Vaccines based on cytomegalovirus (CMV) demonstrate protection in animal models of infectious disease and cancer. Vaccine efficacy is associated with the ability of CMV to elicit and indefinitely maintain high frequencies of circulating effector memory T cells (T-EM) providing continuous, lifelong anti-pathogen immune activity. To allow for the clinical testing of human CMV (HCMV)-based vaccines we constructed and characterized as a vector backbone the recombinant molecular clone TR3 representing a wildtype genome. We demonstrate that TR3 can be stably propagated in vitro and that, despite species incompatibility, recombinant TR3 vectors elicit high frequencies of T-EM to inserted antigens in rhesus macaques (RM). Live-attenuated versions of TR3 were generated by deleting viral genes required to counteract intrinsic and innate immune responses. In addition, we eliminated subunits of a viral pentameric glycoprotein complex thus limiting cell tropism. We show in a humanized mouse model that such modified vectors were able to establish persistent infection but lost their ability to reactivate from latency. Nevertheless, attenuated TR3 vectors preserved the ability to elicit and maintain T-EM to inserted antigens in RM. We further demonstrate that attenuated TR3 can be grown in approved cell lines upon elimination of an anti-viral host factor using small interfering RNA, thus obviating the need for a complementing cell line. In sum, we have established a versatile platform for the clinical development of live attenuated HCMV-vectored vaccines and immunotherapies.

BackgroundThe capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds.MethodsWe generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8(+) T cell response were determined. Mice were subsequently challenged with tumor cells, and the tumor protection was monitored.ResultsImmunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8(+) T cell responses of >0.3% of the total circulating CD8 T cell population fully protects mice against lethal tumor challenge. However, low dose inoculations via the IP or SC route or IN vaccination elicited vaccine-induced CD8(+) T cell responses that did not reach protective thresholds for tumor protection. In addition, whereas weak pre-existing immunity did not alter the protective thresholds of the vaccine-specific T cell response following subsequent immunization with CMV-based vaccine vectors, strong pre-existing immunity inhibited the development of vaccine-induced T cells and their control on tumor progression.ConclusionsThis study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients.

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.

The mechanisms of T cell vaccination (TCV) are still unclear, especially the molecular interactions for recognition of autoreactive T cells by the immune system. Here we investigated the role of CD28:B7 interaction in TCV-induced protection in the murine EAE model. We demonstrate that there is increased expression of both B7-1 and B7-2 on autoreactive Th1 cells compared to Th2 cells. Blockade of B7 on the vaccinating autoreactive T cell surface or blockade of CD28 in recipient mice reduced the protective effect of TCV. Furthermore, we showed that TCV significantly inhibited Ag-specific CD4 and CD8 T cell proliferation and decreased Ag-specific IFN-gamma production by CD4 T cells in mice undergoing TCV, and blocking of B7 on the surface of vaccinating T cells reduced this inhibition on Ag-specific CD4 and CD8 T cell proliferation, more significantly on Ag-specific CD4 T cell proliferation. These data indicated that B7 expression on autoreactive T cells is necessary for the recognition of autoreactive T cells by the immune system and subsequent protection from EAE in mice undergoing TCV.

MS has been hypothesized to result from autoreactive T cell responses against myelin antigens. In this report, we examined myelin-specific CD8 and CD4 T cells for two markers differentially expressed on naïve, memory and chronically stimulated T cells, CD28 and CD57. We observed differential expression on CD8 T cells in response to myelin antigens, but not in response to the recall antigen mumps. We demonstrate these cells display reduced proliferation and this may explain why therapies that limit the proliferation of T cells have had little effect on the course of MS, particularly later in the course of the disease.

Human T cells adopt a CD28−CD57+ phenotype in chronic viral infections and this has been hypothesized to result from continuous stimulation, however this phenotype may be due to direct viral effects on T cells. Employing MS patients before and after chronic in vivo administration of the antigen glatiramer acetate (GA) we examine this hypothesis. Pre-treatment glatiramer acetate-specific CD8 T cells were CD57−Perforin−. This changed to a predominantly CD28−CD57+Perforin+ response after administration of this drug. This phenotype was only observed after chronic stimulation and not in a recall response to mumps. The relevance to GA's mechanism of action is discussed.

