Immunogenicity of a novel enhanced consensus DNA vaccine encoding the leptospiral protein LipL45.

Leptospirosis is a bacterial zoonotic disease caused by an infection with a spirochete belonging to the genus Leptospira. In animals, leptospirosis displays a wide range of pathologies, including fever, abortion, icterus, and uveitis. Conversely, infection in humans is associated with multi-organ injury, resulting in an increased rate of fatalities. Pathogenic leptospires are able to translocate through cell monolayers at a rate significantly greater than that of non-pathogenic leptospires. Thus, vaccine approaches have been focused on targeting bacterial motility, lipopolysaccharides (LPSs), lipoproteins, outer-membrane proteins (OMPs) and other potential virulence factors. Previous studies have indicated that leptospiral proteins elicit long-lasting immunological memory in infected humans. In the study reported here, the efficacy of a synthetic consensus DNA vaccine developed against the Leptospira membrane lipoprotein LipL45 was tested. After in vivo electroporation (EP) mediated intramuscular immunization with a synthetic LipL45 DNA vaccine (pLipL45) immunized mice developed a significant cellular response along with the development of anti-LipL45-specific antibodies. Specifically, the pLipL45 vaccine induced a significant Th1 type immune response, indicated by the higher production of IL-12 and IFN-γ cytokines. The results presented here are the first demonstration that a LipL45 based DNA immunogen has potential as a anti-Leptospira vaccine.

Novel and enhanced anti-melanoma DNA vaccine targeting the tyrosinase protein inhibits myeloid-derived suppressor cells and tumor growth in a syngeneic prophylactic and therapeutic murine model.

Melanoma is the most deadly type of skin cancer, constituting annually ∼ 75% of all cutaneous cancer-related deaths due to metastatic spread. Currently, because of metastatic spread, there are no effective treatment options for late-stage metastatic melanoma patients. Studies over the past two decades have provided insight into several complex molecular mechanisms as to how these malignancies evade immunological control, indicating the importance of immune escape or suppression for tumor survival. Thus, it is essential to develop innovative cancer strategies and address immune obstacles with the goal of generating more effective immunotherapies. One important area of study is to further elucidate the role and significance of myeloid-derived suppressor cells (MDSCs) in the maintenance of the tumor microenvironment. These cells possess a remarkable ability to suppress immune responses and, as such, facilitate tumor growth. Thus, MDSCs represent an important new target for preventing tumor progression and escape from immune control. In this study, we investigated the role of MDSCs in immune suppression of T cells in an antigen-specific B16 melanoma murine system utilizing a novel synthetic tyrosinase (Tyr) DNA vaccine therapy in both prophylactic and therapeutic models. This Tyr vaccine induced a robust and broad immune response, including directing CD8 T-cell infiltration into tumor sites. The vaccine also reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5 and arginase II, factors important for MDSC expansion. This novel synthetic DNA vaccine significantly reduced the melanoma tumor burden and increased survival in vivo, due likely, in part, to the facilitation of a change in the tumor microenvironment through MDSC suppression.

Electroporation-mediated delivery of a naked DNA plasmid expressing VEGF to the porcine heart enhances protein expression.

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly increased cardiac expression of VEGF compared with injection of plasmid alone. This is the first report showing successful intracardiac delivery, through in vivo EP, of a protein expressing plasmid in a large animal.

Epitope mapping and neuroprotective properties of a human single chain FV antibody that binds an internal epitope of amyloid-beta 1-42.

Active and passive immunotherapy targeted at the amyloid-beta (Abeta) peptide has been proposed as therapeutic approach against Alzheimer's disease (AD), and efforts towards the generation and application of antibody-based reagents that are capable of preventing and clearing amyloid aggregates are currently under active investigation. Previously, we selected and characterized a new anti-Abeta1-42 phage-displayed scFv antibody, designated clone b4.4, using a non-immune human scFv antibody library and demonstrated that a peptide based on the sequence of the Ig heavy chain (VH) complementarity-determining region (HCDR3) of this antibody fragment bound to Abeta1-42)and had neuroprotective potential against Abeta1-42 mediated neurotoxicity in rat hippocampal cultured neurons. In the present study, using novel computational methods and in vitro experiments we demonstrated that b4.4 binds to the central region of Abeta1-42. We also demonstrated that this scFv antibody binds to Abeta-derived diffusible ligands (ADDLs) and neutralizes the toxicity of both fibrillar and oligomeric forms of Abeta1-42 tested in vitro in SH-SY5Y cell cultures.

