Novel prostate cancer immunotherapy with a DNA-encoded anti-prostate-specific membrane antigen monoclonal antibody.

Prostate-specific membrane antigen (PSMA) is expressed at high levels on malignant prostate cells and is likely an important therapeutic target for the treatment of prostate carcinoma. Current immunotherapy approaches to target PSMA include peptide, cell, vector or DNA-based vaccines as well as passive administration of PSMA-specific monoclonal antibodies (mAb). Conventional mAb immunotherapy has numerous logistical and practical limitations, including high production costs and a requirement for frequent dosing due to short mAb serum half-life. In this report, we describe a novel strategy of antibody-based immunotherapy against prostate carcinoma that utilizes synthetic DNA plasmids that encode a therapeutic human mAb that target PSMA. Electroporation-enhanced intramuscular injection of the DNA-encoded mAb (DMAb) plasmid into mice led to the production of functional and durable levels of the anti-PSMA antibody. The anti-PSMA produced in vivo controlled tumor growth and prolonged survival in a mouse model. This is likely mediated by antibody-dependent cellular cytotoxicity (ADCC) effect with the aid of NK cells. Further study of  this novel approach for treatment of human prostate disease and other malignant conditions is warranted.

Rapid and Long-Term Immunity Elicited by DNA-Encoded Antibody Prophylaxis and DNA Vaccination Against Chikungunya Virus.

BACKGROUND: Vaccination and passive antibody therapies are critical for controlling infectious diseases. Passive antibody administration has limitations, including the necessity for purification and multiple injections for efficacy. Vaccination is associated with a lag phase before generation of immunity. Novel approaches reported here utilize the benefits of both methods for the rapid generation of effective immunity. METHODS: A novel antibody-based prophylaxis/therapy entailing the electroporation-mediated delivery of synthetic DNA plasmids encoding biologically active anti-chikungunya virus (CHIKV) envelope monoclonal antibody (dMAb) was designed and evaluated for antiviral efficacy, as well as for the ability to overcome shortcomings inherent with conventional active vaccination and passive immunotherapy. RESULTS: One intramuscular injection of dMAb produced antibodies in vivo more rapidly than active vaccination with an anti-CHIKV DNA vaccine. This dMAb neutralized diverse CHIKV clinical isolates and protected mice from viral challenge. Combination of dMAb and the CHIKV DNA vaccine afforded rapid and long-lived protection. CONCLUSIONS: A DNA-based dMAb strategy induced rapid protection against an emerging viral infection. This method can be combined with DNA vaccination as a novel strategy to provide both short- and long-term protection against this emerging infectious disease. These studies have implications for pathogen treatment and control strategies.

Identification of Novel Neutralizing Monoclonal Antibodies against SARS-CoV-2 Spike Glycoprotein.

Coronavirus disease 2019 (COVID-19) is caused by the newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the highly contagious nature of SARS-CoV-2, it has infected more than 137 million individuals and caused more than 2.9 million deaths globally as of April 13, 2021. There is an urgent need to develop effective novel therapeutic strategies to treat or prevent this infection. Toward this goal, we focused on the development of monoclonal antibodies (mAbs) directed against the SARS-CoV-2 spike glycoprotein (SARS-CoV-2 Spike) present on the surface of virus particles as well as virus-infected cells. We isolated anti-SARS-CoV-2 Spike mAbs from animals immunized with a DNA vaccine. We then selected a highly potent set of mAbs against SARS-CoV-2 Spike protein and evaluated each candidate for their expression, target binding affinity, and neutralization potential using complementary ACE2-blocking and pseudovirus neutralization assays. We identified a total of 10 antibodies, which specifically and strongly bound to SARS-CoV-2 Spike, blocked the receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) interaction, and neutralized SARS-CoV-2. Furthermore, the glycomic profile of the antibodies suggested that they have high Fc-mediated effector functions. These antibodies should be further investigated for elucidating the neutralizing epitopes on Spike for the design of next-generation vaccines and for their potential in diagnostic as well as therapeutic utilities against SARS-CoV-2.

