Mikayel Mkrtichyan

Prototype Alzheimer’s Disease Vaccine Using the Immunodominant B Cell Epitope from β-Amyloid and Promiscuous T Cell Epitope Pan HLA DR-Binding Peptide

Immunization of amyloid precursor protein transgenic mice with fibrillar β-amyloid (Aβ) prevents Alzheimer’s disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Aβ, containing the B and T cell self epitopes of Aβ, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Aβ Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Aβ immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Aβ in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Aβ1–15 sequence lacks the T cell epitope of Aβ. Immunization of BALB/c mice with the PADRE-Aβ1–15 epitope vaccine produced high titers of anti-Aβ Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Aβ peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.

Reducing AD-Like Pathology in 3xTg-AD Mouse Model by DNA Epitope Vaccine — A Novel Immunotherapeutic Strategy

Background The development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Aβ antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Aβ42 (Aβ1–11) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype. Methods and Findings We generated pMDC-3Aβ1–11-PADRE construct and immunized 3xTg-AD mouse model starting at age of 3–4 months old. We demonstrated that prophylactic immunizations with the DNA epitope vaccine generated a robust Th2 immune response that induced high titers of anti-Aβ antibody, which in turn inhibited accumulation of Aβ pathology in the brains of older mice. Importantly, vaccination reduced glial activation and prevented the development of behavioral deficits in aged animals without increasing the incidence of microhemorrhages. Conclusions Data from this transitional pre-clinical study suggest that our DNA epitope vaccine could be used as a safe and effective strategy for AD therapy. Future safety and immunology studies in large animals with the goal to achieve effective humoral immunity without adverse effects should help to translate this study to human clinical trials.

Anti-Aβ1–11 Antibody Binds to Different β-Amyloid Species, Inhibits Fibril Formation, and Disaggregates Preformed Fibrils but Not the Most Toxic Oligomers

Different strategies proposed as therapy for Alzheimer disease (AD) have aimed to reduce the level of toxic forms of Aβ peptide in the brain. Here, we directly analyze the therapeutic utility of the polyclonal anti-Aβ1–11 antibody induced in 3xTg-AD mice vaccinated with the second generation prototype epitope vaccine. Substoichiometric concentrations of purified anti-Aβ1–11 antibody prevented aggregation of Aβ42 and induced disaggregation of preformed Aβ42 fibrils down to nonfilamentous and nontoxic species. Anti-Aβ1–11 antibody delayed Aβ42 oligomer formation but ultimately appeared to stabilize nonfibrillar conformations, including oligomer-like assemblies. The reduced oligomer-mediated cytotoxicity observed upon preincubation of Aβ oligomers with the anti-Aβ1–11 antibody in the absence of oligomer disaggregation suggests a possible oligomer rearrangement in the presence of the antibody. These in vitro observations suggest that preventive vaccination may protect from AD or may delay the onset of the disease, whereas therapeutic vaccination cannot disrupt the toxic oligomers and may only minimally alleviate preexisting AD pathology.

DNA prime–protein boost increased the titer, avidity and persistence of anti-Aβ antibodies in wild-type mice

Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-β (Aβ42) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Aβ immune responses in wild-type and amyloid precursor protein transgenic animals. Although DNA vaccines have several advantages over peptide–protein vaccines, they induce lower immune responses in large animals and humans compared with those in mice. The focus of this study was to further enhance anti-Aβ11 immune responses by developing an improved DNA vaccination protocol of the prime–boost regimen, in which the priming step would use DNA and the boosting step would use recombinant protein. Accordingly, we generated DNA and recombinant protein-based epitope vaccines and showed that priming with DNA followed by boosting with a homologous recombinant protein vaccine significantly increases the anti-Aβ antibody responses and do not change the immunoglobulin G1 (IgG1) profile of humoral immune responses. Furthermore, the antibodies generated by this prime–boost regimen were long-lasting and possessed a higher avidity for binding with an Aβ42 peptide. Thus, we showed that a heterologous prime–boost regimen could be an effective protocol for developing a potent Alzheimers disease (AD) vaccine.

Antitumor efficacy of DNA vaccination to the epigenetically acting tumor promoting transcription factor BORIS and CD80 molecular adjuvant

