Design and Immunogenicity of Stabilized HIV-1 Modified Messenger RNA Vaccines

Despite decades of research, a safe and effective vaccine for Human Immunodeficiency Virus-1 (HIV-1) remains elusive. The HIV-1 envelope (Env) glycoprotein expressed on virion surface is the sole target for neutralizing antibodies. HIV-1 sequences are diverse and most neutralizing antibodies are strain-specific. However, broadly neutralizing antibodies (bnAbs) have been isolated from people living with HIV (PLWH). These bnAbs target conserved regions on the Env protein and neutralize heterologous HIV-1 isolates. Thus, inducing bnAbs is a current major goal of HIV-1 vaccine development.

Nucleoside modified, purified mRNA in lipid nanoparticles (mRNA–LNP) has emerged as a novel vaccine platform. The success of using mRNA–LNP forvaccines was demonstrated first by an experimental Zika vaccine and then by two clinically approved COVID-19 vaccines. mRNA vaccine is safe and effective, and is easier to manufacture. The use of mRNA–LNP for HIV-1 vaccine has not been explored.

This thesis focuses on designs of HIV-1 vaccine immunogens encoded by modified mRNA and the investigation of their antigenicity and immunogenicityin animal models. In the first study (Chapter 3), I demonstrated that mRNA can encode immunogenic Env trimers in the forms of full-length membrane-bound gp160 (MBEnv) or Env–ferritin nanoparticles (Env–NP). I tested mRNAs encoding HIV-1 Env forms with various stabilizing mutations and demonstrated in vitro that mRNA-encoded Env forms were well-folded in native-like conformation when optimal stabilizing mutations were included. Following this, I studied the immunogenicity of optimally stablilized MBEnv and Env–NP encoded by mRNA in a bnAb precursor knock-in mouse model. I demonstrated that mRNA immunization initiated bnAb precursor B cell expansion and induced serum autologous tier-2 neutralizing activities. Next-generation sequencing demonstrated the acquisition of critical bnAb mutations and monoclonal antibodies that neutralized heterologous HIV-1 isolates were isolated. In summary, mRNA–LNP can encode complex HIV-1 immunogens and may be of use in design of HIV-1 vaccines.

In the second study (Chapter 4), I studied the top Env–NP mRNA candidate from the first study in rhesus macaques in comparison with the same immunogen in the form of purified recombinant protein. I demonstrated that purified protein Env–NP induced higher binding and neutralizing antibody responses than mRNA did in rhesus macaques. By immunizing bnAb precursor KI mice at a wide dose range, I demonstrated that this mRNA was immunogenic at 20 µg only and was minimally immunogenic at 1 µg dose. In an effort to improve this mRNA vaccine candidate, I demonstrated that mRNA encoding MBEnv was more immunogenic than mRNA encoding Env–NP. Immunohistochemistry staining of mouse inguinal lymph node (vaccine draining) sections showed that mRNA encoding MBEnv expressed higher levels of Env antigens compared to mRNA encoding Env–NP.

Taken together, these two studies demonstrated the importance of stabilizing mutations in mRNA-encoded Env designs and the significance of evaluationof the immunogenicity of mRNA vaccine candidates at proper doses in animals before moving to next steps.