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Richard Broeker1, Monica Nguyen1, and Laura Doepker*2

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Richard Broeker1, Monica Nguyen1, and Laura Doepker*2 Computational Approach for the Discovery of Broadly Neutralizing HIV Antibodies Richard Broeker1, Monica Nguyen1, and Laura Doepker*2 Introduction Methods The human immunodeficiency virus (HIV) is capable of evading the human immune system through rapid Ø Plasmids containing representative sequences from heavy mutations of envelope (Env) proteins, gp120 and gp41, and light chain antibody families were verified using the Figure 3. Schematic of an ELISA where the recombinant antibodies bind to monomeric HIV UGENE sequencing analysis software and transfected into antigens. HIV antigens coat the surface of the well and remaining empty spaces are coated with a and generally outpaces the adaptive antibody response blocking buffer to prevent false antibody binding. Recombinant antibodies were introduced, which (Kirchoff 2010). However, HIV-infected infants have been human embryonic kidney (HEK293F) cells. bound to the HIV antigens. Secondary antibodies, goat anti-human IgG-HRP, were added and bound found to produce broad and potent antibody responses Ø Antibody protein isolation and purification was done using to recombinant antibodies, allowing for detection of binding through fluorescence. an affinity column with protein–coated agarose beads. Tetramethylbenzidine (TMB) was added to activate fluorescence from secondary antibody goat anti- to HIV, known as broadly neutralizing antibodies (bnAbs), human IgG-HRP, and sulfuric acid was added to stop the reaction. which are utilized as possible vaccine targets (Schief et Ø An enzyme-linked immunosorbent assay (ELISA) was al. 2009; Simonich et al. 2016). Computational mining of Figure 1. Antibody representation showing performed to detect antibody binding to monomeric HIV the heavy and light chain pairing, antigen antigens: gp120, gp41, gp140, and p24. human antibody repertoires could be an efficient binding site, and variable/constant region. process for bnAb discovery. In our research, we applied Ø A cell-surface binding assay this approach using a known bnAb-producing HIV- Objectives (CSBA) was performed to infected infant, BF520. Using computational analysis of detect antibody binding to BF520’s repertoire, representative sequences were Env trimers on the surface selected from heavy and light chain antibody families I. Produce and purify of HEK293T cells. Figure 4. Schematic of a CSBA where the recombinant antibodies bind to Env trimers on the that had potential to be bnAbs and paired together to polyclonal antibodies. Figure 2. HIV virion with the Env surface of human (HEK293T) cells. Plasmids encoding for HIV Env trimers were transfected into proteins highlighted. Depiction of HEK293T cells so that HIV Env trimers would be presented on the surface of the cell. Recombinant make polyclonal antibodies: G2L4, G2L5, G7L2, G7K4, II. Test antibody specificity to gp120, gp41, gp140, and p24 HIV III3 antibodies were introduced, which bound to Env trimer. Secondary antibodies, goat anti-human IgG- G7K6. HIV Env proteins. antigens used in ELISA. HRP, bind to recombinant antibodies, which allows for detection of binding through fluorescence. Results Discussion and Conclusion References Ø Selected representative sequences from heavy and light Ø Kirchhoff F. 2010. Immune evasion and counteraction of restriction factors by HIV-1 and other primate chain families were cloned into plasmids and transfected lentiviruses. Cell host & microbe. 8: 55-67. into HEK293F cells. We were successful in producing Ø Schief WR, Andrew Ban YE, Stamatatos L. 2009. viable antibodies for testing. Challenges for structure-based HIV vaccine design. Ø Our ELISA results using one recombinant antibody, G7K4, Curr Opin HIV AIDs. 4: 431-440. showed it binding to MN gp41, allowing for further Ø Simonich CA, Williams KL, Verkerke HP, Williams JA, Nduati R, Lee KK, Overbaugh J. 2016. HIV-1 analysis of the heavy and light chain antibody families neutraliZing antibodies with limited hypermutation that the representative sequences were selected from. from an infant. Cell. 166: 77-87. Ø No significant antibody binding to HIV Env trimers on the surface of human cells occurred in our CSBA. Acknowledgements Ø Although observation of polyclonal antibodies showed limited Env protein binding, this pilot study using a Figure 5. Absorbance (Au) of bound polyclonal antibodies to monomeric HIV Figure 6. Percentage of cells that were positive for binding of polyclonal antibodies computational approach was successful and can possibly We would like to thank Dr. Laura Doepker for guidance and antigens. In grey are the positive control monoclonal and polyclonal antibodies, as to HIV Env trimer presented on the surface of human cells. In grey are the positive be used for the discovery of bnAbs. opportunity for this research, as well as Dr. Jack Vincent. We well as an assay sensitivity control. Only one polyclonal antibody, G7K4, bound to MN and negative control monoclonal and polyclonal antibodies, as well as an assay would also like to thank University of Washington Tacoma1 and gp41, but it did not bind to gp120 ZM109, gp120 C29UG, gp140 SF162, and p24 HIV sensitivity control. No significant binding to positive control HIV Env trimer, SF162 Fred Hutchinson Cancer Research Center2 for providing us the IIIB. and BF520.w14.I3, occurred. Negative control Env trimer, simian immunodeficiency space and materials to carry out this study. virus (SIV), worked as expected..
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