Vacunación con DNA: una perspectiva inmunológica / DNA vaccination: an immunological perspective

La vacunación con DNA es una nueva y prometedora estrategia para la prevención y el tratamiento de muchas enfermedades debido a su habilidad para inducir una respuesta inmunitaria tanto humoral como celular, frente al antígeno codificado por el DNA recombinante. Una vez inoculado en el huésped, el DNA se introduce en las células, donde el antígeno se expresa y se procesa para ser después reconocido por el sistema inmunitario como si se tratase de una infección natural. Esta tecnología presenta grandes esperanzas para ser usada en la inmunoterapia del cáncer y se espera que llegue a ser el método preferido para la próxima generación de vacunas, particularmente adecuadas contra infecciones intracelulares para las que no hay vacunas efectivas. Esta revisión se centra en los mecanismos por los cuales la vacunación con DNA es capaz de inducir una respuesta inmunitaria y los abordajes que se están llevando a cabo para su optimización. También se ofrece una visión general sobre las aplicaciones de la vacunación con DNA (AU)

Soluble CD86 is a costimulatory molecule for human T lymphocytes.

CD86 is an important costimulatory molecule for the priming and activation of naive and memory T cells, respectively. Here, we show that soluble CD86 is detected in human serum. Soluble CD86 is produced by resting monocytes and results from an alternatively spliced transcript (CD86deltaTM) characterized by deletion of the transmembrane domain. Recombinant CD86deltaTM binds to CD28 and CTLA-4 and induces the activation of T cells after stimulation with anti-CD3 mAb. CD86deltaTM also induces IFNgamma production by virus-specific CD8+ memory human T cells stimulated with the Flu M1 peptide. The concentrations of soluble CD86 found in human serum are sufficient to induce biological activity. Soluble CD86 molecule, therefore, appears to be a functional costimulatory molecule playing a potentially important role in immune surveillance.

The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement.

The cooperative antitumor effects of IL-12 and IL-15 gene transfer were studied in the N592 MHC class I-negative small cell lung cancer cell line xenotransplanted in nude mice. N592 cells engineered to secrete IL-15 displayed a significantly reduced tumor growth kinetics, and a slightly reduced tumor take rate, while N592 engineered with IL-12 displayed only minor changes in their growth in nude mice. However, N592 cells producing both cytokines were completely rejected, and produced a potent local bystander effect, inducing rejection of coinjected wild-type tumor cells. N592/IL-12/IL-15 cells were completely and promptly rejected also in NK-depleted nude mice, while in granulocyte-depleted animals a slight delay in the rejection process was observed. Immunohistochemical analyses of the N592/IL-12/IL-15 tumor area in intact nude mice revealed the presence of infiltrating macrophages, granulocytes, and NK cells, and expression of inducible NO synthase and of secondary cytokines such as IL-1beta, TNF-alpha, and IFN-gamma, and at higher levels GM-CSF, macrophage-inflammatory protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted nude mice, numerous macrophages and granulocytes infiltrated the tumor, and a strong expression of macrophage-inflammatory protein-2 and inducible NO synthase was also observed. Finally, macrophages cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited tumor cell growth, further suggesting their role as effector cells in this model.

Synergistic induction of the Tap-1 gene by IFN-gamma and lipopolysaccharide in macrophages is regulated by STAT1.

Proper regulation of the Tap-1 gene is critical for the initiation and continuation of a cellular immune response. Analysis of the Tap-1/low molecular mass polypeptide 2 bidirectional promoter showed that the IFN-gamma activation site element is critical for the rapid induction of the promoter by IFN-gamma following transfection into the human macrophage cell line THP-1. Furthermore, activation of STAT1 binding to this site was important for the synergistic response seen following the stimulation with both IFN-gamma and LPS. Mutation of an IFN-stimulated regulatory element that binds IFN regulatory factor 1 appeared to enhance the response to IFN-gamma and LPS. These data show that STAT1 is necessary for the activation of Tap-1 gene expression in APCs and initiation of cellular immune responses. Furthermore, our data suggest that bacterial products such as LPS may enhance cellular immune responses through augmenting the ability of STAT1 to regulate IFN-gamma-inducible genes.