Cancer testis (CT) antigens are promising candidates for tumor vaccines due to their immunogenicity and tissue‐restricted expression. Recently, we identified a novel cancer testis gene, BORIS, whose expression is restricted to male testis after puberty and is strictly absent in non‐malignant female tissue. BORIS encodes a DNA‐binding protein that shares 11 zing finger (ZF) with transcription factor CTCF and differs at the N‐ and C‐termini. CTCF has been implicated in epigenetic regulation of imprinting, X chromosome inactivation, repression, and activation of cancer testis antigens. BORIS expression has been documented in cancers of diverse histological origin, including, but not limited to breast, prostate, ovary, gastric, liver, endometrial, glia, colon, and esophagus. Interestingly, BORIS induces demethylation and subsequent expression of many cancer‐testis genes, including MAGE‐A1 and NY‐ESO‐1, indicating that it is expressed very early in malignancy and might be an attractive candidate for immunotherapy. In this study we tested BORIS as a vaccine in a very aggressive, highly metastatic, and poorly immunogenic murine model of mammary carcinoma. Immunizations with a DNA encoding the mutant form of murine BORIS antigen (pmBORIS lacking DNA‐binding function) significantly prolonged survival, and inhibited tumor growth in BALB/c mice inoculated with 4T1 cells. Priming with pmBORIS mixed with molecular adjuvant and boosting with adenoviral vector expressing mBORIS was generally more effective, suggesting that the vaccination protocol could be further optimized. This is the first report demonstrating the feasibility of vaccination with a cancer associated epigenetic regulator for the induction of tumor inhibition. J. Cell. Biochem. 98: 1037–1043, 2006.

Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice

BackgroundNew pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Aβ28 conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576).MethodsMannan was purified, activated and chemically conjugated to Aβ28 peptide. Humoral immune responses induced by the immunization of mice with mannan-Aβ28 conjugate were analyzed using a standard ELISA. Aβ42 and Aβ40 amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry.ResultsImmunizations with low doses of mannan-Aβ28 induced potent and long-lasting anti-Aβ humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Aβ antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Aβ28 prevented Aβ plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Aβ28 showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups.ConclusionMannan conjugated to Aβ28 provided sufficient adjuvant activity to induce potent anti-Aβ antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Aβ self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Aβ28 immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Aβ antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

DNA epitope vaccine containing complement component C3d enhances anti-amyloid-β antibody production and polarizes the immune response towards a Th2 phenotype

We have engineered a DNA epitope vaccine that expresses 3 self-B cell epitopes of Abeta(42) (3Abeta(1-11)), a non-self T helper (Th) cell epitope (PADRE), and 3 copies of C3d (3C3d), a component of complement as a molecular adjuvant, designed to safely reduce CNS Abeta. Immunization of mice with 3Abeta(1-11)-PADRE epitope vaccine alone generated only moderate levels of anti-Abeta antibodies and a pro-inflammatory T helper (Th1 phenotype) cellular immune response. However, the addition of 3C3d to the vaccine construct significantly augmented the anti-Abeta humoral immune response and, importantly, shifted the cellular immune response towards the potentially safer anti-inflammatory Th2 phenotype.

Elicitation of T Cell Responses to Histologically Unrelated Tumors by Immunization with the Novel Cancer-Testis Antigen, Brother of the Regulator of Imprinted Sites

Brother of the regulator of imprinted sites (BORIS) was previously described as a transcription factor for epigenetic reprogramming the expression of which is strictly confined to germ cells of adult testes but is aberrantly activated in the vast majority of neoplastic cells. Considering the critical role of BORIS in cancerogenesis and the fact that its expression pattern may preclude thymic tolerance, we generated DNA- and protein-based mouse BORIS antitumor vaccines using a non-DNA-binding version of the BORIS molecule. Clinical use of BORIS as a vaccine Ag would require that certain safety concerns be met. Specifically, administration of the functional BORIS protein would hypothetically pose a risk of BORIS accelerating the progression of cancer. To alleviate such safety concerns, we have developed vaccines based on the BORIS molecule lacking the DNA-binding zinc fingers domain. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4+ T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production in immunized animals. Importantly, potent anticancer CD8+-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. These cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma.

Cancer-testis antigen, BORIS based vaccine delivered by dendritic cells is extremely effective against a very aggressive and highly metastatic mouse mammary carcinoma

Here, we analyze for the first time the immunological and therapeutic efficacy of a dendritic cell (DC) vaccine based on a cancer-testis antigen, Brother of regulator of imprinted sites (BORIS), an epigenetically acting tumor-promoting transcription factor. Vaccination of mice with DC loaded with truncated form of BORIS (DC/mBORIS) after 4T1 mammary tumor implantation induced strong anti-cancer immunity, inhibited tumor growth (18.75% of mice remained tumor-free), and dramatically lowered the number of spontaneous clonogenic metastases (50% of mice remained metastases-free). Higher numbers of immune effector CD4 and CD8 T cells infiltrated the tumors of vaccinated mice vs. control animals. Vaccination significantly decreased the number of myeloid-derived suppressor cells (MDSCs) infiltrating the tumor sites, but not MDSCs in the spleens of vaccinated animals. These data suggest that DC-based mBORIS vaccination strategies have significant anti-tumor activity in a therapeutic setting and will be more effective when combined with agents to attenuate tumor-associated immune suppression.

DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen.