Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

Inhibition of type I allergic responses with nanogram doses of replicon-based DNA vaccines.

BACKGROUND: Allergic diseases have become a major public health problem in developed countries; yet, no reliable, safe and consistently effective treatment is available. DNA immunization has been shown to prevent and balance established allergic responses, however, the high dose of conventional DNA vaccines necessary for the induction of anti-allergic reactions and their poor immunogenicity in primates require the development of new allergy DNA vaccines. We evaluated protective and therapeutic effects of a Semliki-Forest Virus replicase-based vs a conventional DNA vaccine in BALB/c mice using the model allergen beta-galactosidase. METHODS: Immunoglobulin (Ig)E suppression was determined by a basophil release assay as an in vitro correlate for allergen-specific crosslinking capacity of IgE reflecting the in vivo situation in an allergic individual. Th1 memory responses were measured by cytokine detection via enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT). RESULTS: Nanogram amounts of a replicase-based vector triggered a Th1 response comparable with that achieved with the injection of 20,000-times more copies of a conventional DNA plasmid, and induced IgE suppression in both a protective and a therapeutic setting. CONCLUSIONS: Replicase-based DNA vaccines fulfill the stringent criteria for an allergy DNA vaccine, i.e. low dose, strong Th1 immunogenicity and memory, lack of 'therapy-induced' IgE production and anaphylactic side effects. Moreover, by triggering apoptosis in transfected cells, their unique 'immunize and disappear' feature minimizes the hypothetical risks of genomic integration or induction of autoimmunity.

Mining the melanosome for tumor vaccine targets: P.polypeptide is a novel tumor-associated antigen.

To identify novel, tumor-specific target antigens for vaccine development, we studied immune responses to P.polypeptide, an M(r) 110,000 integral melanosomal membrane protein associated with the Prader-Willi syndrome. Together with expressed sequence tag (EST) and serial analyses of gene expression (SAGE) library analyses, reverse transcription-PCR and Northern blotting verified that P.polypeptide expression was limited to melanoma and melanocytes. A single dominant epitope corresponding to positions 427-435 (IMLCLIAAV) was identified using allele-specific epitope forecasting combined with work in HLA-A*0201/K(b) transgenic mice. This epitope was then used to generate de novo human P.polypeptide-specific CD8+ T cells capable of recognizing P.polypeptide expressing human tumor cell lines in an HLA-A*0201-restricted fashion. Thus, P.polypeptide may be valuable in the creation of novel therapeutic anticancer vaccines.

The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.

In healthy cells, fusion and fission events participate in regulating mitochondrial morphology. Disintegration of the mitochondrial reticulum into multiple punctiform organelles during apoptosis led us to examine the role of Drp1, a dynamin-related protein that mediates outer mitochondrial membrane fission. Upon induction of apoptosis, Drp1 translocates from the cytosol to mitochondria, where it preferentially localizes to potential sites of organelle division. Inhibition of Drp1 by overexpression of a dominant-negative mutant counteracts the conversion to a punctiform mitochondrial phenotype, prevents the loss of the mitochondrial membrane potential and the release of cytochrome c, and reveals a reproducible swelling of the organelles. Remarkably, inhibition of Drp1 blocks cell death, implicating mitochondrial fission as an important step in apoptosis.

Nucleic acid for the treatment of cancer: genetic vaccines and DNA adjuvants.

Despite some interesting pilot experiments more than a century ago, nucleic acid has only recently been added to the list of agents used for the prevention and therapy of cancer. Two distinct features of nucleic acids are used for this purpose: in DNA and RNA vaccines, genetic information for pathogen- or tumor-derived antigens is delivered to the host who then produces the encoded antigen and initiates an immune response. In DNA adjuvants, immunostimulatory sequences (CpG motifs) present in DNA of bacterial origin are used. Such sequences are delivered in the form of oligonucleotides or within the sequence of DNA vaccine. In addition, CpG oligonucleotides by themselves have successfully been used to stimulate the immune system in an antigen-independent manner for the treatment of experimental tumors. DNA and RNA vaccines for the treatment and prevention of cancer and other diseases suffer from two some shortcomings: insufficient immunogenicity and--in the case of RNA--low stability. A variety of strategies are being explored to improve the efficacy of nucleic acid vaccines (genetic vaccines) especially for self-antigens in the case of cancer. Among the most recent improvements are self-replicating RNA vaccines and replicase-based DNA-vaccines in which antigen expression is under the control of an alphaviral replicase. Despite highly promising results in many animal tumor models the efficacy of nucleic acid vaccines and adjuvants in the clinic remains to be seen.

Removal of the circumsporozoite protein (CSP) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP-specific Th2 type immune responses and improvesprotection against malaria infection.

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.

Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein.

The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.

Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice.