The membrane form of the type II IL-1 receptor accounts for inhibitory function.

IL-1 signaling is mediated by the type I IL-1R (IL-1RI). The nonsignaling type II receptor has a regulatory function, since it reduces IL-1 effects by scavenging free IL-1 molecules. This regulatory function has been demonstrated only for the soluble form, released from the membrane receptor by action of specific proteases, but is still ill-defined for the membrane receptor itself. To assess the function of membrane IL-1RII, a modified IL-1RII cDNA was constructed, in which the cleavable domain was replaced with the corresponding uncleavable sequence of the epidermal growth factor receptor. The human keratinocyte line HaCaT, which does not express wild-type IL-1RII (wtIL-1RII), was stably transfected with this modified cDNA (unconventionally cleavable IL-1RII (uIL-1RII)). Cells transfected with uIL-1RII expressed the membrane form of IL-1RII, but were unable to produce the 60-kDa soluble receptor. Upon analysis of IL-1 responsiveness, parental HaCaT and vector-transfected cells (E27), expressing IL-1RI and the accessory chain IL-1R accessory protein, were responsive to IL-1. Conversely, cells overexpressing wtIL-1RII (811) or uIL-1RII (9D4) showed comparable reduction in responsiveness to both IL-1alpha (bound by membrane and soluble receptors) and IL-1beta (recognized by the membrane receptor only), suggesting that the membrane form of the IL-1RII is mainly responsible for IL-1 inhibition. In contrast with wtIL-1RII, uIL-1RII did not interact with IL-1R accessory protein. Thus, the membrane form of IL-1RII possesses strong IL-1-inhibitory activity, independent of sequestration of the accessory protein and circumscribed to its ligand sink function.

In vitro and in vivo transfection of p21 gene enhances cyclosporin A-mediated inhibition of lymphocyte proliferation.

Cyclosporine has potent antiproliferative properties, some of which may be via the induction of the cyclin inhibitor p21. In this study, we describe the effects of in vitro and in vivo transfection of p21 in lymphoid and nonlymphoid cells. For in vitro studies, p21 sense plasmid DNA was transfected in A-549 cells (lung adenocarcinoma cell line) and Jurkat cells (human lymphoid cell line). This in vitro transfection of p21 resulted in the inhibition of spontaneous and mitogen-induced cellular proliferation ([3H]thymidine uptake) and also augmented the antiproliferative effects of cyclosporine. In vivo transfection of p21 was accomplished in mice via the i.m. injection of p21 sense plasmid DNA complexed with cationic lipids. As was the case in the cell lines, p21 mRNA was augmented in heart, lung, liver, and spleen 7 days after i.m. injection of p21 sense plasmid DNA. The mitogen (anti-CD3)-induced proliferation of splenocytes from p21-overexpressing mice was significantly decreased, and again this effect was augmented by cotreatment with cyclosporine. These novel findings demonstrate the potential of targeting the cell cycle directly to inhibit alloimmune activation in organ transplantation. This may serve as an alternate strategy to induce immunosuppression, perhaps with less toxicity than that which is seen with conventional immunosuppressive agents.

Transduction of dendritic cells with Bcl-xL increases their resistance to prostate cancer-induced apoptosis and antitumor effect in mice.

We have shown that prostate cancer (PCa) causes apoptosis of dendritic cells (DC), which might block the development of specific antitumor immune responses. Analysis of murine prostatic carcinoma tissues revealed the significant decrease in intratumoral DC number during tumor progression. We demonstrated that the cytokine-mediated increase in DC survival was accompanied by an elevated expression of the anti-apoptotic protein Bcl-xL. Next, we evaluated the resistance to tumor-induced apoptosis and the antitumor efficiency of genetically engineered DC overexpressing Bcl-xL. DC were transduced with an adenoviral vector encoding the murine Bcl-xL gene and injected intratumorally. Data analysis revealed that treatment of PCa-bearing mice with Bcl-xL-transduced DC resulted in significant inhibition of tumor growth compared with the administration of nontransduced DC. Thus, our data suggest that the protection of DC from PCa-induced apoptosis might significantly increase the efficacy of DC-based therapies in cancer even in the absence of available tumor-specific Ags.

