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Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100(F)Y, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100(F)Y fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100(F)Y, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies.
BACKGROUND - Expression of microbial protein sequences in eukaryotic cells transfected by transcriptional/translational permissive cDNA constructs can induce systemic humoral and cellular responses in vivo. Two methods of in vivo transfection have been described to date. One method uses large quantities of naked DNA injected into skeletal muscle. The second method uses relatively small quantities of DNA complexed to gold particles for bollistic penetration of the plasma membrane of keratinocytes. The major disadvantage of the bolistic method is that instrumentation is required which is not generally available.
OBJECTIVES - The objectives of this study were to determine whether the use of DNA complexed with a cationic lipopolyamine could reduce the quantity of DNA required to induce systemic humoral responses following muscle transfection and whether similar DNA/lipopolyamine complexes could induce mucosal humoral responses following airway exposure.
STUDY DESIGN - Balb/c mice were exposed by nasal aerosol or intramuscular inoculation to a mammalian transcriptional/translational permissive DNA construct containing the entire sequence for the HIV-1 envelope polyprotein. Experimental animals were further segregated by the number of exposures at 3-week intervals and whether the DNA was complexed to dioctadecylamidoglycylspermine (DOGS) at a 5:1 molar charge ratio of DOGS/DNA.
RESULTS - DOGS facilitated in vivo transfection of mouse muscle reduced the quantity of DNA required for a systemic humoral response to surface expressed HIV-envelope proteins by one order of magnitude. Exposure of airway mucosa to both 10 micrograms and 1 microgram quantities of DNA complexed to DOGS produced systemic humoral responses to HIV-envelope as well as mucosal antibodies in pulmonary and colonic epithelia. Molecular modeling of DOGS/DNA complexes at the 5:1 charge ratio used in this study indicates that the DNA component is not exposed to aqueous solvents and may be relatively resistant to degradation under common biological environments.
CONCLUSION - Facilitated transfer of DNA by DOGS to transcriptional/translational competent cells offers several distinct advantages to the use of DNA alone. Since significantly smaller amounts of DNA are required, the potential for the induction of antibodies against DNA itself lessens the likelihood for the development of a lupus-like syndrome. More importantly, however, is the apparent ability to transfect mucosal cells which results in the development of specific mucosal immune responses. This procedure may allow the development of general methods for the induction of mucosal immunity at the level of entry for mucosal pathogens without the disadvantages inherent in live attenuated vectors.