Chandrasekhar Satishchandran

DNA Vaccines Encoding Interleukin-8 and RANTES Enhance Antigen-Specific Th1-Type CD4+ T-Cell-Mediated Protective Immunity against Herpes Simplex Virus Type 2 In Vivo

ABSTRACT Chemokines are inflammatory molecules that act primarily as chemoattractants and as activators of leukocytes. Their role in antigen-specific immune responses is of importance, but their role in disease protection is unknown. Recently it has been suggested that chemokines modulate immunity along more classical Th1 and Th2 phenotypes. However, no data currently exist in an infectious challenge model system. We analyzed the modulatory effects of selected chemokines (interleukin-8 [IL-8], gamma interferon-inducible protein 10 [IP-10], RANTES, monocyte chemotactic protein 1 [MCP-1], and macrophage inflammatory protein 1α [MIP-1α]) on immune phenotype and protection against lethal challenge with herpes simplex virus type 2 (HSV-2). We observed that coinjection with IL-8 and RANTES plasmid DNAs dramatically enhanced antigen-specific Th1 type cellular immune responses and protection from lethal HSV-2 challenge. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vitro and in vivo T-cell subset deletion. Thus, IL-8 and RANTES cDNAs used as DNA vaccine adjuvants drive antigen-specific Th1 type CD4+ T-cell responses, which result in reduced HSV-2-derived morbidity, as well as reduced mortality. However, coinjection with DNAs expressing MCP-1, IP-10, and MIP-1α increased mortality in the challenged mice. Chemokine DNA coinjection also modulated its own production as well as the production of cytokines. These studies demonstrate that chemokines can dominate and drive immune responses with defined phenotypes, playing an important role in the generation of protective antigen-specific immunity.

Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments

A novel DNA assembly method, chain reaction cloning (CRC), is described. CRC enables the ordered assembly of multiple DNA fragments in a single step. The power of the technique was demonstrated by the directed in vitro assembly of a plasmid comprised of six DNA fragments from a pool of 12 available fragments. The odds of obtaining the correct plasmid clone in a single step, using conventional techniques, is less than 1 in 191000000. Using CRC, the desired plasmid was recovered at a frequency of one in two. Ligation is no longer the rate limiting step in cloning, and limitless possibilities exist for the reconstruction of complex genomes.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

RNA interference and its potential applications to chronic HBV treatment: results of a Phase I safety and tolerability study

BACKGROUND RNA interference (RNAi) provides an attractive tool to modulate biological systems, and ultimately, to treat human diseases. We describe early results from a Phase Ib, first-in-human safety and tolerability study of an RNAi-based therapy, NUC B1000, among patients with mild to moderate chronic HBV. METHODS Three subjects received a single 5 mg DNA dose of NUC B1000 as part of a planned dose escalation study. RESULTS All participants reported pharyngitis, chills, myalgia and fever approximately 4-7 h after dosing. All subjects were asymptomatic after a single antipyretic dose with no symptom recurrences. Measurements of interferon (IFN)-α and -γ, interleukin (IL)-10, 12 18, 8 and 6, and tumour necrosis factor-α performed before and after dosing revealed cytokine increases before study drug administration. After drug administration, IFN-γ and IL-10 increased in two patients; IL-8 increased in one. Most increases returned to pretreatment levels within 1 week. Two patients were subsequently successfully treated with entecavir indicating that NUC B1000 does not compromise subsequent antiviral therapy. CONCLUSIONS Thus far, NUC B1000 appears safe and well-tolerated; safety and efficacy studies across a larger, more diverse patient spectrum using increasing doses are needed to determine its appropriate role in the antiviral armamentarium.

Using RNA interference to modulate gene expression

Abstract RNA interference (RNAi) has emerged as one of the most promising technologies among those directed towards regulating gene expression in animals. The presence of a double-stranded RNA (dsRNA) in eukaryotic cells triggers this post-transcriptional gene-silencing mechanism, leading to a sequence-specific degradation of the target mRNA. RNAi offers unique advantage over other technologies due to its ability both to amplify catalytically the initial trigger signal to silence the target RNA sequence and to systemically spread the silencing signal to other cells. Various strategies currently being developed to employ RNAi technology for rapid functional genomic analyses and therapeutic applications will be reviewed in this paper.

Induction of HSV-gD2 specific CD4+ cells in Peyer's patches and mucosal antibody responses in mice following DNA immunization by both parenteral and mucosal administration

A DNA vaccine encoding glycoprotein D (gD) of herpes simplex virus type 2 (pHSV-gD2) was injected via parenteral and mucosal routes to determine the optimal route of delivery for immune stimulation. Generation of distal mucosal immunity following parenteral vaccination was also evaluated. While all routes of DNA vaccine administration resulted in systemic cellular and humoral responses, the intra-muscular (i.m.) and intra-dermal (i.d.) routes of delivery produced the highest responses. Furthermore, i.m. and i.d. routes produced mucosal humoral responses that were comparable to those obtained via mucosal routes. Specific pHSV-gD2 PCR signals were detected in the Peyers patches (PP) within hours following vaccination and antigen specific IgA was detected in secretions and supernatants from gut fragment cultures. Furthermore, antigen specific CD4(+) cells were found in PP. Collectively these results suggest that the DNA vaccine stimulated a response in the PP, a major inductive site for mucosal responses.

Preclinical Safety of DNA Vaccines

DNA or genetic vaccines are currently being evaluated for safety and efficacy in human clinical trials in the areas of infectious disease and cancer. Since DNA vaccines induce antibodies and cytotoxic T lymphocytes (CTLs), they are currently being evaluated in humans for both prevention and therapy of HSV-2, HIV-1, and HBV infections, for prevention of influenza and malaria, and therapy of cutaneous T-cell lymphoma (CTCL) and colorectal cancer.