CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

296LB STRUCTURE-GUIDED IMPROVEMENT OF HIV-1 BROADLY NEUTRALIZING ANTIBODIES Qingbo Liu , Yen-Ting Lai, Peng Zhang, Peter D. Kwong, Paolo Lusso NIAID, Bethesda, MD, USA Background: Broadly neutralizing antibodies (bNAb) are a promising alternative to antiretroviral drugs for prevention and treatment of HIV-1 infection. We recently reported the identification of a second, quaternary CD4- binding site (CD4BS2) on the native HIV-1 Env trimer spike, and found that some anti-CD4BS NAbs like VRC03 and VRC06 mimic the quaternary binding mode of CD4, establishing contacts with a neighboring gp120 protomer through the extended framework 3 (FR3) loop of their heavy chain. In contrast, some bNAbs like N6 and VRC01 contain a shorter FR3 loop and interact with only a single gp120 protomer. The aim of this study was to investigate if bNAbs like N6 and VRC01 could benefit from establishing quaternary contacts and if this could be achieved by elongation of their FR3 loop. Methods: All wild-type (WT) and mutated antibodies were expressed in 293FS cells and purified by protein-A columns; trimer binding was measured by ELISA and surface plasmon resonance (SPR); the crystal structure of antibody- trimer complex was solved by x-ray diffraction and molecular replacement; neutralization was determined by the TZM-bl assay against a global panel of HIV-1 isolates. Results: The key role of the FR3 loop in the quaternary interaction of VRC03 and VRC06 was confirmed by FR3-loop truncation and testing on CD4BS2 mutants. Thus, we employed structure-based engineering to engraft the long FR3 loop of VRC03 into several potent CD4-supersite antibodies. Three of 4 chimeric antibodies (derived from VRC01, VRC07 and N6) showed enhanced neutralizing capacity against the majority of HIV-1 strains tested. Binding of chimeric antibodies to the Env trimer was also increased. SPR analysis showed that the quaternary contact stabilized the interaction between the modified antibody and the Env trimer by prolonging their dissociation rate. The crystal structure of one engineered antibody, N6 70-03, was solved in complex with the BG505 SOSIP.664 trimer, showing that the FR3 loop of chimeric N6 70-03 interacts both with CD4BS2 and with the V3-loop base in the neighboring gp120 protomer. Conclusion: Engraftment of the FR3 loop enabled potent anti-CD4BS antibodies to establish quaternary contact with a neighboring protomer of the HIV-1 Env timer, leading to increased potency. These modified bNAbs are potential candidates for application in HIV-1 therapy and prevention.

In some cases, structural diversification of the clades appears to be relatively recent; epitopes were similarly distributed in the 1980’s but then decayed at different rates during the next three decades. The in-host volatility of each epitope was clade-specific and correlated strongly with its rate of population decay in the same clade. Conclusion: HIV-1 Env structure is highly dynamic in the population. Epitopes appear to have defined and clade-specific longevities. The in-host volatility of structural features guides their longitudinal divergence and accounts for their population-level diversification patterns over the course of the pandemic. The conserved clade-specific rates of diversification help us to predict the future distribution of epitopes, to guide design of population-targeted immunogens. 295 OPENPRIMER, A PRIMER DESIGN TOOL FOR AMPLIFYING B CELL CDNA ENCODING HUMAN ANTIBODIES Matthias Döring 1 , Christoph Kreer 2 , Nathalie Lehnen 2 , Nico Pfeifer 3 , Florian Klein 2 1 Max Planck Institute for Informatics, Saarbrücken, Germany, 2 Cologne University Hospital, Cologne, Germany, 3 Max Planck Institute for Infection Biology, Berlin, Germany Background: Deciphering the human antibody response requires the amplification of cDNA from B cells using multiplex polymerase chain reaction (mPCR), which necessitates primers targeting a variety of immunoglobulin gene segments with or without additional somatic mutations. Broadly neutralizing antibodies (bNAbs) against HIV-1 carry high levels of somatic mutations, which can impede the successful amplification of their cDNA. Therefore, we set out to design novel primers that target the putatively less mutated immunoglobulin leader region in order to enhance the probability of covering antibody genes even with high levels of somatic mutations. Methods: To design novel sets of primers, we developed a primer design algorithmwith three stages: (1.) Construction of a degenerate set of primers by computing consensus sequences of hierarchically-clustered target region substrings, (2.) filtering of the initial primer set by applying constraints on up to eleven physicochemical properties, and (3.) optimization of the remaining primer candidates by solving an instance of the set cover problem using either a greedy algorithm or an integer linear programming formulation. The theoretical significance of the designed primer sets was computed by applying Fisher’s exact test on the constraint fulfillment matrix. The designed primer sets for the heavy chain were validated by performing mPCR on individual clones of germline immunoglobulin cDNA and comparing in silico and in vitro coverage events. Results: We have designed novel primer sets that bind with at most one mismatch to the leaders of IGHV (n=14), IGKV (n=7), and IGLV (n=8) such that all antibody variants are covered (Figure 1). The newly designed primer sets fulfilled significantly more physicochemical constraints than existing primer sets from the literature (p-values of 3.2e-22, 7.7e-12, and 7.5e-14, respectively). The experimental validation of the designed primer sets showed that in silico and in vitro coverages agreed well, with both sensitivity and specificity exceeding 80%. Conclusion: We have developed openPrimeR , an open-source computational tool for designing, evaluating, and comparing primer sets for mPCR. Using the tool, we constructed primer sets that could facilitate the discovery of new bNAbs. Since the tool is very general, it could be used to analyze and design primer sets in many other application scenarios. openPrimeR (http:// openprimer.mpi-inf.mpg.de) will be available in Bioconductor release 3.6.

Poster Abstracts

297 THE CODEVELOPMENT OF ANTIBODY GLYCOSYLATION AND FUNCTIONALITY IN HIV INFECTION

Madeleine Jennewein 1 , Jennifer Mabuka 1 , Bruce D. Walker 1 , Thumbi Ndungú 2 , Galit Alter 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 KwaZulu-Natal Research Institute for TB and HIV, Durban, South Africa Background: Increasing evidence points to a role for antibody-mediated effector function in preventing and controlling HIV infection. Antibody effector functions are regulated both by changes in antibody subclass/isotype selection and changes in antibody glycosylation. However, it us unknown how antibody effector function evolves following infection, nor how the humoral immune response naturally is tuned to recruit the antiviral activity of the innate immune system. Methods: Using a set of unique hyper-acute HIV-infection samples from 15 South-African women who were identified within 48 hours of infection (the FRESH cohort), systems serology was performed to evaluate both the functional and biophsycial changes in p24-, gp120-, and gp41- specific antibody responses

CROI 2018 104