A novel synthetic DNA vaccine elicits protective immune responses against Powassan virus.

Powassan virus (POWV) infection is a tick-borne emerging infectious disease in the United States and North America. Like Zika virus, POWV is a member of the family Flaviviridae. POWV causes severe neurological sequalae, meningitis, encephalitis, and can cause death. Although the risk of human POWV infection is low, its incidence in the U.S. in the past 16 years has increased over 300%, urging immediate attention. Despite the disease severity and its growing potential for threatening larger populations, currently there are no licensed vaccines which provide protection against POWV. We developed a novel synthetic DNA vaccine termed POWV-SEV by focusing on the conserved portions of POWV pre-membrane and envelope (prMEnv) genes. A single immunization of POWV-SEV elicited broad T and B cell immunity in mice with minimal cross-reactivity against other flaviviruses. Antibody epitope mapping demonstrated a similarity between POWV-SEV-induced immune responses and those elicited naturally in POWV-infected patients. Finally, POWV-SEV induced immunity provided protection against POWV disease in lethal challenge experiments.

Neutralization of hepatitis B virus by a novel DNA-encoded monoclonal antibody.

Hepatitis B virus (HBV) causes a potentially life-threatening liver infection that frequently results in life-long chronic infection. HBV is responsible for 887,000 deaths each year, most resulting from chronic liver diseases and hepatocellular carcinoma. Presently, there are 250 million chronic HBV carriers worldwide who are at a high risk for developing cirrhosis and hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer with a strong association with HBV infection. HBV transmission through blood transfusions and perinatal transfer from infected mother to child have been common routes of infection. In the present study, we describe the development of a synthetic DNA plasmid encoding an anti-HBV human monoclonal antibody specific for the common "a determinant region" of HBsAg of hepatitis B virus and demonstrate the ability of this platform at directing in vivo antibody expression. In vivo delivery of this DNA encoded monoclonal antibody (DMAb) plasmid in mice resulted in expression of human IgG over a period of one month following a single injection. Serum antibody was found to recognize the relevant conformational epitope from plasma purified native HBsAg as well as bound HBV in HepG2.2.15 cells. The serum DMAb efficiently neutralized HBV and prevented infection of HepaRG cells in vitro. Additional study of these HBV-DMAb as a possible therapy or immunoprophylaxis for HBV infection is warranted.

Synthetic nucleic acid antibody prophylaxis confers rapid and durable protective immunity against Zika virus challenge.

Significant concerns have arisen over the past 3 y from the increased global spread of the mosquito-borne flavivirus, Zika. Accompanying this spread has been an increase in cases of the devastating birth defect microcephaly as well as of Guillain-Barré syndrome in adults in many affected countries. Currently there is no vaccine or therapy for this infection; however, we sought to develop a combination approach that provides more rapid and durable protection than traditional vaccination alone. A novel immune-based prophylaxis/therapy strategy entailing the facilitated delivery of a synthetic DNA consensus prME vaccine along with DNA-encoded anti-ZIKV envelope monoclonal antibodies (dMAb) were developed and evaluated for antiviral efficacy. This immediate and persistent protection strategy confers the ability to overcome shortcomings inherent with conventional active vaccination or passive immunotherapy. A collection of novel dMAbs were developed which were potent against ZIKV and could be expressed in serum within 24-48 h of in vivo administration. The DNA vaccine, from a previous development, was potent after adaptive immunity was developed, protecting against infection, brain and testes pathology in relevant mouse challenge models and in an NHP challenge. Delivery of potent dMAbs protected mice from the same murine viral challenge within days of delivery. Combined injection of dMAb and the DNA vaccine afforded rapid and long-lived protection in this challenge model, providing an important demonstration of the advantage of this synergistic approach to pandemic outbreaks.

Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Abeta antibodies.

BACKGROUND: Prior work from our group found that acid dissociation (pH 2.5 incubation) of serum from APP transgenic mice vaccinated against Abeta increased the apparent anti-Abeta titers, suggesting antibody masking by antigen in the ELISA assay. Subsequently, we found that pH 2.5 incubation of serum from unvaccinated non-transgenic mice showed antibody binding to Abeta1-42, but no increase when other proteins, including shorter Abeta peptides, coated the ELISA plate. To investigate further the effects of low pH incubation on apparent anti-Abeta1-42 signals, we examined normal sera from nonTg unvaccinated mice, nonTg mice vaccinated with Abeta peptide (to produce authentic anti-Abeta antibodies) or a monoclonal antibody against Abeta (6E10) using competitive-inhibition ELISA and Abeta epitope mapping assays. In addition, we examined use of a less stringent low pH procedure at pH 3.5, to ascertain if it had the same effects as the pH 2.5 procedure. RESULTS: We believe there are three distinct effects of pH 2.5 incubation.; A) an artifactual increase in binding to full length Abeta by mouse immunoglobulin which has low affinity for Abeta, B) an inactivation of anti-Abeta antibodies that is time dependent and C) unmasking of high affinity anti-Abeta antibodies when high levels of circulating Abeta is present in APP transgenic mice. All three reactions can interact to produce the final ELISA signal. Incubation of sera from unvaccinated nonTg mice at pH 2.5 enhanced ELISA signals by process A. Conversely, pH 2.5 incubation of sera from vaccinated nonTg mice with caused a time dependent reduction of antibody signal by process B (overcoming the increase caused by A). The artifactual anti-Abeta ELISA signal enhanced by pH 2.5 incubation of normal mouse sera could not be effectively competed by low to moderate concentrations of Abeta, nor bind to shorter Abeta peptides in a manner similar to authentic anti-Abeta antibodies. Incubation of mouse sera at pH 3.5 caused neither an apparent increase in anti-Abeta ELISA signal, nor an inactivation of the ELISA signals resulting from either vaccination or monoclonal antibodies. However, incubation at pH 3.5 was able to completely reverse the reduction in ELISA signal caused by Abeta complexing with antibodies in sera from vaccinated mice or monoclonal anti-Abeta antibodies. CONCLUSION: Incubation at pH 3.5 is sufficient to dissociate Abeta bound to anti-Abeta antibodies without producing artifactual increases in the signal, or inactivating authentic antibody binding. Thus, use of pH 3.5 is a considerable improvement over pH 2.5 incubation for unmasking anti-Abeta antibodies in ELISA assays to measure antibodies in APP transgenic mouse sera.

Protocols for Developing Novel Chikungunya Virus DNA Vaccines.

To date, there have been several million infections by the Chikungunya virus (CHIKV), a mosquito-transmitted emerging pathogen that is considered to be taxonomically an Old World RNA virus. Although original CHIKV outbreaks were restricted to India, East Asian countries, Northern Italy, and France, a recent sharp rise had been identified in 41 countries or territories in the Caribbean, Central America, South America, and North America. A total of 1,012,347 suspected and 22,579 laboratory-confirmed CHIKV cases have been reported from these areas, which signals an increasing risk to the US mainland. Unlike past epidemics that were usually associated with Ae. aegypti transmission, the Caribbean outbreak was associated with Ae. albopictus transmission as the principal mosquito vector. In addition, the substantial increase in the number of deaths during this epidemic, as well as incidence of neurologic disease, suggests that CHIKV may have become more virulent. Currently, there are no licensed vaccines or therapeutics available for CHIKV or its associated disease pathologies. Therefore, development of new vaccines and therapies that could confer immunity and/or treat clinical symptoms of CHIKV is greatly desired. This chapter describes the use of entirely cutting edge technologies/methodologies developed by our group for the development and evaluation of novel DNA vaccines against CHIKV.

In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine.