Recombinant attenuated Toxoplasma gondii expressing the Plasmodium yoelii circumsporozoite protein provides highly effective priming for CD8+ T cell-dependent protective immunity against malaria.

The protozoan parasite Toxoplasma gondii elicits strong cell-mediated immunity against itself as well as nonspecific resistance against other pathogens and tumors. For this reason, we asked whether recombinant Toxoplasma could be utilized as an effective vaccine vehicle for inducing immunity against heterologous microbial infections. The circumsporozoite protein (PyCSP) of Plasmodium yoelii was engineered into a T. gondii temperature-sensitive strain (ts-4), a mutant that induces complete protection against virulent Toxoplasma challenge. When administered to mice in a single dose, a recombinant ts-4 (CSC3) that both secretes and expresses surface PyCSP induced strong anti-CSP Ab responses, with an isotype distribution pattern similar to that stimulated by the T. gondii carrier. When challenged with P. yoelii sporozoites during the first month after CSC3 vaccination, these animals displayed substantial levels of nonspecific resistance attributable entirely to the T. gondii carrier. Nevertheless, after the nonspecific protection had waned, high levels (up to 79%) of specific immunity against sporozoite challenge were achieved by boosting the animals with recombinant vaccinia virus expressing PyCSP. These CSC3-primed PyCSP-vaccinia-boosted mice displayed high frequencies of splenic PyCSP-specific IFN-gamma-producing cells, as well as CD8+ T cell-dependent cytolytic activity. In vivo depletion of CD8+ lymphocytes at the time of challenge completely ablated protective immunity in the T. gondii-primed/vaccinia-boosted animals, while neutralization of IFN-gamma or IL-12 caused a partial but significant reduction in resistance. Together these findings establish the efficacy of recombinant attenuated Toxoplasma as a vaccine vehicle for priming CD8+-dependent cell-mediated immunity.

Expression of human complement receptor 2 (CR2, CD21) in Cr2-/- mice restores humoral immune function.

Complement receptor type 2 (CR2, CD21) is expressed by both human and murine B cells and has been demonstrated to play a pivotal role in the humoral immune response. We have reconstituted Cr2-/- mice with an 80-kb human genomic fragment (designated P1-5) containing the full-length human CR2 (hCR2) gene. Transfection of P1-5 into the mouse A20 B cell line confirmed that it would direct expression of the hCR2 protein in mouse B cells. Immunoprecipitation analysis in these cells revealed that hCR2 coassociates with mouse CD19. After creation of transgenic mice using P1-5, we found significant expression of hCR2 on peripheral blood and splenic B cells by flow cytometric analysis. RT-PCR analysis of tissues and purified cell populations from transgene-positive mice revealed that hCR2 expression was restricted to B cells and the spleen in a pattern that matches mouse CR2. To rigorously assess the functional capabilities of hCR2, the transgene was bred onto Cr2-/- mice, which have a notable defect in response to SRBC Ag. We found that Cr2-/- mice expressing hCR2 had a substantial restoration of the humoral immune response to SRBC as compared with nontransgenic Cr2-/- littermate controls. Overall, this study suggests that hCR2 is able to substitute for mouse CR2 in the murine immune system. Therefore, hCR2-transgenic mice offer a valuable model system to further examine immunologic roles as well as structure-function relationships important for hCR2 function in primary cells in vivo.

Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c.

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b. DAF and CD1c. DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c. DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b. DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c. DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b. DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.

Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression.

IL-10 is a unique cytokine because it is anti-inflammatory and immunosuppressive. IL-10 is regulated at the level of transcription, but the critical motifs and the relevant transcription factors controlling this gene have remained elusive to date. We now report that a sequence at -120 bp in the human IL-10 promoter binds Stat3 but no other Stat proteins. Mutation of this motif abrogates LPS-induced trans-activation. Overexpression of dominant negative Stat3 suppresses promoter activity, while wild-type Stat3 leads to an enhancement of this activity. Our results show that Stat3, by binding to a single motif in the IL-10 promoter, is controlling expression of the human IL-10 gene.

