Immunogen design to enhance Influenza and SARS-CoV-2 vaccine efficacy

Abstract

Influenza and SARS-CoV-2 are pleomorphic viruses that cause respiratory diseases in humans and present significant public health threats. Vaccination has been essential in controlling the spread of these infections and reducing associated morbidity and mortality. However, both the viruses continuously evolve by mutations, enabling them to escape immune recognition. As a result, vaccines must be frequently updated to match with circulating strains and ensure their continued effectiveness. These challenges have driven accelerated efforts to develop broadly protective vaccines. Chapter 1 offers a concise overview of the Influenza and SARS-CoV-2 viruses, focusing on the structure and function of their major surface glycoproteins and the regions targeted by antibodies. It reviews the current vaccine strategies used to combat these respiratory infections, discusses their limitations, and concludes with an exploration of novel approaches and recent efforts to enhance vaccine efficacy. Chapters 2-7 outline strategies to improve the efficacy of influenza vaccines, while Chapters 8-10 detail our efforts in developing a broadly protective SARS-CoV-2 vaccine. Influenza viruses are characterized by two key surface glycoproteins: Hemagglutinin (HA) and Neuraminidase (NA). HA plays a crucial role in receptor binding and viral fusion with the host cell membrane. Due to its critical role in viral entry, HA is the primary target of neutralizing antibodies produced during both infection and vaccination, making it a central focus for vaccine development. Influenza vaccines are typically standardized for HA content and include components from the most common circulating strains, such as Influenza A H1N1, H3N2, and Influenza B Yamagata, Victoria lineages. However, HA is under significant immune pressure, which leads to the emergence of escape mutations, reducing the effectiveness of seasonal vaccines. To improve vaccine efficacy and broaden protection, incorporating more conserved antigens, such as NA and M2e, has been proposed. However, adding these antigens to existing quadrivalent vaccines introduces technical challenges, increases costs, and complicates manufacturing. Chapters 2, 3, and 4 explore potential solutions to these issues. Chapter 2 discusses the design of recombinant HA ectofusions, where the HA ectodomains from Influenza A subtypes H1N1 and H3N2 were genetically fused using a flexible GSA linker. Two variants were created: one with a single foldon trimerization domain at the Cterminus of the H3 HA ectodomain, and another with two foldon trimerization domains—one at the C-terminus of each H1 and H3 ectodomain. The resulting immunogens were expressed in mammalian cells, biophysically characterized, and shown to be well-folded. The immunogens demonstrated high immunogenicity and protective efficacy against both H1N1 and H3N2 viruses, comparable to an equimolar mixture of the H1 and H3 trimeric ectodomains. Chapter 3 focuses on the design of recombinant HA ectofusions for influenza B, where the HA ectodomains from the Yamagata and Victoria lineages were genetically fused. The immunogens were expressed and characterized, with both ectofusions providing superior protection against the Victoria virus challenge compared to an equimolar mixture of the HV and HY ectodomains. Notably, the ectofusion containing a single foldon trimerization domain provided protection similar to the equimolar mixture of influenza B HA ectodomains, while the ectofusion with two foldons demonstrated reduced efficacy. While the results from Chapters 2 and 3 demonstrate that recombinant HA ectofusions are a promising strategy to reduce the number of HA components needed in seasonal influenza vaccines, there are several limitations. These designed HA ectofusions would still only provide strain-specific immunity, requiring annual updates. If only one HA strain needs to be updated, the entire ectofusion would needed to be re-synthesized/cloned, expressed, and characterized— a process that is both time-consuming and labour-intensive. Furthermore, the yields of these ectofusions are lower than those of individual ectodomains, which would lead to higher manufacturing costs. To address these challenges, Chapter 4 introduces an alternative strategy where different HA subtypes are co-displayed using de novo-designed coiled-coil oligomeric sequences. We engineered two previously reported helical hairpin designs that form a trimers of dimers, with monomeric helix of the helical hairpin (inner and outer helices) connected by short linking loops. The H1 and H3 HA ectodomains were genetically fused to the inner and outer helices, respectively, with the connecting loop truncated. When co-expressed in mammalian cells, the engineered H1 and H3 constructs formed a stable dodecameric complex in solution. This H1H3 5IZS dodecameric complex was immunogenic and provided protection comparable to the H1H3 cocktail. Additionally, the designed HA constructs were expressed in good yields (~ 40 mg/L for the HA-outer helix and ~ 25 mg/L for the HA-inner helix), demonstrating that this approach successfully assembles different HA ectodomains into a heterodimeric complex through in vitro assembly. This strategy also offers the potential for integrating other conserved antigens into vaccine formulations, potentially enhancing vaccine efficacy. Foldon is widely used as a trimerization domain to present antigens in their native trimeric form, which is essential for inducing strong immune responses. However, a drawback of foldon is that it can provoke an immune response against the foldon domain itself. To overcome this, our lab has recently developed a disulfide-linked oligomerization domain. Chapter 5 explores the effects of this novel disulfide-linked oligomerization domain on the expression, biophysical properties, immunogenicity, and protective efficacy of various HA ectodomains. The results revealed that, compared to foldon, the disulfide-linked oligomerization domain improved expression levels