Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity.

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy.

Many tumor-associated Ags represent tissue differentiation Ags that are poorly immunogenic. Their weak immunogenicity may be due to immune tolerance to self-Ags. Prostatic acid phosphatase (PAP) is just such an Ag that is expressed by both normal and malignant prostate tissue. We have previously demonstrated that PAP can be immunogenic in a rodent model. However, generation of prostate-specific autoimmunity was seen only when a xenogeneic homolog of PAP was used as the immunogen. To explore the potential role of xenoantigen immunization in cancer patients, we performed a phase I clinical trial using dendritic cells pulsed with recombinant mouse PAP as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly vaccinations of xenoantigen-loaded dendritic cells with minimal treatment-associated side effects. All patients developed T cell immunity to mouse PAP following immunization. Eleven of the 21 patients also developed T cell proliferative responses to the homologous self-Ag. These responses were associated with Ag-specific IFN-gamma and/or TNF-alpha secretion, but not IL-4, consistent with induction of Th1 immunity. Finally, 6 of 21 patients had clinical stabilization of their previously progressing prostate cancer. All six of these patients developed T cell immunity to human PAP following vaccination. These results demonstrate that xenoantigen immunization can break tolerance to a self-Ag in humans, resulting in a clinically significant antitumor effect.

Dendritic cells injected via different routes induce immunity in cancer patients.

Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-gamma production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.

Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations.

Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.

We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.

Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization: implications for immunotherapy of prostate cancer.

Prostatic acid phosphatase (PAP) is uniquely expressed in prostatic tissue and prostate cancer. In this study, the immunogenicity of PAP was investigated in a male rat model. We show that immunization with recombinant rat or human PAP in CFA leads to a significant Ab response, but does not generate CTL or result in autoimmune prostatitis. In contrast, immunization with recombinant vaccinia expressing human PAP, but not rat PAP, generates a CTL response and tissue-specific prostatitis in the absence of detectable PAP-specific Abs. These findings suggest that a cellular immune response to PAP, rather than Abs, mediates destructive autoimmune prostatitis. Thus, xenogeneic forms of PAP are a new tool for the induction of prostate-specific immunity and may prove useful for the immunotherapy of prostate cancer.

Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide.

Protection against most intracellular pathogens requires T cells that recognize pathogen-derived peptides in association with MHC class I molecules on the surface of infected cells. However, because exogenous proteins do not ordinarily enter the cytosol and access the MHC class I-processing pathway, protein-based vaccines that induce class I-restricted CTL responses have proved difficult to design. We have addressed this problem by conjugating proteins, such as OVA, to a short cationic peptide derived from HIV-1 tat (residues 49-57). When APC were exposed in vitro to such protein conjugates, they processed and presented the peptides in association with MHC class I molecules and stimulated CD8+ Ag-specific T cells. Moreover, Ag-specific CTLs were generated in vivo by immunizing mice with histocompatible dendritic cells that had been exposed to protein-tat conjugates.