An epithelial cell line that can stimulate alloproliferation of resting CD4+ T cells, but not after IFN-gamma stimulation.

It has previously been shown that IFN-gamma-induced up-regulation of HLA class II on the surface of epithelial cells is not sufficient to induce proliferation of allospecific CD4+ T cells in vitro. To further investigate this phenomenon, a human epithelial bladder carcinoma, T24, was induced to constitutively express HLA class II without IFN-gamma stimulation, by permanent transfection with the full-length class II transactivator (CIITA) gene. Proliferation of allospecific T cells to transfected and wild-type cells with and without prior activation with saturating levels of IFN-gamma for 4 days was examined. IFN-gamma-activated T24 did not induce any response from CD4+ T cells. However, T24.CIITA induced significant levels of alloproliferation, which could be abrogated by pretreatment of T24.CIITA with a mAb to LFA-3. Prestimulation of T24. CIITA with saturating levels of IFN-gamma for 4 days also prevented allospecific CD4+ T cell proliferation. These findings suggest that epithelial cells may be intrinsically able to process and present alloantigen and provide adequate costimulation. We propose that IFN-gamma has a secondary, as yet unidentified, effect that acts to negatively regulate this response, at least in some epithelial cells.

Involvement of an ATP-dependent peptide chaperone in cross-presentation after DNA immunization.

Immunization with plasmid DNA holds promise as a vaccination strategy perhaps useful in situations that currently lack vaccines, since the major means of immune induction may differ from more conventional approach. In the present study, we demonstrate that exposure of macrophages to plasmid DNA encoding viral proteins or OVA generates Ag-specific material that, when presented in vitro by dendritic cells to naive T cells, induces primary CTL response or elicits IL-2 production from an OVA peptide-specific T-T hybridoma. The immunogenic material released was proteinaceous in nature, free of apoptotic bodies, and had an apparent m.w. much larger than a 9-11-aa CTL-recognizable peptide. The macrophage-released factor(s) specifically required a hydrolyzable ATP substrate and was inhibited by procedures that removed or hydrolyzed ATP; in addition, anti-heat-shock protein 70 antiserum abrogated the activity to a large extent. These results indicate the possible involvement of a heat-shock protein 70-linked peptide chaperone in a cross-priming method of immune induction by DNA vaccination. Such a cross-priming process may represent a principal mechanism by which plasmid DNA delivered to cells such as myocytes effectively shuttle Ag to DC or other APC to achieve CTL induction in vivo.

A role for the region encompassing the c" strand of a TCR V alpha domain in T cell activation events.

The distinct strand topology of TCR V alpha domains results in a flatter surface in the region encompassing the c" strand than the corresponding region in Ig V domains. In the current study a possible role for this region in T cell activation has been investigated by inserting a potential glycosylation site at V alpha residue 82. This residue is in proximity to the c" strand and distal to the putative interaction site for cognate peptide:MHC ligand. An additional N-linked carbohydrate at this position would create a protrusion on the V alpha domain surface, and this may interfere with TCR aggregation and/or recruitment of signaling molecules. The modified TCR has been expressed in transfected T cells, and the phenotype following stimulation has been compared with that of cells expressing the wild-type TCR. The mutation has significant effects on activation-induced cell death and TCR internalization, but, unexpectedly, does not affect IL-2 secretion. Furthermore, analyses with tetrameric, peptide:MHC class II complexes suggest that the mutation decreases the ability of the TCR to aggregate into a configuration compatible with avid binding by these multivalent ligands.

Repression of IL-2 promoter activity by the novel basic leucine zipper p21SNFT protein.

IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. Described here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene.

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses.

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.

An IFN-gamma-inducible transcription factor, IFN consensus sequence binding protein (ICSBP), stimulates IL-12 p40 expression in macrophages.

