccine.