| dc.description.abstract | Vaccines have proven effective against numerous viral diseases, yet the development of a successful vaccine for Human Immunodeficiency Virus (HIV-1) remains elusive. A key immunogen for vaccine design is the surface envelope glycoprotein (Env) of HIV-1, particularly gp120, which is prominently displayed on the viral surface. The formidable challenge in eliciting a potent neutralizing antibody response against HIV-1 arises from the extensive variability in the Env sequence. Beyond sequence diversity, HIV-1 employs various defense mechanisms to evade the host immune system. The gp120 subunit of the Env protein is one of the most heavily glycosylated viral proteins known, with approximately half of its molecular mass attributed to glycans. This extensive glycosylation serves to obscure vital neutralization epitopes, although some broadly neutralizing antibodies (bNAbs) target glycan-dependent epitopes to achieve virus neutralization. Moreover, the presence of lengthy, variable immunodominant loops in gp120 strategically directs the immune response away from conserved epitopes, further complicating vaccine development against HIV-1. Certain conserved epitopes are cryptic and are exposed only after gp120 binds to CD4 and eventually remain exposed to the immune system for a very short period of time. Structural characterization of gp120 is exceptionally challenging due to its high flexibility and the labile nature of the gp120:gp41 complex. Shed gp120 adopts various non-native conformations that may not be present in the native Env, leading to the elicitation of non-neutralizing antibodies. Despite these defense mechanisms, approximately 20-30% of HIV-1 patients manage to generate a broad neutralization response, with about 1% achieving high potency. While the identification of bNAbs and their corresponding epitopes has occurred, the challenge lies in inducting similar bNAbs through immunization. This underscores the complexity of inducing a robust and broadly effective immune response against HIV-1. Monomeric gp120 stands out as a potential immunogen due to its surface accessibility and immunogenicity observed in natural infections. However, initial immunization attempts using full-length monomeric gp120 proved unsuccessful in eliciting neutralizing antibodies and did not demonstrate protective efficacy in a human clinical trial. This suggests a suboptimal presentation of neutralization epitopes on monomeric gp120. Given the importance of inducing neutralizing antibodies for an effective vaccine, there is an urgent need for rational design approaches to direct the immune response towards specific epitopes targeted by known bNAbs. The primary goal of any rational immunogen design methodology for HIV-1 is to enhance the exposure of conserved neutralization epitopes while simultaneously minimizing the exposure of variable, non-neutralizing epitopes to the immune system. This can be achieved by stabilizing the immunogen, which eventually helps to minimize the exposure of non-neutralizing epitopes and focus the immune responses toward the conserved neutralizing epitopes.
Chapter 1 provides a concise overview of the HIV-1 virus and outlines the structural organization of its Env protein, along with a description of difficulties in generating an effective HIV-1 vaccine and discusses recent advances in rational, structure-based HIV-1 vaccine design. It also provides an overview of next-generation sequencing and yeast surface display. The outer domain (OD) of HIV-1 Env glycoprotein gp120 contains the site for the binding of its cellular receptor CD4 and contains epitopes for a large number of recently discovered bNAbs. Therefore, OD is considered to be an important candidate for structure-based vaccine design and for designing minimal gp120 immunogens. We have previously reported the design and characterization of a non-glycosylated, E. coli-expressed outer domain immunogen (ODEC) that bound CD4 and bNAb b12 with micromolar affinity and elicited a modest neutralizing antibody response in rabbits (Bhattacharyya et al., 2010; Rathore et al., 2018).
In Chapter 2, saturation suppressor libraries of OD were generated separately in the background of two destabilizing mutations, which abolished the binding with the conformation-specific antibody VRC01. Then, the library was screened against VRC01 antibody to identify putative stabilizing mutations that help regain binding with VRC01, using yeast surface display (YSD) coupled to fluorescence-activated cell sorting (FACS) and deep sequencing. Mutants obtained after deep sequencing were incorporated into WT OD and then biophysically characterized to confirm that they were stabilizing mutations. We found a couple of mutations that improve OD stability without hampering the binding to different monoclonal broadly neutralizing antibodies. Further incorporation of stabilizing mutations in the background of the previously described cyclically permutated trimeric gp120 derivative (Kesavardhana et al., 2017; Saha et al., 2012), hCMP-V1CycP_JRCSFgp120, improves its stability without hampering binding to different monoclonal antibodies. Next, we screened stabilized OD, WT OD, and hCMP-V1cycP_JRCSFgp120 formulated with five different adjuvants, followed by immunization in guinea pigs. Immunization studies showed that stabilized OD improves ELISA endpoint OD and gp120 titers relative to WT OD. Two adjuvant groups showed higher OD and gp120 titer compared to other adjuvant groups. Sera also neutralized the Tier-2 homologous JRCSF pseudoviruses with ID50 values ranging from 50-500 and showed heterologous Tier-2 neutralization against some subtype-B viruses. Currently, we are studying the neutralization breadth for all the sera from different groups.
In Chapter 3, we used deep mutational scanning to probe residue-specific contributions needed for six-helix bundle (6HB) formation of HIV-1. A yeast surface two-hybrid (YS2H) system was used to reconstitute gp41 6HB on the yeast surface by expressing N36 and C34 peptides derived from the N-terminal heptad repeat (NHR) and C-terminal heptad repeats (CHR) of gp41, which together form the 6HB during viral (HIV) and cellular membrane fusion. We generated separate saturation mutagenesis libraries of each peptide and displayed these on the yeast cell surface, followed by FACS and deep sequencing of the sorted populations. After deep sequencing, we scored the mutants based on their mutational effects on their binding to cognate peptides. The scores elucidate residue-specific contributions needed for six-helix bundle formation and identify residues of each peptide crucial for 6HB formation. We identified a couple of mutations in each peptide that disrupt the formation of 6HB, as well as mutations that enhance the formation of 6HB. This methodology can be applied to other proteins or peptides to elucidate interacting residues where structural information is not available. In Chapter 4, we describe an epitope mapping methodology. A library of single cysteine mutations is introduced at surface-exposed residues in the HIV-1 envelope glycoprotein (Env), the primary target of neutralizing antibodies (Nabs). Mutant viruses are chemically labeled with a bulky Cys-reactive reagent (maleimide-PEG2-biotin). Labeling blocks the binding of NAbs to epitopes but has no effect on non-epitope residues. Epitopes are inferred using assays that monitor viral infectivity in the absence and presence of NAbs, followed by deep sequencing. We successfully applied this technique to distinguish residues at the epitope from residues lying outside the epitope for several NAbs. The methodology is also able to accurately map epitopes for polyclonal NAbs from HIV-1-infected individuals and immunized animals (Datta et al., 2020).
In Chapter 5, we used charge scanning mutagenesis to map epitopes of bNAbs. In previous studies (Chandra et al., 2021, 2023; Das et al., 2020) and Chapter 3, we have seen that charged substitutions were binding defective, exclusively at interacting positions, and were well tolerated at the exposed non-interacting sites. For further validation of the hypothesis and to employ this methodology in epitope mapping, we used YSD to display individual OD variants carrying charged mutations at epitope and non-epitope sites of VRC01 and b12 bNAbs. The binding was checked with the respective bNAbs. Epitope mutants showed no binding to reduced binding, whereas non-epitope mutants did not hamper the binding with the respective bNAbs. Next, we made a library of single-charged mutants of the HIV-1 Env. Individual viruses carrying the charged mutations on the Env surface will be made from the library, and using infectivity assays followed by deep sequencing, the viral library will be used to map epitopes of different polyclonal sera isolated from HIV-1 infected individuals (elite neutralizer). | en_US |