Significant concerns have been raised owing to the rapid global spread of infection and disease caused by the mosquito-borne Zika virus (ZIKV). Recent studies suggest that ZIKV can also be transmitted sexually, further increasing the exposure risk for this virus. Associated with this spread is a dramatic increase in cases of microcephaly and additional congenital abnormalities in infants of ZIKV-infected mothers, as well as a rise in the occurrence of Guillain Barre' syndrome in infected adults. Importantly, there are no licensed therapies or vaccines against ZIKV infection. In this study, we generate and evaluate the in vivo efficacy of a novel, synthetic, DNA vaccine targeting the pre-membrane+envelope proteins (prME) of ZIKV. Following initial in vitro development and evaluation studies of the plasmid construct, mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. Vaccinated animals were found to generate antigen-specific cellular and humoral immunity and neutralisation activity. In mice lacking receptors for interferon (IFN)-α/ß (designated IFNAR-/-) immunisation with this DNA vaccine induced, following in vivo viral challenge, 100% protection against infection-associated weight loss or death in addition to preventing viral pathology in brain tissue. In addition, passive transfer of non-human primate anti-ZIKV immune serum protected IFNAR-/- mice against subsequent viral challenge. This study in NHP and in a pathogenic mouse model supports the importance of immune responses targeting prME in ZIKV infection and suggests that additional research on this vaccine approach may have relevance for ZIKV control and disease prevention in humans.

Intracranially administered anti-Abeta antibodies reduce beta-amyloid deposition by mechanisms both independent of and associated with microglial activation.

Active immunization against the beta-amyloid peptide (Alphabeta) with vaccines or passive immunization with systemic monoclonal anti-Abeta antibodies reduces amyloid deposition and improves cognition in APP transgenic mice. In this report, intracranial administration of anti-Alphabeta antibodies into frontal cortex and hippocampus of Tg2576 transgenic APP mice is described. The antibody injection resulted initially in a broad distribution of staining for the antibody, which diminished over 7 d. Although no loss of immunostaining for deposited Abeta was apparent at 4 hr, a dramatic reduction in the Alphabeta load was discernible at 24 hr and was maintained at 3 and 7 d. A reduction in the thioflavine-S-positive compact plaque load was delayed until 3 d, at which time microglial activation also became apparent. At 1 week after the injection, microglial activation returned to control levels, whereas Alphabeta and thioflavine-S staining remained reduced. The results from this study suggest a two-phase mechanism of anti-Alphabeta antibody action. The first phase occurs between 4 and 24 hr, clears primarily diffuse Alphabeta deposits, and is not associated with observable microglial activation. The second phase occurs between 1 and 3 d, is responsible for clearance of compact amyloid deposits, and is associated with microglial activation. The results are discussed in the context of other studies identifying coincident microglial activation and amyloid removal in APP transgenic animals.

Gene-based vaccines and immunotherapeutic strategies against neurodegenerative diseases: Potential utility and limitations.

There has been a recent expansion of vaccination and immunotherapeutic strategies from controlling infectious diseases to the targeting of non-infectious conditions including neurodegenerative disorders. In addition to conventional vaccine and immunotherapeutic modalities, gene-based methods that express antigens for presentation to the immune system by either live viral vectors or non-viral naked DNA plasmids have been developed and evaluated. This mini-review/commentary summarizes the advantages and disadvantages, as well as the research findings to date, of both of these gene-based vaccination approaches in terms of how they can be targeted against appropriate antigens within the Alzheimer and Parkinson disease pathogenesis processes as well as potentially against targets in other neurodegenerative diseases. Most recently, the novel utilization of these viral vector and naked DNA gene-based technologies includes the delivery of immunoglobulin genes from established biologically active monoclonal antibodies. This modified passive immunotherapeutic strategy has recently been applied to deliver passive antibody immunotherapy against the pathologically relevant amyloid ß protein in Alzheimer disease. The advantages and disadvantages of this technological application of gene-based immune interventions, as well as research findings to date are also summarized. In sum, it is suggested that further evaluation of gene based vaccines and immunotherapies against neurodegenerative diseases are warranted to determine their potential clinical utility.

Anti-human α-synuclein N-terminal peptide antibody protects against dopaminergic cell death and ameliorates behavioral deficits in an AAV-α-synuclein rat model of Parkinson's disease.