CD4+ T cells play a central role in the induction and persistence of CD8+ T cells in several models of autoimmune and infectious disease. To improve the efficacy of a synthetic peptide vaccine based on the self-Ag, gp100, we sought to provide Ag-specific T cell help. To identify a gp100 epitope restricted by the MHC class II allele with the highest prevalence in patients with malignant melanoma (HLA-DRB1*0401), we immunized mice transgenic for a chimeric human-mouse class II molecule (DR4-IE) with recombinant human gp100 protein. We then searched for the induction of CD4+ T cell reactivity using candidate epitopes predicted to bind to DRB1*0401 by a computer-assisted algorithm. Of the 21 peptides forecasted to bind most avidly, murine CD4+ T cells recognized the epitope (human gp10044-59, WNRQLYPEWTEAQRLD) that was predicted to bind best. Interestingly, the mouse helper T cells also recognized human melanoma cells expressing DRB1*0401. To evaluate whether human CD4+ T cells could be generated from the peripheral blood of patients with melanoma, we used the synthetic peptide h-gp10044-59 to sensitize lymphocytes ex vivo. Resultant human CD4+ T cells specifically recognized melanoma, as measured by tumor cytolysis and the specific release of cytokines and chemokines. HLA class II transgenic mice may be useful in the identification of helper epitopes derived from Ags of potentially great clinical utility.

The promise of nucleic acid vaccines.

Establishing the effective use of 'naked' nucleic acids as vaccines would undoubtedly be one of the most important advances in the history of vaccinology. While nucleic acids show much promise for use as vaccine vectors in experimental animals, not a single naked nucleic acid vector has been approved for use in humans. Indeed, data from human clinical trials is scant: nucleic acid vaccines have not been clearly demonstrated to have any convincing efficacy in the prevention or treatment of infectious disease or cancer. Here we illustrate possible mechanisms underlying effective nucleic acid vaccination. We focus on progress that has been made in the improvement of their function. Additionally, we identify promising new strategies and try to forecast future developments that could lead to the real success of nucleic acid vaccines in the prevention and treatment of human disease.

Enhancement of tumor-specific immune response with plasmid DNA replicon vectors.

To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.

DNA and RNA-based vaccines: principles, progress and prospects.

DNA vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. Here we review the current understanding of the mechanisms underlying the activities of these new vaccines. We focus on recent strategies designed to enhance their function including the use of immunostimulatory (CpG) sequences, dendritic cells (DC), co-stimulatory molecules and cytokine- and chemokine-adjuvants. Although genetic vaccines have been significantly improved, they may not be sufficiently immunogenic for the therapeutic vaccination of patients with infectious diseases or cancer in clinical trials. One promising approach aimed at dramatically increasing the immunogenicity of genetic vaccines involves making them 'self-replicating'. This can be accomplished by using a gene encoding RNA replicase, a polyprotein derived from alphaviruses, such as Sindbis virus. Replicase-containing RNA vectors are significantly more immunogenic than conventional plasmids, immunizing mice at doses as low as 0.1 microg of nucleic acid injected once intramuscularly. Cells transfected with 'self-replicating' vectors briefly produce large amounts of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super-activate DC. Thus, the enhanced immunogenicity of 'self-replicating' genetic vaccines may be a result of the production of pro-inflammatory dsRNA, which mimics an RNA-virus infection of host cells.

Cancer therapy using a self-replicating RNA vaccine.

'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.

Plasmodium falciparum malaria blood stage parasites preferentially inhibit macrophages with high phagocytic activity.

Exposure to malaria blood stage antigens results in several defects of macrophages/monocytes one of which is an irreversible reduction of phagocytic activity. In the present study we analysed phagocytic activity of subpopulations of human monocyte-derived-macrophages (MDM) based on the capacity of individual cells to ingest FITC-labelled microbeads. The results demonstrate that malaria infection affected predominantly MDM subpopulations with high level of phagocytosis. This population decreased during parasitaemia, however, during recovery from the infection the highly phagocytic cells replaced the damaged cells. The exposure of MDM cultures to blood stage antigens showed that the highly active macrophages from persons with active malaria infection decreased further, while the population increased during recovery. Furthermore, we observed that while ingestion of a few parasitized RBC (3 schizonts) stimulated phagocytosis, larger amounts or longer exposure periods eventually paralysed the entire phagocytic system. Accordingly, by selectively blocking actively phagocytizing macrophages, the malaria parasite prevents both specific and non-specific immune responses, which are initiated by macrophages as phagocytes and professional antigen presenting cells.

Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites.

The circumsporozoite protein (CSP) is a target for effector Ab and cell mediated immunity against malaria parasites; DNA vaccination can induce both types of effector response. The immunogenicity and efficacy of two DNA plasmids expressing different amounts of Plasmodium berghei CSP were evaluated by immunizing BALB/c mice i.m. or epidermally and by varying the number of immunizations (one to three doses) and the interval between immunizations. Expanding the interval gave the strongest effect, increasing efficacy and antibody boosting, and, in the case of epidermal vaccination, promoting a switch in CSP-specific IgG isotypes from IgG1 to a balance with IgG2a. The strongest humoral immune response and the greatest level of protection were induced by vaccinating epidermally with high expresser plasmid, using a gene gun to administer three doses at 6-wk intervals. For this group, the mean, repeat-specific, prechallenge antibody titer among mice not infected after challenge was significantly higher than that in infected mice, but the mean prechallenge titers for antibody reactive with whole sporozoites were not significantly different. The interval-dependent induction of IgG2a antibodies by epidermal vaccination contradicts the widely held belief that antibody responses induced by this method are restricted to those that are Th2 dependent.