CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

are relatively weak stimulants of pDC and the mechanism of HIV-1 recognition by pDC and pDC activation is not clearly defined. Using two HIV-1 isolates, designated R3A and R3B, we aimed to determine the critical elements of the HIV-1 viral particle required for IFN-I induction in pDC. Methods: Human PBMC and pDC were used to study the IFNa induction. IFNa was measured by Elisa. Viral production was quantified by p24-Elisa. Ala-substitution mutagenesis was used to analyze the Nef-functional domains. Results: In this study we showed that two highly similar HIV-1 variants isolated from a rapid progressor had distinct activity to stimulate pDC and induce IFNa, which in turn correlated with their relative pathogenic activity. The highly pathogenic HIV-1 isolate R3A induced robust IFNa production in pDC, while R3B did not. The viral determinant of efficient IFNa induction was mapped to R3A Env and its V1V2 region with enhanced CD4 binding activity. Interestingly, the Nef gene in R3A was also required for the IFNa induction from pDC. To define which Nef domain or activity are required for increased IFNa induction, we analyzed a panel of R3A Nef functional mutants. We showed that Nef domains involved in CD4 downregulation are necessary for R3A to induce IFNa in pDC. Conclusions: Our data indicate that HIV-1 induced pDC activations depends on (1) the efficacy of envelop to bind CD4 receptor and (2) the Nef activity involved in CD4 downregulation. Our work provides new insights into the mechanism by which HIV-1 stimulates IFNa in pDC and describes novel function of Nef protein which contributes to increased IFNa production and therefore to the pathogenesis of HIV-1 infection. 226 Naturally Occurring Polymorphisms in HIV-1 Nef Impair Its Functions and Decrease Viral Replication Capacity Thomas Vollbrecht 2 ; Lorelei Bornfleth 1 ; Patricia Frohnen 1 ; Martha J. Lewis 1 1 David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA, US; 2 University of California San Diego (UCSD), La Jolla, CA, US Background: The multifunctional accessory HIV-1 protein Nef plays a pivotal role in viral pathogenesis. Key functions of Nef include the downregulation of CD4 and MHC class-I molecules from the surface of HIV-1 infected cells. Previously, we have identified new polymorphisms in Nef from nine subjects under selection pressure by HIV specific CTL. Here we investigate the effect of these polymorphisms on the ability of Nef to downregulate CD4 and MHC class-I molecules, as well as the impact of these polymorphisms on viral replication capacity. Methods: The polymorphisms N52A, N52S, A84D, Y135F, G140R, S169I, H171N, H171P, H71G, D175E, D175N, and V180E were created individually within the plasmid AA1305, a NL43-based proviral vector with the murine CD24 (HSA) reporter gene in vpr, to generate VSV-G pseudotyped replication defective reporter viruses. Additionally, we introduced these mutations into the plasmid p8310, a NL4-3 based half-genome plasmid to generate replication competent reporter viruses. CEMx174.T1 cells were transduced with the replication defective individual viruses to determine the downregulation of MHC class-I and CD4 by flow cytometry. Viral replication capacity was assessed using gDNA and qPCR from PBMC transduced with replication competent reporter viruses. Results: We found in total seven polymorphisms (N52S, A84D, Y135F, G140R, S169I, H171N, H171P) resulting in a significant reduction of HLAA*02 downregulation and five polymorphisms (A84D, G140R, S169I, H171N, H171P) with a significant decrease in CD4 downregulation compared to wild type NL4-3 Nef. Nef with either of two polymorphisms, A84D and G140R was defective for downregulation of HLA-A*02 and CD4. Additionally, virus with the G140R polymorphism also exhibited a significantly decreased viral replication capacity. Conclusions: Our study shows that the majority of the tested polymorphisms exert an inhibitory effect on Nef’s functions. The two polymorphisms A84D and G140R block CD4 and MHC class-I downregulation by Nef. Additionally, the G140R polymorphism also results in a decreased viral replication capacity in primary cells. All tested variants were natural occurring polymorphisms that were previously identified in primary isolates. Our findings lead the way for better Nef targeting by vaccines and for the development of highly effective Nef inhibitors. These results demonstrate new potential targets for immunization or pharmacological intervention strategies. 