IL-12 is a cytokine that links innate and adaptive immunity. Its subunit p40 is induced in macrophages following IFN-gamma/LPS stimulation. Here we studied the role for IFN consensus sequence binding protein (ICSBP), an IFN-gamma/LPS-inducible transcription factor of the IFN regulatory factor (IRF) family in IL-12 p40 transcription. Macrophage-like cells established from ICSBP-/- mice did not induce IL-12 p40 transcripts, nor stimulated IL-12 p40 promoter activity after IFN-gamma/LPS stimulation, although induction of other inducible genes was normal in these cells. Transfection of ICSBP led to a marked induction of both human and mouse IL-12 p40 promoter activities in ICSBP+/+ and ICSBP-/- cells, even in the absence of IFN-gamma/LPS stimulation. Whereas IRF-1 alone was without effect, synergistic enhancement of promoter activity was observed following cotransfection of ICSBP and IRF-1. Deletion analysis of the human promoter indicated that the Ets site, known to be important for activation by IFN-gamma/LPS, also plays a role in the ICSBP activation of IL-12 p40. A DNA affinity binding assay revealed that endogenous ICSBP is recruited to the Ets site through protein-protein interaction. Last, transfection of ISCBP alone led to induction of the endogenous IL-12 p40 mRNA in the absence of IFN-gamma and LPS. Taken together, our results show that ICSBP induced by IFN-gamma/LPS, acts as a principal activator of IL-12p40 transcription in macrophages.

Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro.

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.

Novel protein transfection of primary rat cortical neurons using an antibody that penetrates living cells.

An Ab-based system to deliver functional proteins into neurons was developed using the murine mAb, mAb 3E10. This was achieved by covalently conjugating catalase to the Ab so that the conjugate retained high activity for the degradation of hydrogen peroxide. Three-dimensional fluorescence microscopy was used to demonstrate penetration of the Ab into the nucleus of living primary cortical neurons. The Ab conjugate localized in both the cytoplasm and nucleus. Retention of catalase activity after penetration and distribution of conjugate was demonstrated by reduction in cell death following exposure of treated neurons to hydrogen peroxide. These studies illustrate the potential of this method for the intracellular delivery of therapeutic proteins.

Enhancement of immunogenicity of tumor cells by cotransfection with genes encoding antisense insulin-like growth factor-1 and B7.1 molecules.

Insulin-like growth factor-1 (IGF-1) is expressed in many tumor cell lines and has a role in both normal cell proliferation and in the growth of cancers. Tumor cells transfected with a vector encoding an IGF-1 antisense cDNA transcriptional cassette driven by the mouse metallothionein-1 promoter become immunogenic and lose their tumorigenicity in syngeneic animals. The enhanced immunogenicity is associated with an up-regulation in the expression of major histocompatibility complex class I molecule on cell surfaces. Blockade of the expression of IGF-1 in tumor cells by the IGF-1 antisense RNA approach is not uniformly effective in the induction of antitumoral protective immunity in low and nonimmunogenic tumor model systems. Here, we report that the immunogenicity of hepa 1-6 hepatoma and SMCC-1 colon carcinoma cells, which are poorly immunogenic and unresponsive to antisense IGF-1 gene transfer, can be induced by cotransfection with genes encoding antisense IGF-1 and mouse B7.1 molecules. The tumor cells modified in this manner become strongly immunogenic and can be used as a cellular vaccine to induce a protective immune response in vivo. Immunization with the transfected tumor cells also results in regression of the established hepa 1-6 hepatoma and SMCC-1 colon cancer. The immunity is tumor-specific and is mediated by CD3+ CD8+ T cells. Cytotoxic T lymphocytes generated in vitro by priming naive spleen cells and in vivo by immunizing mice with the double-transfected tumor cells specifically lysed autologous tumors cells and were effective in adoptive immunotherapy. The data suggest that modification of tumor cells in vitro by cotransfection with genes encoding antisense IGF-1 and B7.1 molecules may open a new avenue for cancer immunogene therapy.