1. Design of soluble protein constructs displaying the extracellular domains of the HIV coreceptors CCR5 and CXCR4. The HIV-1 Env glycoprotein has been studied extensively at the atomic level (X-ray crystallography and high resolution cryo-EM), including constructs of the gp120 glycoprotein (the receptor-binding subunit) in unliganded form and bound to soluble CD4 and specific monoclonal antibodies. However the complex of gp120 bound to coreceptor (CCR5 or CXCR4) has thus far proven refractory to such analyses, in large part due to the intricate membrane associations of these molecules (they are GPCRs with 7 TM segments). In FY2015, we extended our design and analysis of soluble protein constructs containing extracellular domains of CCR5 known to be critical for coreceptor function, with particular emphasis on a recombinant protein containing sCD4 attached by a polypeptide linker to the CCR5 N-terminal region. By optimizing features of the construct (e.g. linker length) and its expression (e.g. to maximize tyrosine sulfation), we have shown in assays of Env-mediated cell fusion and pseudovirus neutralization that the CCR5 N-terminal moiety interacts specifically with its cognate site on gp120. Efforts are underway to further analyze these interactions using surface plasmon resonance, as a precursor to collaborative efforts to crystalize this protein construct in complex with gp120, hopefully leading to atomic level structural analysis. 2. Targeting killing of HIV-infected cells. We have proposed that complementing combination anti-retroviral therapy (cART) with an immunotoxin designed to selectively kill HIV-infected cells might offer a means to cure acute infection. In collaboration with Dr. Ira Pastan, NCI, we have conducted studies of a new immunotoxin based on Pseudomonas aeruginosa exotoxin A, employing a targeting domain from an optimized broadly neutralizing antibody isolated by the NIAID Vaccine Research Center. Our studies demonstrate excellent breadth and potency against genetically diverse HIV-1 isolates. To test the concept of curing acute infection in a nonhuman primate model, we have begun a collaborative study with investigators at the US MHRP and the VRC to test whether immunotoxin administration can complement cART to achieve a cure of acute SHIV infection of rhesus macaques, based on prevention of virus emergence after cessation of all treatment. At present, we are analyzing sera from animals intended for the study to test for pre-existing anti-PE antibodies; the SHIV challenge/treatment protocol will begin pending the results of this screen. As a potential functional cure of chronic HIV infection, we are extending our studies with chimeric antigen receptors (CARs) containing novel bispecific CD4-based targeting motifs. Our results with CARs containing CD4 linked to the carbohydrate recognition domains (CRDs) of various human C-type lectins indicate the greatly enhance potencies of the CRDs from human mannose-binding lectin 2 (MBL-2) and langerin, compared to those from DC-SIGN and L-SIGN. We have generated a CD4-MBL2 CAR construct in which all components are based entirely of sequences from rhesus macaques. Pending the expected confirmation of the in vitro efficacy of this CAR in virus suppression assays using SHIV-162P and rhesus PBMC, collaborative in vivo studies will be initiated with Dr. Mario Roederer of the VRC. Another collaborative study has been initiated with Dr. Victor Garcia-Martinez of UNC to test the human CD4-MBL2 CAR in the BLT humanized mouse model.
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