As a means of developing therapies that target the pathogenic T cells in multiple sclerosis (MS) without compromising the immune system or eliciting systemic side effects, we investigated the use of T-bet-specific antisense oligonucleotides and small interfering RNAs (siRNA) to silence T-bet expression in autoreactive encephalitogenic T cells and evaluated the biological consequences of this suppression in experimental autoimmune encephalomyelitis, a model for MS. The T-bet-specific AS oligonucleotide and siRNA suppressed T-bet expression, IFNgamma production, and STAT1 levels during antigen-specific T cell differentiation. In vitro suppression of T-bet during differentiation of myelin-specific T cells and in vivo administration of a T-bet-specific antisense oligonucleotide or siRNA inhibited disease. T-bet was shown to bind the IFNgamma and STAT1 promoters, but did not regulate the IL-12/STAT4 pathway. Since T-bet regulates IFNgamma production in CD4(+) T cells, but to a lesser extent in most other IFNgamma-producing cells, T-bet may be a target for therapeutics for Th1-mediated diseases.

Glatiramer acetate (GA; Copaxone) is a random copolymer of glutamic acid, lysine, alanine, and tyrosine that is used therapeutically in patients with multiple sclerosis (MS). To investigate the mechanism of the drug’s immunomodulatory effect, we used immunophenotypic approaches to characterize the precise nature of GA-induced T cell responses. We demonstrate here that healthy individuals and untreated MS patients exhibit prominent T cell proliferative responses to GA. However, these responses are different in distinct subsets of T cells. Whereas GA-induced CD4+ T cell responses are comparable in healthy individuals and MS patients, CD8+ T cell responses are significantly lower in untreated MS patients. Treatment with GA results in upregulation of these CD8+ responses with restoration to levels observed in healthy individuals. Both CD4+ and CD8+ GA-specific responses are HLA-restricted. GA therapy also induces a change in the cytokine profile of GA-specific CD4+ and CD8+ T cells. This study provides the first direct immunophenotypic evidence, to our knowledge, of GA-specific CD8+ T cell responses and their upregulation during the course of therapy, which may suggest a role for these responses in the immunomodulatory effects of the drug.

The expression of neural regulatory molecules by immune cells that infiltrate the nervous system upon injury may be a mechanism for cross-regulation between the nervous system and the immune system. Several lines of evidence implicate nerve growth factor (NGF) signaling through its receptors (TrkA and p75(NGFR)) as a potential source of communication between the two systems. We observed changes in NGF mRNA expression and protein secretion by T lymphocytes polarized toward the Th2 phenotype. The presence of NGF did not affect T cell proliferation or cytokine production in vitro. Mice treated with NGF by i. p. injection following induction of experimental autoimmune encephalomyelitis, an inflammatory, demyelinating disease of the central nervous system, showed a delayed onset of disease and lower clinical scores during the course of disease. These data suggest a role for NGF signaling in the regulation of the immune response, possibly by enhancing sympathetic innervation of lymphoid tissues.

