Active immunization with tau epitope in a mouse model of tauopathy induced strong antibody response together with improvement in short memory and pSer396-tau pathology.

Abnormal tau hyperphosphorylation and its aggregation into neurofibrillary tangles are a hallmark of tauopathies, neurodegenerative disorders that include Alzheimer's disease (AD). Active and passive Tau-immunotherapy has been proposed as a therapeutic approach to AD with mixed results. One of the limitations of active immunotherapy may be associated with the mediocre immunogenicity of vaccines that are not inducing therapeutically potent titers of antibodies. The aim of this study was to test the efficacy of an anti-tau vaccine, AV-1980R/A composed of N terminal peptide of this molecule fused with an immunogenic MultiTEP platform and formulated in a strong adjuvant, AdvaxCpG in a Tg4510 mouse model of tauopathy. Experimental mice were immunized with AV-1980R/A and a control group of mice were injected with adjuvant only. Nontransgenic and tetracycline transactivator (tTA) transgenic littermates were included as baseline controls to contrast with the tau phenotype. Active immunization with AV-1980R/A induced very strong anti-tau humoral immune responses in both nontransgenic and transgenic mice with evidence of IgG in brains of AV-1980R/A vaccinated mice. These experimental animals displayed an improvement in short-term memory during a novel object recognition test. However, impairments in other behavioral tasks were not prevented by AV-1980R/A vaccinations. At the same time, high titers of anti-tau antibodies reduced hyperphosphorylated pSer396 tau but did not lower the level of other phosphorylated tau species in the brains of AV-1980R/A vaccinated mice. These data indicate that active immunotherapy with an N-terminal Tau epitope was only partially effective in improving cognition and reducing pathology in the stringent Tg4510 mouse model of tauopathy.

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

Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-beta (Abeta(42)) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Abeta 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-Abeta(11) 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-Abeta 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 Abeta(42) peptide. Thus, we showed that a heterologous prime-boost regimen could be an effective protocol for developing a potent Alzheimer's disease (AD) vaccine.

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.

Inflammatory changes parallel the early stages of Alzheimer disease.

Alzheimer disease (AD) is the most prominent cause of dementia in the elderly. To determine changes in the AD brain that may mediate the transition into dementia, the gene expression of approximately 10,000 full-length genes was compared in mild/moderate dementia cases to non-demented controls that exhibited high AD pathology. Including this latter group distinguishes this work from previous studies in that it allows analysis of early cognitive loss. Compared to non-demented high-pathology controls, the hippocampus of AD cases with mild/moderate dementia had increased gene expression of the inflammatory molecule major histocompatibility complex (MHC) II, as assessed with microarray analysis. MHC II protein levels were also increased and inversely correlated with cognitive ability. Interestingly, the mild/moderate AD dementia cases also exhibited decreased number of T cells in the hippocampus and the cortex compared to controls. In conclusion, transition into AD dementia correlates with increased MHC II(+) microglia-mediated immunity and is paradoxically paralleled by a decrease in T cell number, suggesting immune dysfunction.

Murine monoclonal antibodies biologically active against the amino region of HIV-1 gp120: isolation and characterization.

The human immunodeficiency virus (HIV)-1 envelope glycoprotein is synthesized as a precursor (gp160) and subsequently cleaved to generate the external gp120 and transmembrane gp41 glycoproteins. Both gp120 and gp41 have been demonstrated to mediate critical functions of HIV, including viral attachment and fusion with the cell membrane. The antigenic variability of the HIV-1 envelope glycoprotein has presented a significant problem in the design of appropriate and successful vaccines and offers one explanation for the ability of HIV to evade immune surveillance. Therefore, the development and characterization of functional antibodies against conserved regions of the envelope glycoprotein is needed. Because of this need, we generated a panel of murine monoclonal antibodies (MuMabs) against the HIV-1 envelope glycoprotein. To accomplish this, we immunized Balb/C mice with a recombinant glycoprotein 160 (gp160) that was synthesized in a baculovirus expression system. From the growth-positive hybridomas, three MuMabs were generated that demonstrated significant reactivity with recombinant gp120 but failed to show reactivity against HIV-1 gp41, as determined by enzyme-linked immunosorbent assay (ELISA). Using vaccinia constructs that synthesize variant truncated subunits of gp160, we were able to map reactivity of all three of the Mabs (ID6, AC4, and AD3) to the first 204 residues of gp120 (i.e., the N terminus of gp120) via Western blot analysis. Elucidation of the epitopes for these Mabs may have important implications for inhibition of infection by HIV-1. Our initial attempts to map these Mabs with linear epitopes have not elucidated a specific antigenic determinant; however, several physical characteristics have been determined that suggest a continuous surface epitope. Although these antibodies failed to neutralize cell-free or cell-associated infection by HIV-1, they did mediate significant antibody-dependent cellular cytotoxicity (ADCC) activity, indicating potential therapeutic utility. In summary, these data suggest the identification of a potentially novel site in the first 200 aa of gp120 that mediates ADCC.

