Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

Abstract

Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.

Expression of enhancing-activity-free neutralizing antibody against dengue type 1 virus in plasmid-inoculated mice

“…The in vivo expression of antibodies against infectious diseases from a plasmid vector was first demonstrated in a human immunodeficiency virus type 1-mouse model, but the long-term persistence of the neutralizing antibody was not reported [40]. We have successfully demonstrated the maintenance of detectable neutralizing antibody (>90% plaque reduction) against DENV-1 for 3 months in mice inoculated with an antibodyexpressing plasmid, when serum samples were diluted 1:2.…”

“…1) was used and attempts to increase antibody production in mice with p7F4-IL2ss, pIRES7F4HL, or pIRES7F4LH also failed (data not shown). Further studies are required to improve the expression cassette and plasmid delivery rate, for codon optimization, and to increase the antibody half-life, as reported elsewhere [40,43,44]. Because the model antibody used in the present study (7F4) was specific for DENV-1 [27], further studies are required to identify an appropriate human neutralizing antibody clone that is broadly cross-reactive with all four DENV serotypes.…”

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

“…Specifically, the Kozak sequence (GCCACC) has been demonstrated to be necessary to preced the ATG start codon in order to drive protein translation 55,56 . As well, incorporation of an IgE leader sequence (MDWTWILFLVAAATRVHS) has been demonstrated to enhance translation of the The sequence was then synthesized commercially 50,54 and cloned into the pVax1 expression vector (Invitrogen) between restriction sites KpnI and NotI. The production of the DNA construct for vaccine use was performed by Aldevron (Fargo, North Dakota, US).…”

“…The Cluster X program (Version 1.81) was used to align the sequences and select the consensus amino acid sequence. The consensus sequence was optimized for expression, including codon and RNA optimization (Gene Optimizer™, GeneArt, Regensburg, Germany) 54 . A Kozak sequence as well as an IgE leader sequence was incorporated in the N terminal region of the construct in order to enhance the expression and ultimately immunogenicity of the plasmid based vaccine.…”

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

“…In that study it was shown that after delivery of the mAb DNA, mice were able to generate in their sera long-term expression of antibodies that were able to neutralize HIV-1 in vitro. 57 These proof-ofconcept studies suggest that this non-viral DNA plasmid delivery method might have applications in terms of targeting mAbs against a¡syn and Ab as well as other potential antigens. Even though this method has a likely better safety profile than viral vector based delivery systems, further optimization of the strategy is needed in order to generate the higher expression levels that were attained by AAV delivery methods.…”