The protein α-synuclein (α-Syn) has a central role in the pathogenesis of Parkinson's disease (PD) and immunotherapeutic approaches targeting this molecule have shown promising results. In this study, novel antibodies were generated against specific peptides from full length human α-Syn and evaluated for effectiveness in ameliorating α-Syn-induced cell death and behavioral deficits in an AAV-α-Syn expressing rat model of PD. Fisher 344 rats were injected with rAAV vector into the right substantia nigra (SN), while control rats received an AAV vector expressing green fluorescent protein (GFP). Beginning one week after injection of the AAV-α-Syn vectors, rats were treated intraperitoneally with either control IgG or antibodies against the N-terminal (AB1), or central region (AB2) of α-Syn. An unbiased stereological estimation of TH+, NeuN+, and OX6 (MHC-II) immunostaining revealed that the α-Syn peptide antibodies (AB1 and AB2) significantly inhibited α-Syn-induced dopaminergic cell (DA) and NeuN+ cell loss (one-way ANOVA (F (3, 30) = 5.8, p = 0.002 and (F (3, 29) = 7.92, p = 0.002 respectively), as well as decreasing the number of activated microglia in the ipsilateral SN (one-way ANOVA F = 14.09; p = 0.0003). Antibody treated animals also had lower levels of α-Syn in the ipsilateral SN (one-way ANOVA F (7, 37) = 9.786; p = 0.0001) and demonstrated a partial intermediate improvement of the behavioral deficits. Our data suggest that, in particular, an α-Syn peptide antibody against the N-terminal region of the protein can protect against DA neuron loss and, to some extent behavioral deficits. As such, these results may be a potential therapeutic strategy for halting the progression of PD.

Evaluation of an α synuclein sensitized dendritic cell based vaccine in a transgenic mouse model of Parkinson disease.

In order to develop a cell-based vaccine against the Parkinson disease (PD) associated protein α-synuclein (α-Syn) 3 peptides were synthesized based upon predicted B cell epitopes within the full length α-Syn protein sequence. These peptide fragments as well as the full length recombinant human α-Syn (rh- α-Syn) protein were used to sensitize mouse bone marrow-derived dendritic cells (DC) ex vivo, followed by intravenous delivery of these sensitized DCs into transgenic (Tg) mice expressing the human A53T variant of α-Syn. ELISA analysis and testing of behavioral locomotor function by rotometry were performed on all mice after the 5th vaccination as well as just prior to euthanasia. The results indicated that vaccination with peptide sensitized DCs (PSDC) as well as DCs sensitized by rh-α-Syn induced specific anti-α-Syn antibodies in all immunized mice. In terms of rotometry performance, a measure of locomotor activity correlated to brain dopamine levels, mice vaccinated with PSDC or rh- α-Syn sensitized DCs performed significantly better than non-vaccinated Tg control mice during the final assessment (i.e. at 17 months of age) before euthanasia. As well, measurement of levels of brain IL-1α, a cytokine hypothesized to be associated with neuroinflammation, demonstrated that this proinflammatory molecule was significantly reduced in the PSDC and rh- α-Syn sensitized DC vaccinated mice compared to the non-vaccinated Tg control group. Overall, α-Syn antigen-sensitized DC vaccination was effective in generating specific anti- α-Syn antibodies and improved locomotor function without eliciting an apparent general inflammatory response, indicating that this strategy may be a safe and effective treatment for PD.

A synthetic consensus anti-spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates.

First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

T-cell responses induced in normal volunteers immunized with a DNA-based vaccine containing HIV-1 env and rev.