Characterization of a novel human immunodeficiency virus type 1 neutralizable epitope within the immunodominant region of gp41.

Previously, we generated human monoclonal antibodies using peripheral blood mononuclear cells from an asymptomatic human immunodeficiency virus type 1 (HIV-1)-seropositive donor. One of these monoclonal antibodies (designated clone 3, CL3) recognized 10 amino acids (GCSGKLICTT) within the immunodominant region (cluster I) of the transmembrane envelope glycoprotein gp41 and neutralized infection of target cells with different laboratory isolates. Because the epitope recognized by CL3 has two cysteine residues that could potentially produce a disulfide loop in gp41, we analyzed binding of our monoclonal antibody to the cyclic and linear motif of the peptide sequence IWGCSGKLICTTAVP (residues 600-614). The CL3 antibody did not bind to the synthetic cyclic peptide but did recognize the linear form. Two polyclonal rabbit sera against both the linear and cyclic peptides were then generated. Both antisera bound to viral glycoproteins gp41 and gp160, but neither sera neutralized HIV-1 laboratory isolates. Using a set of alanine-substituted IWGCSGKLICTTAV peptides, we analyzed binding of polyclonal antisera and CL3. The profile of binding of polyclonal antisera to these peptides was different from that of CL3 to the same peptides. This suggests that CL3 recognized a unique neutralizable core epitope, which was not immunogenic in either the cyclic or the linear IWGCSGKLICTTAVP peptides used as immunogens in the rabbits.

Macrophage colony-stimulating factor can modulate immune responses and attract dendritic cells in vivo.

Studies have indicated that professional APCs in the periphery, such as dendritic cells and macrophages, play an important role in initiating DNA vaccine-specific immune responses. To engineer the immune response induced by DNA vaccines in vivo we investigated the modulatory effects of codelivering growth factor genes for the hematopoietic APCs along with DNA vaccines. Specifically, we examined the effects on the antigen-specific immune responses following the codelivery of the gene expression cassettes for M-CSF, G-CSF, and GM-CSF along with HIV-1 DNA immunogen constructs. We observed that coimmunization with GM-CSF increased the antibody response and resulted in a significant enhancement of lymphoproliferative response. Furthermore, among all coinjection combinations, we found that M-CSF coinjections resulted in a high level of CTL enhancement. This enhancement of CTL responses observed from the coinjection with M-CSF was CD8+ T cell dependent and was associated with the presence of CD11c+ cells at the site of injection and with the antigen-specific induction of the beta-chemokine MIP-1beta, suggesting a role for this chemokine in CTL induction. These results suggest that hematopoietic growth factors should be further studied as potential adjuvants for in vivo modulators of immune responses.

Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-gamma, IL-12, or IL-18 gene adjuvants.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To engineer the immune response in vivo towards more T-helper (Th)1-type cellular responses, we investigated the co-delivery of inteferon (IFN)-gamma, interleukin (IL)-12, and IL-18 genes along with DNA vaccine constructs. We observed that both antigen-specific humoral and cellular immune responses can be modulated through the use of cytokine adjuvants in mice. Most of this work has been performed in rodent models. There has been little confirmation of this technology in primates. We also evaluated the immunomodulatory effects of this approach in rhesus macaques, since non-human primates represent the most relevant animal models for human immunodeficiency virus (HIV) vaccine studies. As in the murine studies, we also observed that each Th1 cytokine adjuvant distinctively regulated the level of immune responses generated. Co-immunization of IFN-gamma and IL-18 in macaques enhanced the level of antigen-specific antibody responses. Similarly, co-delivery of IL-12 and IL-18 also enhanced the level of antigen-specific Th proliferative responses. These results extend this adjuvant strategy in a more relevant primate model and support the potential utility of these molecular adjuvants in DNA vaccine regimens.

CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo.

Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-gamma production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs.

Intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates T cells in vivo.

The potential roles of adhesion molecules in the expansion of T cell-mediated immune responses in the periphery were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the adhesion molecules intracellular adhesion molecule-1 (ICAM-1), lymphocyte function associated-3 (LFA-3), and vascular cell adhesion molecule-1 (VCAM-1) along with DNA immunogens, and we analyzed the resulting antigen-specific immune responses. We observed that antigen-specific T-cell responses can be enhanced by the coexpression of DNA immunogen and adhesion molecules ICAM-1 and LFA-3. Coexpression of ICAM-1 or LFA-3 molecules along with DNA immunogens resulted in a significant enhancement of T-helper cell proliferative responses. In addition, coimmunization with pCICAM-1 (and more moderately with pCLFA-3) resulted in a dramatic enhancement of CD8-restricted cytotoxic T-lymphocyte responses. Although VCAM-1 and ICAM-1 are similar in size, VCAM-1 coimmunization did not have any measurable effect on cell-mediated responses. These results suggest that ICAM-1 and LFA-3 provide direct T-cell costimulation. These observations are further supported by the finding that coinjection with ICAM-1 dramatically enhanced the level of interferon-gamma (IFN-gamma) and beta-chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation normal T-cell expression and secreted (RANTES) produced by stimulated T cells. Through comparative studies, we observed that ICAM-1/LFA-1 T-cell costimulatory pathways are independent of CD86/CD28 pathways and that they may synergistically expand T-cell responses in vivo.

Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses from the codelivery of Thl cytokines (interleukin-2 [IL-2] and IL-12), Th2 cytokines (IL-4 and IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes along with a DNA vaccine construct encoding for simian immunodeficiency virus (SIV) gag/pol proteins. We observed that coinjection with IL-2, IL-4, IL-10, and GM-CSF resulted in increased levels of antigen-specific antibodies. In addition, we found that coinjection with cytokine genes drove the immune responses toward a more Thl or Th2 phenotype. We also observed that coadministration of IL-2, IL-12, and GM-CSF genes resulted in a dramatic enhancement of Th proliferation responses. Moreover, coimmunization with IL-12 genes resulted in a dramatic enhancement of antigen-specific cytotoxic T lymphocyte (CTL) responses. These results support the potential utility of molecular adjuvants in DNA vaccine regimens.

Engineering DNA vaccines via co-delivery of co-stimulatory molecule genes.

DNA immunization has been investigated as a potential immunization strategy against infectious diseases and cancer. To enhance a DNA vaccine's ability to induce CTL response in vivo, we co-administered CD80 and CD86 expression cassettes along with HIV-1 immunogens. This manipulation resulted in a dramatic increase in MHC class I-restricted and CD8+ T-cell-dependent CTL responses in both mice and chimpanzees. This strategy of engineering vaccine producing cells to be more efficient T-cell activators could be an important tool for optimizing antigen-specific T-cell-mediated immune responses in the pursuit of more rationally designed vaccines and immune therapies.

CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines.

The potential roles of CD8(+) T-cell-induced chemokines in the expansion of immune responses were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the alpha-chemokines IL-8 and SDF-1alpha and the beta-chemokines MIP-1alpha, RANTES, and MCP-1 along with DNA immunogens and analyzed the resulting antigen-specific immune responses. In a manner more similar to the traditional immune modulatory role of CD4(+) T cells via the expression of Th1 or Th2 cytokines, CD8(+) T cells appeared to play an important role in immune expansion and effector function by producing chemokines. For instance, IL-8 was a strong inducer of CD4(+) T cells, indicated by strong T helper proliferative responses as well as an enhancement of antibody responses. MIP-1alpha had a dramatic effect on antibody responses and modulated the shift of immune responses to a Th2-type response. RANTES coimmunization enhanced the levels of antigen-specific Th1 and cytotoxic T lymphocyte (CTL) responses. Among the chemokines examined, MCP-1 was the most potent activator of CD8(+) CTL activity. The enhanced CTL results are supported by the increased expression of Th1 cytokines IFN-gamma and TNF-alpha and the reduction of IgG1/IgG2a ratio. Our results support that CD8(+) T cells may expand both humoral and cellular responses in vivo through the elaboration of specific chemokines at the peripheral site of infection during the effector stage of the immune response.

Monoclonal antibodies define a cellular antigen involved in HTLV-I infection.

The exact mechanism by which the human T cell leukemia viruses (HTLV) infects target cells remains unclear; although some molecules have been identified to be important in viral infection and entry. To investigate these phenomena, we generated a panel of monoclonal antibodies (MAb) against a B cell line (BJAB-WH) which is highly permissive for infection with HTLV. These MAb have been used to further characterize the membrane molecules important for HTLV infection. Three of these MAb designated 4.2.3, 3.3.10, and 11.2.3 were capable of inhibiting syncytium formation induced in human B and T cell lines (i.e., BJAB-WH and SupT-1, respectively) by co-culture with HTLV-I infected MT-2 cells. All of these MAbs immunoprecipitated a 80-85 kDa antigen from the lysates of metabolically labeled BJAB-WH but not from BJAB-CC/84, a noninfectible target cell. The binding of these MAb with different HTLV target cells was analyzed and compared with binding of polyclonal monospecific antisera to the same cell lines. A 80-85 kDa membrane glycoprotein was isolated with an immunoaffinity chromatographic column constructed with MAbs 4.2.3 and 3.3.10. This cellular antigen was capable of inhibiting HTLV I/MT-2 induced fusion. This is the first direct demonstration that a 80-85 kDa cellular glycoprotein is directly involved in HTLV I/II infection and syncytium formation.

Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens.

Immunization with nucleic acids has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. We hypothesize that immunization with DNA could be enhanced by directing specific immune responses induced by the vaccine based on the differential correlates of protection known for a particular pathogen. Recently we and others reported that specific immune responses generated by DNA vaccine could be modulated by co-delivery of gene expression cassettes encoding for IL-12, granulocyte-macrophage colony-stimulating factor and the co-stimulatory molecule CD86. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses following the co-delivery of pro-inflammatory cytokine (IL-1 alpha, TNF-alpha, and TNF-beta), Th1 cytokine (IL-2, IL-12, IL-15, and IL-18), and Th2 cytokine (IL-4, IL-5 and IL-10) genes. We observed enhancement of antigen-specific humoral response with the co-delivery of Th2 cytokine genes IL-4, IL-5, and IL-10 as well as those of IL-2 and IL-18. A dramatic increase in antigen-specific T helper cell proliferation was seen with IL-2 and TNF-alpha gene co-injections. In addition, we observed a significant enhancement of the cytotoxic response with the co-administration of TNF-alpha and IL-15 genes with HIV-1 DNA immunogens. These increases in CTL response were both MHC class I restricted and CD8+ T cell dependent. Together with earlier reports on the utility of co-immunizing using immunologically important molecules together with DNA immunogens, we demonstrate the potential of this strategy as an important tool for the development of more rationally designed vaccines.

Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes.

Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes. In contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

Mucosal immunization with a DNA vaccine induces immune responses against HIV-1 at a mucosal site.

Mucosal immunity is the first defense system in protection against mucosal infection by sexually transmitted diseases and subsequent systemic dissemination of infection. Development of vaccines which can induce protective mucosal immunity would have great promise for preventing sexually transmitted diseases including AIDS. DNA vaccines have recently shown certain advantages over other types of vaccines in safety and elicitation of specific immune responses. We have hypothesized that direct delivery of a DNA plasmid coding the HIV-1 envelope (pcMN160) via mucosal routes will stimulate mucosal immunity against HIV-1. The expression of DNA plasmid inoculated intravaginally was detected in various tissues. Intravaginal inoculation of pcMN160 elicits production of vaginal immunoglobulins which specifically bind to the HIV-1 envelope and neutralize HIV-1 infectivity in vitro. These results indicate the feasibility of inducing mucosal immunity following mucosal inoculation of DNA vaccines. When coupled with systemic inoculation of appropriate DNA constructs, effective mucosal and systemic immunity may be generated.

Nucleic acid immunization of chimpanzees as a prophylactic/immunotherapeutic vaccination model for HIV-1: prelude to a clinical trial.

Vaccine development strategies have often utilized recombinant envelope glycoproteins which usually generate strong humoral immune responses but which do not generate strong cytotoxic T lymphocytes (CTL). A recent novel experimental vaccination approach involves the technology known as nucleic acid immunization in which DNA plasmids expressing a gene of interest is injected intramuscularly in experimental animals. These expressed proteins then are presented to the immune system with the subsequent development of strong antibody and cellular (particularly CTL) immune responses. These types of immune responses have been elicited in rodents as well as nonhuman primates including chimpanzees. Results from studies on nucleic acid immunization of HIV-1 infected chimpanzees with envelope glycoprotein expressing constructs indicated that this method was able to decrease substantially HIV-1 viral load in these chimpanzees. These data are useful for the development and implementation of human phase I clinical trials with HIV constructs expressing various genes from the HIV-1 genome.

In vitro and in vivo tumor growth suppression by HIV-1 Vpr.

We have previously reported that the human immunodeficiency virus type 1 (HIV-1) regulatory gene vpr induces differentiation of rhabdomyosarcoma (embryonal muscle tumor cell line) cells, an effect that is accompanied by reduced proliferative capacity of the transfected cells. In this report, we examine the effect of Vpr expression on several different tumor cell lines derived from unique lineages. These tumor cells display different patterns of modulated oncogenes including both ras and p53 mutations. Here we demonstrate that the growth of tumor cells in vitro and in vivo is arrested by the expression of HIV-1 Vpr. Expression of Vpr in several human tumor cell lines upon transfection resulted in an accumulation of cells in the G2/M phase of cell cycle with altered cellular morphology, including an increase in adherence, and growth arrest, consistent with a differentiated phenotype. Vpr expression in B78/H1 cells results in a marked reduction in colony formation in vitro and an associated reduction in melanin synthesis by the cells. Vpr-transfected melanoma cells inoculated into syngenic C57BL/6 mice showed a markedly reduced ability to form tumors in vivo. These results suggest that this retroviral regulatory gene has broad tumor suppressor effects, likely mediated by transcriptional regulation of the state of the host cell.