Regression of subcutaneous B16 melanoma tumors after intratumoral delivery of an IL-15-expressing plasmid followed by in vivo electroporation.

In vivo electroporation has been used to efficiently deliver drugs and 'therapeutic' genes to tumors, including melanoma lesions. This study reports on the effect of intratumoral delivery of an optimized DNA plasmid expressing interleukin-15 (pIL-15) on established murine melanoma tumors. IL-15 has been demonstrated to have a pivotal role in the function of memory CD8+ T cells and natural killer cells, which are critical for tumor immunosurveillance. In this study, C57BL/6 mice were injected with B16.F10 melanoma cells and randomized into different experimental groups: untreated (P-V-E-), treated with pIL-15 (P+) or backbone plasmid (V+), with or without electroporation (E+ or E-). Treatment was performed intratumorally with 50 microg of plasmid on days 0, 4 and 7 and tumor volume/size, tumor regression and long-term survival were measured. At day 100 after initiation of treatment, the percentage of mice surviving with complete tumor regression in the P-V+E+, P+V-E-, P+V-E+ and P-V-E- treatment groups were 0, 12.5, 37.5 and 0%, respectively. These results demonstrate the ability of pIL-15 to mediate B16 melanoma regression, with the effect being significantly enhanced by electroporative delivery. This is the first description of the ability of a naked DNA plasmid expressing IL-15 to alone mediate complete regression of B16 melanoma tumors and underscores the potential clinical use of these plasmids for the treatment of malignant tumors when delivered with in vivo electroporation.

Expression of Streptococcus mutans wall-associated protein A gene in Chinese hamster ovary cells: prospect for a dental caries DNA vaccine.

The Streptococcus mutans strain GS-5 wall-associated protein A (Wap-A) is a precursor to the extracellular antigen A (AgA), a recognized candidate dental caries vaccine. The full-length wapA gene (wapA-E) and a C-terminal truncated version (wapA-G) encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The resulting constructs were propagated in the Escherichia coli Top10. To investigate the expression of the S. mutans genes in mammalian cells, the above constructs were used to transfect Chinese hamster ovary (CHO) cells in the presence of the cationic lipid pfx-8. Transient expression of the wapA-E and wapA-G genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis using a rabbit antiserum to S. mutans cell wall. Immunochemical staining of the transfected CHO cells showed expression of WapA mainly in the cells and budding vesicles, whereas AgA was found mainly in the transfected cells and extracellular medium. The expression of S. mutans proteins in CHO cells, in either vesicles or soluble form, suggested an antibody response to the above DNA constructs. Work is under way to test the efficacy of these as DNA vaccines against S. mutans.

Protection from immunodeficiency virus challenges in rhesus macaques by multicomponent DNA immunization.

Multicomponent DNA vaccines were used to elicit immune responses, which can impact viral challenge in three separate rhesus macaque models. Eight rhesus macaques were immunized with DNA vaccines for HIV env/rev and SIV gag/pol and were challenged intravenously with 10 animal infective doses (AID(50)) of cell-free SHIV IIIB. Three of eight immunized rhesus macaques were protected, exhibiting no detectable virus. Animals protected from nonpathogenic SHIVIIIB challenge were rested for extended periods of time and were rechallenged first with pathogenic SIV(mac239) and subsequently with pathogenic SHIV89.6P viruses. Following the pathogenic challenges, all three vaccinated animals were negative for viral coculture and antigenemia and were negative by PCR. In contrast, the control animals exhibited antigenemia by 2 weeks postchallenge and exhibited greater than 10 logs of virus/10(6) cells in limiting dilution coculture. The control animals exhibited CD4 cell loss and developed SIV-related wasting with high viral burden and subsequently failed to thrive. Vaccinated animals remained virus-negative and were protected from the viral load, CD4 loss, disease, and death. We observed strong Th1-type cellular immune responses in the protected macaques throughout the study, suggesting their important roles in protection. These studies support the finding that multicomponent DNA vaccines can directly impact viral replication and disease in a highly pathogenic challenge system, thus potentially broadening our strategies against HIV.

Duration and specificity of humoral immune responses in mice vaccinated with the Alzheimer's disease-associated beta-amyloid 1-42 peptide.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by overproduction of beta-amyloid (Abeta), which is formed from amyloid precursor protein (APP), with the subsequent pathologic deposition of Abeta in regions of the brain important for memory and cognition. Recently, vaccination of murine models of AD that exhibit Abeta deposition has halted or delayed the usual progression of the pathology of AD. Our group has demonstrated that vaccination of a doubly transgenic mouse model (expressing mutant APP and presenilin-1) with the Abeta 1-42 peptide protects these mice from the memory deficits they would ordinarily develop. This report further characterizes the Abeta 1-42 peptide vaccine in mice. Anti-Abeta response time course analysis indicated that at least three vaccinations (each 100 microg) were necessary to elicit a significant anti-Abeta titer. Subsequent vaccinations resulted in half-maximal antibody titers of at least 10,000, and these titers were maintained for at least 5 months after the final boost. Peptide binding competition studies indicated that the highest humoral responses are generated against the N terminus of the Abeta peptide. Also, measurement of specific murine Ig isotypes in Abeta-vaccinated mice demonstrated a predominant IgG(1) and IgG(2b) response, suggesting a type 2 (Th2) T-helper cell immune response, which drives humoral immunity. Finally, lymphocyte proliferation assay experiments using Abeta peptides and splenocytes from vaccinated mice demonstrated that the vaccine specifically stimulates T-cell epitopes present within the Abeta peptide.

Number of Abeta inoculations in APP+PS1 transgenic mice influences antibody titers, microglial activation, and congophilic plaque levels.

There have been several reports on the use of beta-amyloid (Abeta ) vaccination in different mouse models of Alzheimer's disease (AD) and its effects on pathology and cognitive function. In this report, the histopathologic findings in the APP+PS1 doubly transgenic mouse were compared after three, five, or nine Abeta inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Abeta antibody titers. After three inoculations, the antibody titer of transgenic mice was substantially lower than that found in nontransgenic animals. However, after nine inoculations, the levels were considerably higher in both genotypes and no longer distinguishable statistically. The number of inoculations influenced CD45 expression, an indicator of microglial activation. There was an initial upregulation, which was significant after five inoculations, but by nine inoculations, the extent of microglial activation was equivalent to that in mice given control vaccinations. Along with this increased CD45 expression, there was a correlative reduction in staining by Congo red, which stains compact plaques. When data from the mice from all groups were combined, there was a significant correlation between activation of microglia and Congo red levels, suggesting that microglia play a role in the clearance of compact plaque.

Therapeutic immunization of HIV-infected chimpanzees using HIV-1 plasmid antigens and interleukin-12 expressing plasmids.

OBJECTIVE: To assess HIV-1 DNA vaccination and co-immunization with interleukin (IL)-12 and IL-10 as immunotherapy in the HIV-1 infected chimpanzee model system. METHODS: Four chimpanzees that were infected with HIV-1-IIIB for longer than 4 years and remained symptom free were immunized with HIV-1 plasmid vaccines. Two chimpanzees were immunized with DNA plasmids that encoded env/rev, gag/pol along with a plasmid that encoded both chains of human IL-12. A third animal was immunized with HIV-1 DNA vaccine constructs and co-immunized with an IL-10 expressing plasmid. Finally a control animal received the HIV-1 DNA vaccine constructs alone. RESULTS: There was no evidence of systemic toxicity associated with the administration of the DNA vaccines or the cytokine-expressing plasmids. We observed that the IL-12/HIV-1 DNA vaccinated animals had enhanced proliferative responses to multiple HIV-1 antigens at multiple time points. The animal that was co-immunized with HIV-1 and IL-10 did not have any changes in the proliferative responses. Finally, the control chimpanzee demonstrated moderate increases in the proliferative responses to HIV-1 antigens. The animal that received HIV-1 vaccines alone and the animals co-immunized with IL-12 all had declines in viral load over the course of the study, however, the decrease in viral loads were transient in all animals. CONCLUSION: Immunization of HIV-1 infected chimpanzees with DNA based vaccines containing the env, gag and pol genes can transiently boost the env specific proliferative responses. Co-administration of IL-12 expressing plasmids further leads to transient boosting of the proliferative response to the core protein, p24 as well. However, at these doses the impact on viral load is minimal.