OBJECTIVE: An effective HIV-1 vaccine will likely need to induce strong cell-mediated immunity in humans. Therefore, we examined the ability of a DNA HIV-1 vaccine to induce a T-cell response in HIV-1 seronegative humans. DESIGN: Individuals were enrolled in a phase I clinical trial of safety and immune responses to an env/rev-containing plasmid at doses of 100, 300 or 1000 microg. Peripheral blood mononuclear cells (PBMC) samples were analyzed by standard lymphocyte proliferation, cytotoxic T lymphocyte (CTL) and ELISPOT techniques. RESULTS: PBMCs from subjects immunized with doses as low as 300 microg proliferated in vitro to env (four of six) or (three of six) proteins. Importantly, when the dose of vaccine was increased to 1000 microg of DNA, lymphocytes secreted IFN-gamma in an ELISPOT assay following in vitro stimulation with env (three of six) or rev (four of six) proteins. CONCLUSION: We observed HIV-1 DNA plasmid vaccines induce CD4 T-helper cell responses in humans. We observed a discrepancy in the CD4 versus CD8 response suggesting the importance of analyzing both compartments in clinical evaluation. Furthermore, this report demonstrates the high level of immunogenicity of and its importance as a component of a prophylactic vaccine for HIV-1.

Vaccine development against HIV-1: current perspectives and future directions.

The development of an efficacious vaccine against the human immunodeficiency virus (HIV) is of great urgency, because it is accepted that vaccination is the only means capable of controlling the AIDS pandemic. The foundation of HIV vaccine development is the analysis of immune responses during natural infection and the utilization of this knowledge for the development of protective immunization strategies. Initial vaccine development and experimentation are usually in animal models, including murine, feline, and nonhuman primates. Experimental vaccine candidates are closely studied for both efficacy and safety before proceeding to human clinical trials. There are a number of different therapeutic and prophylactic vaccine strategies currently being studied in human clinical trials. Vaccine strategies that are being tested, or have previously been tested, in humans include subunit, DNA plasmid, and viral vector, and combinations of these various strategies. Some of the results of these trials are promising, and additional research has focused on the development of appropriate chemical and genetic adjuvants as well as methods of vaccine delivery to improve the host immune response. This review summarizes the vaccine strategies that have been tested in both animal models and human clinical trials.

Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice.

BACKGROUND: In prior work we detected reduced anti-Abeta antibody titers in Abeta-vaccinated transgenic mice expressing the human amyloid precursor protein (APP) compared to nontransgenic littermates. We investigated this observation further by vaccinating APP and nontransgenic mice with either the wild-type human Abeta peptide, an Abeta peptide containing the "Dutch Mutation", E22Q, or a wild-type Abeta peptide conjugated to papillomavirus virus-like particles (VLPs). RESULTS: Anti-Abeta antibody titers were lower in vaccinated APP than nontransgenic mice even when vaccinated with the highly immunogenic Abeta E22Q. One concern was that human Abeta derived from the APP transgene might mask anti-Abeta antibodies in APP mice. To test this possibility, we dissociated antigen-antibody complexes by incubation at low pH. The low pH incubation increased the anti-Abeta antibody titers 20-40 fold in APP mice but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-Abeta titers were still lower in transgenic mice vaccinated with wild-type Abeta or E22Q Abeta relative to non-transgenic mice. Importantly, the dissociated anti-Abeta titers were equivalent in nontransgenic and APP mice after VLP-based vaccination. Control experiments demonstrated that after acid-dissociation, the increased antibody titer did not cross react with bovine serum albumin nor alpha-synuclein, and addition of Abeta back to the dissociated serum blocked the increase in antibody titers. CONCLUSIONS: Circulating human Abeta can interfere with ELISA assay measurements of anti-Abeta titers. The E22Q Abeta peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against Abeta abrogate the effects of Abeta self-tolerance.

Characterization of a novel human anti-HIV-1 gp41 IgM monoclonal antibody designated clone 37.

