CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

significantly decreased the extracellular production of p24 antigen from human PBMCs from two individuals infected with three different HIV-1 strains (X4, R5, X4R5). PBMC viability was high in the presence of STO-609 indicating that the reduction in virus infectivity was not due to STO-609 cytotoxicity. These results suggest CaMKK2 is critical for HIV replication. The goal of the proposed research is to determine at what step STO-609 inhibits HIV-1 replication. Methods: Gag/Pol and Env were co-expressed in 293T cells. The levels of intracellular Gag expression and extracellular p24 release was assessed by western blot and p24 ELISA, respectively. To evaluate Gag intracellular trafficking, Gag interactions with the autophagy marker protein LC3, and the ESCRT (endosomal sorting complex required for transport) proteins Alix and Tsg101 required for Gag trafficking to and efficient release of virus particles from the plasma membrane, were evaluated by immunoprecipitation. Results: The levels of intracellular Gag expression, p55 and the proteolytically processed p41 and p24, were similar in the absence or presence of STO-609. In contrast, p24 release and detection in the media of Gag-expressing cells were greatly reduced in cells cultured in the presence of STO-609 compared to cells cultured in the absence of STO-609. We next evaluated whether STO-609 inhibits Gag trafficking to the plasma membrane. In the absence or presence of STO-609, we identified that Gag interacts with the membrane-inserted LC3 II autophagy protein, and the ESCRT proteins Alix, and Tsg101 suggesting STO-609 does not inhibit Gag trafficking to the plasma membrane. Conclusion: These results suggest that STO-609 does not affect Gag expression, trafficking to the plasma membrane, or proteolytic processing but inhibits p24 release from cells. This is an unexpected and potentially exciting finding, and we hypothesize that activation of HIV in latently-infected cells cultured in the presence of STO-609 will allow expression of HIV proteins while preventing HIV budding and release. We will focus our studies in the next year on testing this hypothesis in HIV-infected PBMCs. Proving our hypothesis may lead to strategies for enhanced detection of HIV-infected cells by the immune system and may be useful in the “kill” arm of the “shock and kill” strategy to cure HIV. 173 PROGRESS IN DEVELOPING HIGHLY POTENT SECOND-GENERATION MATURATION INHIBITORS Phuong Pham 1 , Emiko Urano 1 , Sherimay Ablan 1 , Justin A. Kaplan 1 , T.J. Nitz 2 , David E. Martin 3 , Carl Wild 3 , Ritu Gaur 2 , Eric O. Freed 1 1 National Cancer Institute, Frederick, MD, USA, 2 South Asian University, Chanakyapuri, New Delhi, India, 3 DFH Pharma, Inc, Gaithersburg, MD, USA Background: Maturation inhibitors (MIs) block HIV replication by disrupting conversion of CA-SP1 to mature CA resulting in the formation of non-infectious viral particles. MIs likely bind at or near the CA-SP1 cleavage site in the assembled immature Gag complex. Proof-of-concept for MIs was established with bevirimat (BVM), which was found safe and effective in reducing viral load. However, a single amino acid polymorphism in the SP1 region of Gag reduced susceptibility to BVM. Methods: We identified a set of C-28 alkyl amine BVM analogs that exhibited enhanced activity against BVM-resistant polymorphs. The structure-activity relationship (SAR) of these analogs was determined. Variables included side chain length, amine substitution and heteroatom incorporation. Activity against BVM-sensitive and -resistant polymorphic viruses was determined by measuring CA-SP1 processing. Resistance selection studies were carried out against subtype B and C isolates. Results: Effect of side-chain length was examined using aminoalkylamines with 2, 3 or 4 carbon atom linkers between amine moieties proximal to the triterpene template. Antiviral activity increased with linker length and was highest for the aminobutylamines. Amine substitution was examined using a series of N-substituted aminoalkylamines. Significant reduction in activity was observed on conversion of the C-28 proximal amine from secondary to tertiary. Alkyl substitution of the distal amine enhanced activity, with the dimethyl amine blocking CA-SP1 processing by 61%. Heteroatom incorporation was examined by substituting the terminal cyclohexyl group in the aminopropylalkyl side chain with piperidine, N-methylpiperidine, morpholine or N-methylpiperazine. Resistance mutations mapped to the major homology region of capsid and to positions in SP1 not previously observed in BVM selection experiments. Conclusion: Side chain length and amine substitution had significant effects on activity against BVM-resistant polymorphs while heteroatom introduction did not. The amine proximal to the triterpene core plays a key role in activity,

most likely by serving as a hydrogen bond donor that stabilizes compound/ target interaction. The effect of side chain length could reflect the extended nature of the Gag target with a longer C-28 side chain positioning compound/ target residues to better interact. These findings, coupled with the novel resistance mutations identified, provide new insights into the target and mechanism of action of MIs. 174 THE VIROME OF A NEWWORLD PRIMATE UNVEILS A RETROVIRUS THAT CAUSES IMMUNOSUPPRESSION Cláudia Priscila Ramos Muniz 1 , Dawn M. Dudley 2 , Liliane T. Cavalcante 3 , Gislaine Curty 1 , Stefanie V. Santos 4 , Alcides Pissinatti 5 , David H. O’Connor 2 , Marcelo Alves Soares 1 , André Felipe Andrade dos Santos 3 1 Instituto Nacional de Cancer, Rio de Janeiro, Brazil, 2 University of Wisconsin, Madison, WI, USA, 3 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 4 Universidade Federal de São Paulo, São Paulo, Brazil, 5 Centro de Primatologia do Rio de Janeiro, Rio de Janeiro, Brazil Background: The natural reservoirs for some viruses are still controversial, with several hypotheses raising the possibility of non-human primates (NHPs) cross-species transmission of viruses to human. For this purpose, it is extremely important to characterize viral infections in NHP. A colony of Brachyteles arachnoides, a NewWorld primate (NWP) species hosted at Centro de Primatologia of Rio de Janeiro (CPRJ), Brazil, has been affected by a disease of unknown cause. In the last years, NHPs from this colony showed clinical signs similar to those observed in immunosuppression diseases. Immunohistochemistry analysis of deceseased animals revealed that the causa mortis was due to the consequences of a viral infection from the Retroviridae family. Transmission electron microscopy (TEM) images of liver and lung samples showed viral particles with siman foamy virus and simian retrovirus type D morphologies. In order to identify the viral agents that possibly led these NHPs to death, specimen 2506 was selected for this study Methods: Saliva sample was collected and treated to digest unprotected nucleic acid. An RT-PCR reaction was performed to obtain cDNA from the RNA viruses while preserving DNA from DNA viruses. DNA libraries were constructed using the Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced in a MiSeq Illumina platform. PCR and Sanger sequencing were performed to complete sequence gaps to acquire the complete genome. Phylogenetic analyzes were conducted using maximum likelihood in MEGA6.0. Results: The complete genomes of a simian foamy virus (SFV) and a simian retrovirus (SRV) that infected the specimen were sequenced. Both findings corroborate with TEM images obtained from NHPs from this colony. Phylogenetic analyses showed that SFV from B. arachnoides grouped with NWP SFV, corroborating with the cospeciation hypothesis for this virus. No pathology has been associated with SFV infection so far, but it is known that it may be an opportunistic agent. Phylogenetic analysis showed the SRV found grouped with SRV from Asian monkeys. The clinical signs observed in the NWP specimen were similar to those found in sick Asian monkeys infected by SRV. Conclusion: For the first time, the complete genomes from SFV and SRV infecting B. arachnoides were obtained. The SRV described in this study is the first exogenous retrovirus able to cause immunosuppression identified so far in a NWP, leading to its death. 175LB PRIMER ID SEQUENCING TO STUDY THE DEVELOPMENT OF CXCR4-USING HIV-1 VARIANTS Shuntai Zhou 1 , Ean Spielvogel 1 , Sabrina Clark 1 , Adaora Adimora 1 , Catalina Ramirez 1 , Andrew Edmonds 1 , Igho Ofotokun 2 , Seble Kassaye 3 , Margaret Fischl 4 , Stephen J. Gange 5 , Alan Landay 6 , Maria Villacres 7 , Jack DeHovitz 8 , Kathryn Anastos 9 , Ronald Swanstrom 1 1 University of North Carolina Chapel Hill, Chapel Hill, NC, USA, 2 Emory University, Atlanta, GA, USA, 3 Georgetown University, Washington, DC, USA, 4 University of Miami, Miami, FL, USA, 5 Johns Hopkins University, Baltimore, MD, USA, 6 Rush University, Chicago, IL, USA, 7 University of Southern California, Los Angeles, CA, USA, 8 State University of New York at Albany, Rensselaer, NY, USA, 9 Montefiore Medical Center, Bronx, NY, USA Background: HIV-1 uses CD4 as the main receptor and either CCR5 (R5 variants) or CXCR4 (X4 variants) as a coreceptor to enter the host cells. X4 variants are usually seen in the late stage of HIV-1 infection and are related to rapid disease progression. X4 and R5 variants have substantial sequence differences especially at the V3 region. However, the evolution pathways that drive development of X4 variants are unclear. We performed Primer ID deep

Poster Abstracts

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CROI 2018