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated, autoimmune disorder characterized by central nervous system (CNS) inflammation and demyelination, features reminiscent of the human disease, multiple sclerosis (MS). In addition to the signal the encephalitogenic T cell receives through the T cell receptor (TCR), a second signal, termed costimulation, is required for complete T cell activation. The B7 family of cell surface molecules expressed on antigen presenting cells (APC) is capable of providing this second signal to T cells via two receptors, CD28 and CTLA-4. Our studies have shown that costimulation provided by B7 molecules to its ligand CD28 is important in the initiation of the autoimmune response in EAE. Further, it appears the costimulation provided by B7-1 is important in disease development, while B7-2 may play an important regulatory role. We and others later showed that B7/CTLA-4 interaction plays a critical role in down-regulating the immune response. Previous work has shown that activated T cells and T cells of a memory phenotype are less dependent on costimulation than naive T cells. T cells reactive with myelin components that are involved in the pathogenesis of EAE and possibly MS would be expected to have been activated as part of the disease process. Building upon our prior work in the EAE model, we have tested the hypothesis that myelin-reactive T cells, which are relevant to the pathogenesis of CNS inflammatory demyelination, can be distinguished from naive myelin-reactive T cells by a lack of dependence upon costimulation for activation and that the costimulatory requirements of these myelin-reactive T cells change during the course of disease. Our studies in the EAE model have also addressed the mechanisms of extrathymic (peripheral) T cell tolerance following intravenous (i.v.) administration of high dose antigen. It is believed that TCR signaling in the absence of costimulation is a vital component of peripheral tolerance mechanisms. However, recent evidence suggests that peripheral tolerance of antigen-specific T cells induced in vivo may require CTLA-4 engagement of the tolerized T cells. We have begun to examine the molecular mechanisms of tolerance induction following intravenous and intraperitoneal administration of myelin antigens in the EAE model and test the hypothesis that tolerance induction is dependent on the B7:CD28/CTLA-4 pathway. The results from our studies will enhance our understanding of the role that myelin-reactive T cells may play in the pathogenesis of MS. We have determined that MBP-reactive T cells in MS patients are less dependent upon CD28 costimulation than in normal controls, suggesting that these T cells were previously primed in vivo. Characterization of these CD28-independent myelin-specific T cells will have broad implications for a variety of immunologically based therapies in diseases such as MS.

Given the critical role of cytokines in the regulation of an inflammatory response, we investigated whether certain cytokines are expressed in the brains of normal mice during maturation that could contribute to the immune-privileged nature of the CNS or potentially influence an immune-mediated illness such as experimental allergic encephalomyelitis. The gene expression of IFNγ (Th1 cytokine) and IL-4 (Th2 cytokine) was analyzed in the brain of several strains of mice. IFNγ was not detectable. However, IL-4 was present in the brains of neonatal mice, but not adult mice. Resident CNS cells are believed to be the source of the IL-4, because mice deficient in T cells (SCID and RAG2−/−) expressed the IL-4 gene in the CNS. Further analysis indicated that the gene expression of the Th2 cytokine transcription factor, GATA-3, correlated with IL-4 and IL-10 expression in the brain. Since GATA-3-deficient mice have an abnormal CNS, brain-derived Th2 cytokines may play an important role in CNS development, as well as potentially contribute to the immune-privileged nature of the brain.

Recent evidence suggests that co-stimulation provided by B7 molecules through CTLA-4 is important in establishing peripheral tolerance. In the present study, we examined the kinetics of tolerance induction and T cell differentiation following i.p. administration of myelin basic protein (MBP) Ac1-11 in mice transgenic for a TCR V(beta)8.2 gene derived from an encephalitogenic T cell clone specific for MBP Ac1-11. Examination of the lymph node cell response after antigen administration demonstrated a dependence on CTLA-4 for i.p. tolerance induction. Examination of splenocyte responses suggested that i.p. antigen administration induced a T(h)2 response, which was potentiated by anti-CTLA-4 administration. Interestingly, i.p. tolerance was able to inhibit the induction of experimental autoimmune encephalomyelitis and anti-CTLA-4 administration did not alter this phenotype, suggesting that CTLA-4 blockade did not block tolerance induction. Thus, T cell differentiation and the dependence on CTLA-4 for tolerance induction following i.p. antigen administration differs between lymph node and spleen in a model of organ-specific autoimmunity.

Previous studies have shown complex roles for the B7 receptors in providing both positive and negative regulation of experimental autoimmune encephalomyelitis (EAE). B7 blockade can ameliorate clinical EAE by indirectly interfering with CD28 signaling. However, B7 blockade can also result in disease exacerbation, presumably by interfering with regulatory B7:CTLA-4 interactions. Therefore, we have directly targeted T cell CD28 with specific mAbs both during initial Ag priming and after the onset of clinical signs of EAE. We found that CD28 blockade ameliorated EAE during the efferent and afferent limbs of the immune response. Disease amelioration at disease onset was associated with suppression of TNF-alpha production. Finally, Ab blockade of T cell CD28 during the first disease episode resulted in significant attenuation of the subsequent disease course, with no significant relapses. In contrast to previous studies targeting APC B7 with CTLA4-Ig, reagents targeting CD28 can block ongoing disease. Therefore, the present results suggest a clinically relevant therapeutic scenario for human diseases, such as multiple sclerosis.