227 Dynamic Range of Down-Regulation of HIV-1 Entry Receptors by Primary Nef Isolates Mako Toyoda 1 ;Yoko Ogata 1 ; Macdonald Mahiti 1 ; Florencia Pereyra 3 ;Toshiyuki Miura 2 ; BruceWalker 3 ; Zabrina L. Brumme 4 ; Mark A. Brockman 4 ;Takamasa Ueno 1 1 Kumamoto University, Kumamoto, Japan; 2 University of Tokyo, Tokyo, Japan; 3 Ragon Institute of MGH, MIT and Harvard University, Cambridge, MA, US; 4 Simon Fraser University, Burnaby, Canada Background: HIV-1 Nef down-regulates the viral entry receptor CD4 as well as the co-receptors CCR5 and CXCR4 from the surface of infected cells, leading to promotion of viral replication through super-infection resistance and other pathways. In vitro mutagenesis of laboratory strains have identified various sequence motifs within Nef that modulate these functions. However, it remains unclear whether primary Nef sequences isolated from patients with different disease status also modulate down-regulation of these receptors via these same sequence motifs. Methods: Nef clones were amplified from plasma viral RNA of 45 elite controllers (EC) and 46 chronic progressors (CP) and cloned into an expression plasmid. Additional mutations and reversions were introduced into these primary Nef clones to identify functionally important residues and motifs. The resultant plasmids were transfected into TZM-bl cells and CEM cells. Steady-state Nef expression level and cell surface expression of viral receptors were analyzed by Western blot and flow cytometry, respectively. Results: Nef clones from EC showed significantly impaired activity in down-regulation of CD4 and CCR5, compared to those from CP (p<0.001), whereas the difference in down-regulation activity of CXCR4 was not statistical significance (p=0.05). The ability to down-regulate CD4 and CCR5 correlated positively in both EC and CP (R>0.7, p<0.001), suggesting that both activities are simultaneously required in vivo. Nef codon-function analyses failed to identify residues significantly associated with Nef functions. Instead, mutagenesis studies on three EC Nef clones showing substantially diminished functions revealed that multiple residues were involved in altered Nef functions and protein expression level. Specifically, polymorphisms at the highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and within known CTL epitope regions were responsible for reduced Nef functions in these clones. Conclusions: Reduced ability of Nef to down-regulate viral entry receptors appear to be mediated by multiple sequence motifs in primary Nef isolates, including those not identified in in vitro mutagenesis studies. Very rare polymorphisms as well as polymorphisms at immune-reactive sites were involved in this phenomenon in EC. Results suggest that functional attenuation of Nef in EC is mediated by complex polymorphism networks in individual Nef sequences. 228 Differential Down-Regulation of HLA Class I Allotypes by HIV-1 Nef Primary Isolates Macdonald Mahiti 1 ; Xiaofei Jia 2 ; MakoToyoda 1 ; Francis Mwimanzi 1 ; BruceWalker 3 ; Zabrina L. Brumme 4 ; Mark Brockman 4 ;Yong Xiong 2 ; Takamasa Ueno 1 1 Kumamoto University, Kumamoto, Japan; 2 Yale University, New Haven, CT, US; 3 Ragon Institute of MGH, MIT and Harvard University, Boston, MA, US; 4 Simon Fraser University, Burnaby, Canada Background: HIV-1 Nef, in conjunction with the host adaptor protein 1 (AP1), binds to the cytoplasmic region of HLA-A and HLA-B molecules and down-regulates them from the cell surface, thus allowing virus-infected cells to evade immune detection. Polymorphic residues within the HLA class I cytoplasmic region may affect Nef binding and subsequent down-regulation. However, the impact of HLA polymorphisms on recognition by primary Nef isolates, and the specific Nef regions responsible for flexible or differential down- regulation of various HLA-A and HLA-B molecules remain incompletely known.

Poster Abstracts

212

CROI 2015