High-level expression of a truncated wall-associated protein A from the dental cariogenic Streptococcus mutans.

Streptococcus mutans plays a primary role in the formation of dental caries. Previously, in our laboratory, an S. mutans genomic library was prepared, and the wapA gene was cloned into the shuttle vector, pSA4/4B2. To generate overexpression of wapA and to facilitate efficient purification of the WapA protein for use as an immunogen, an expression vector with the strong tac promoter was used. In order to answer questions regarding the optimization of solubility and expression based on gene size or the hydrophobicity of the protein product, 12 truncated constructs of the wapA gene were prepared using PCR. The truncated products were subcloned into the pGEX-6P-1 glutathione S-transferase (GST) fusion vector and expressed in E. coli BL21. The fusion proteins were analyzed by SDS-PAGE and confirmed by analysis with anti-GST and anti-WapA antibodies. Our study suggests that abrogation of the wapA promoter is necessary for expression of this gene in this expression system. Deletion of the signal peptide and the hydrophobic C terminus of WapA increased expression compared with the full-length construct, and truncation at the protease cleavage site of the C-terminal region greatly increased the stability of the protein without a loss in reactivity with the anti-WapA antibody. Western immunoblot analysis with anti-WapA antiserum clearly showed that the majority of the epitopes of the GST-WapA fusions are located in the N-terminal region of WapA. The immunogenicity of the various WapA fusion products is being examined in mice and rats to further map the immunologically dominant regions of the protein. This method effectively increased the expression of WapA and should contribute to the further understanding of gene expression of E. coli, as well as aid in the characterization of this protein for future immunologic evaluation.

Immunopathological factors for vertical transmission of HIV-1.

With the global rise in human immunodeficiency virus-1 (HIV-1) infection in women of childbearing age, there has also been an alarming rise in the number of mother-to-child transmissions of HIV-1. Although drug therapies such as zidovudine as well as nevirapine have been demonstrated to significantly decrease the incidence of vertical transmission of HIV-1, these therapeutic regimens are still not widely available in some developing countries where maternal-to-child transmission of HIV-1 continues to occur at an alarming rate. Therefore, the continued studies of mechanisms and correlates of vertical transmission of HIV-1 are warranted. The current status of immunological and virological correlates of vertical transmission are summarized in this review. In addition, information concerning recent therapeutic agents for the prevention of HIV-1 vertical transmission is presented.

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.

Modulation of antigen-specific humoral responses in rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants.

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.

A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease.

Vaccinations with amyloid-beta peptide (A beta) can dramatically reduce amyloid deposition in a transgenic mouse model of Alzheimer's disease. To determine if the vaccinations had deleterious or beneficial functional consequences, we tested eight months of A beta vaccination in a different transgenic model for Alzheimer's disease in which mice develop learning deficits as amyloid accumulates. Here we show that vaccination with A beta protects transgenic mice from the learning and age-related memory deficits that normally occur in this mouse model for Alzheimer's disease. During testing for potential deleterious effects of the vaccine, all mice performed superbly on the radial-arm water-maze test of working memory. Later, at an age when untreated transgenic mice show memory deficits, the A beta-vaccinated transgenic mice showed cognitive performance superior to that of the control transgenic mice and, ultimately, performed as well as nontransgenic mice. The A beta-vaccinated mice also had a partial reduction in amyloid burden at the end of the study. This therapeutic approach may thus prevent and, possibly, treat Alzheimer's dementia.

Engineering of DNA vaccines using molecular adjuvant plasmids.

These studies support the view that additional goals of enhancing DNA vaccine technology will probably be at several levels. The ability to deliver antigens more efficiently to professional APCs is likely to have important implications for our studies of basic principles of immunology. Furthermore, there are simple practical approaches to vaccine enhancement that can be tested with the present group of DNA vaccines. These studies should include the use of cytokine molecular adjuvants as well as possible co-stimulatory molecules. It is expected that the delivery of these "adjuvanted" DNA vaccines will require additional safety evaluation; however, it is clear that studies can be easily designed to address the important safety issues associated with these novel vaccine adjuvants. Overall, the results indicate that further more precise quantitative studies and combination studies examining these additional promising adjuvant candidates are warranted.

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.