Design of viral entry inhibitors and vaccines

Abstract

Full text Embargo upto 12/12/2026 Human Immunodeficiency Virus type 1 (HIV-1) remains one of the most persistent global health threats, despite significant efforts in the development of vaccines and major strides in antiretroviral therapy. The envelope glycoprotein (Env) is the major component for the infection to occur and is composed of the surface unit gp120 and the transmembrane unit gp41. While Env has long been recognized as a prime target for both vaccine design and therapeutic inhibition, its high genetic variability, conformational plasticity, and extensive glycosylation make it a challenging candidate for eliciting broad and lasting immune responses. In particular, the gp41 subunit, responsible for membrane fusion during viral entry, represents a structurally conserved region with therapeutic potential that has yet to be fully harnessed. One of the chapters focus on quantitating residue specific interactions in the gp41 fusion machinery of HIV-1. By employing deep mutational scanning strategies, key residues in the N terminal and C-terminal heptad repeat regions were systematically probed to identify mutations that perturb the stability of the six-helix bundle intermediate. These insights can be used to rationally engineer synthetic peptide inhibitors with improved biophysical characteristics, offering avenues for antiviral development against HIV-1 virus. In addition to therapeutic targeting, the thesis explores immunogen design strategies aimed at enhancing the stability, yield, and humoral immune response against HIV-1 Env. Using a ferritin-like nanoparticle scaffold to display trimeric envelope proteins, the immunogenicity of multivalent formulations was assessed in murine models. The nanoparticle presentation increased early antibody titers and improved antigen recognition, although the durability of the response remained limited. These findings underscore the importance of scaffold architecture and immunogen stability in designing next-generation HIV vaccines. The thesis also focuses on vaccine development strategies against other enveloped viruses, such as SARS-CoV-2 and Respiratory Syncytial Virus (RSV). Both viruses rely on surface glycoproteins spike and F protein, respectively, for host cell entry. Stabilization of the prefusion state, has been instrumental in eliciting potent neutralizing responses, but mutations used in these licensed vaccines are protected by intellectual property. Thus, a novel strategy of prefusion stabilization of both the surface glycoproteins of these viruses using aspartic acid substitutions to destabilize the postfusion form was utilised. The strategy was found to stabilize the prefusion conformation, increasing the yields of proteins and in the case of spike, without decreasing immunogenicity and protective efficacy. Finally, the thesis evaluates the use of mRNA-based platforms for the delivery of stabilized viral antigens, which offer rapid and scalable alternatives to traditional vaccine technologies. As part of this work, various mRNA constructs encoding engineered antigens of the Spike protein from SARS-CoV-2 and Hemagglutinin from the Influenza virus were generated and formulated into lipid nanoparticles. The resultant formulations were biophysically characterized, and expression efficiency was evaluated. Immunogenicity and protective efficacy of these mRNA-LNPs were also evaluated in small animals. The data support the feasibility of mRNA delivery systems for tailored vaccine candidates against rapidly evolving pathogens. Collectively, the research done in this thesis contributes to a growing body of knowledge aimed at combating global viral threats. By combining structural virology, rational protein engineering, and emerging delivery technologies, it offers new directions for combating chronic viral infections and strengthening pandemic preparedness.