Human monoclonal antibodies (HuMAbs) demonstrate great potential for passive immunotherapy against HIV-1. The gp41 transmembrane envelope glycoprotein of HIV has an important role in the pathogenicity of AIDS and importantly displays considerably less hypervariability than the gp120 surface envelope HIV glycoprotein, which makes it particularly a better candidate for the development of passive and active immunotherapies. The general aim of this study was to develop HuMAbs to HIV surface glycoproteins and particularly gp41. Peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-seropositive long-term nondisease progressing patient. B-cells from this individual were then immortalized by Epstein-Barr virus (EBV) transformation, and antibody production was stabilized by fusion of transformed cells with a heteromyeloma. Subsets of the human heterohybridomas so generated were analyzed by ELISA. The hybridoma with the highest binding by immunoassay against gp160 was further analyzed. This hybridoma, designated as clone 37 (C37), was determined to be an IgM Kappa antibody and overlapping peptides of HIV envelope proteins (derived from the MN tissue culture line adapted HIV isolate) were used to map the specific binding domain of this HuMAb. Overlapping peptides designated 2026 (SWSNKSLDDIWNN, AA614-626), and 2027 (DDIWNNMTWMQWEREIDNYT, AA621-640) within the HIV-1 gp41 transmembrane glycoprotein were demonstrated to bind to C37 indicating that the specific binding domain for the antibody was DDIWNN. High affinity binding of C37 by ELISA to recombinant gp41 was demonstrated as well. Few IgM HuMAbs against HIV have been generated and characterized. Theoretically, because of the pentameric binding nature of IgM antibodies as well as their very efficient ability to activate complement, such reagents could have potential as anti-HIV agents.

HIV-1 Vpr: enhancing sensitivity of tumors to apoptosis.

Cancers can adapt several evasive functions including apoptosis evasion, self-sufficiency in growth signals, insensitivity to anti-growth signals, sustained angiogenesis, limitless replication potential, tissue invasion and metastasis. The invariable hurdle for development of therapies against such aberrant conditions requires both selective and potent cytotoxicity. Analysis of HIV-1 Vpr's apoptotic and anti-proliferative activity have revealed potentially important implications for cancer therapy. Accordingly, we have reviewed the properties of Vpr that will likely contribute to its efficacious function as an anti-tumor agent. Among these are its ability to induce cell cycle arrest, inhibit inflammation, provoke p53 independent apoptosis, and selective killing of rapidly dividing cells.

Vaccination induced changes in pro-inflammatory cytokine levels as an early putative biomarker for cognitive improvement in a transgenic mouse model for Alzheimer disease.

Several pieces of experimental evidence suggest that administration of anti-ß amyloid (Aß) vaccines, passive anti-Aß antibodies or anti-inflammatory drugs can reduce Aß deposition as well as associated cognitive/behavioral deficits in an Alzheimer disease (AD) transgenic (Tg) mouse model and, as such, may have some efficacy in human AD patients as well. In the investigation reported here an Aß 1-42 peptide vaccine was administered to 16-month old APP+PS1 transgenic (Tg) mice in which Aß deposition, cognitive memory deficits as well as levels of several pro-inflammatory cytokines were measured in response to the vaccination regimen. After vaccination, the anti-Aß 1-42 antibody-producing mice demonstrated a significant reduction in the sera levels of 4 pro-inflammatory cytokines (TNF-α, IL-6, IL-1 α, and IL-12). Importantly, reductions in the cytokine levels of TNF-α and IL-6 were correlated with cognitive/behavioral improvement in the Tg mice. However, no differences in cerebral Aß deposition in these mice were noted among the different control and experimental groups, i.e., Aß 1-42 peptide vaccinated, control peptide vaccinated, or non-vaccinated mice. However, decreased levels of pro-inflammatory cytokines as well as improved cognitive performance were noted in mice vaccinated with the control peptide as well as those immunized with the Aß 1-42 peptide. These findings suggest that reduction in pro-inflammatory cytokine levels in these mice may be utilized as an early biomarker for vaccination/treatment induced amelioration of cognitive deficits and are independent of Aß deposition and, interestingly, antigen specific Aß 1-42 vaccination. Since cytokine changes are typically related to T cell activation, the results imply that T cell regulation may have an important role in vaccination or other immunotherapeutic strategies in an AD mouse model and potentially in AD patients. Overall, these cytokine changes may serve as a predictive marker for AD development and progression as well as having potential therapeutic implications.