and facilitated the formation of more stable, higher-order oligomers in solution. However, the immunogenicity of the engineered HA constructs was subtype-specific. The H1 and HY constructs exhibited enhanced protective efficacy, whereas the H3 and HV constructs were less protective than their foldon counterparts. Neuraminidase (NA) is the second most abundant surface glycoprotein on the Influenza virus. Titers of NA-specific antibody that inhibit its enzymatic activity, measured by neuraminidase inhibition (NAI) titers, are well-established correlates of protection against influenza. Studies have shown that including NA in seasonal influenza vaccines can improve efficacy, especially when there is a mismatch between the vaccine strain and the circulating virus, as NA is suggested to be less prone to antigenic drift than hemagglutinin (HA). Chapters 6 and 7 describe our efforts in designing recombinant NA immunogens. One major challenge with recombinant NA immunogens is their low yield and instability. To address these issues, Chapter 6 explores two strategies: site saturation mutagenesis and computational prediction tools to identify mutants with enhanced stability. Using a site saturation library of N1 NA head (NNK library), we found that yeast surface display (YSD) was not suitable for screening the N1 NA library. Hence, we used mammalian cell surface display and demonstrated that site-specific integration of single mutants into the mammalian cell genome, using a mammalian landing pad approach, is a more effective platform for screening NA mutant libraries. FACS screening of the initial library revealed an enrichment of mutants with higher binding affinity than the wild-type NA. However, further identification and in vitro testing of individual mutants is required to assess their expression levels and immunogenicity. Additionally, using Large Language Models (LLM) and ThermoMPNN, we identified two mutants with significantly improved expression. One of these mutants also exhibited enhanced protective efficacy in mice compared to the wild-type N1 NA head mutants. Chapter 7 focuses on the design of N2 and NB NA immunogens. We explored various combinations of signal sequences and tetramerization domains to identify the optimal combination for achieving high expression of active, tetrameric NA. Building on the results from the N1 NA study (Chapter 6), which showed that tetrameric NA elicits a potent immune response, we determined that both N2 and NB NA immunogens, when paired with an Ig signal sequence and the Tetrabrachion (TB) tetramerization domain, demonstrated good expression in mammalian cells and formed stable, active tetramers in solution. Immunization studies are currently ongoing. Chapters 8, 9, and 10 detail our efforts to develop vaccine candidates targeting SARS-CoV- 2 and its variants of concern (VOCs). The SARS-CoV-2 was responsible for the respiratory disease outbreak that triggered the COVID-19 pandemic, which began in early 2020 and lasted until 2023. However, the virus continues to circulate endemically in human populations. During this time, several variants of SARS-CoV-2 emerged, each accumulating mutations— especially in the spike protein—that enhanced transmissibility and allowed the virus to partially evade immune responses induced by previous infection or vaccination. Thus, we tried to develop broadly protective vaccine candidates against ancestral SARS-CoV-2 and its variants. Chapter 8 explores the role of the conserved S2 subunit of the SARS-CoV-2 spike protein in providing broader protection against SARS-CoV-2 variants. We developed a stabilized S2 immunogen, as well as two genetically fused immunogens combining stabilized Receptor Binding Domain (RBD) and S2. These immunogens were expressed, characterized, and evaluated for their immunogenicity and protective efficacy in various animal models. The results demonstrated that mice immunized with the stabilized S2 immunogen were protected against the Beta variant, despite lacking neutralizing activity. This suggested the involvement of non-neutralizing immune mechanisms in protection. The genetically fused RBD and S2 (RS2/S2R) immunogen exhibited significantly higher immunogenicity compared to RBD or S2 alone and was able to neutralize a wide range of VOCs, including clade 1a pseudoviruses. Importantly, the RS2 immunogen was more immunogenic than the stabilized full-length spike protein in hamsters, providing superior protective efficacy against the heterologous Beta variant. Moreover, the RS2 immunogen demonstrated exceptional stability. Lyophilized RS2 remained stable at temperatures up of to 90 °C for at least 60 minutes and could be stored at 37 °C for one month without any loss of antigenicity, immunogenicity, or protective efficacy. Both RS2 and S2R achieved impressive yields of approximately 800-900 mg/L in mammalian cells—about 5.5 times higher than the yield of stabilized spike protein—highlighting their potential for large-scale vaccine production. Chapter 9 presents the design and evaluation of several RS2 derivatives—trimeric RS2, extended RS2, and Receptor binding motif (RBM)-deleted RS2—developed to improve immunogenicity. We fused a previously designed disulfide-linked trimerization domain to either the N- or C-terminus of RS2 and investigated its effects on expression levels, biophysical characteristics, and immunogenic potential. The results showed that placing the trimerization domain at the N-terminus led to the formation of homogeneous trimers in solution, whereas the C-terminal fusion resulted in a heterogeneous mixture of monomers and trimers. Although trimerization or oligomerization is often expected to enhance immunogenicity, the trimeric RS2 demonstrated immunogenicity and protective efficacy comparable to the wild-type (WT) at the tested dose. In the initial RS2 design, we included only heptad repeat 1 (HR1) and the connecting domain of the S2 region, as the structures of HR2 and the surrounding regions were not yet resolved. To investigate the role of HR2 in enhancing immunogenicity, we developed an extended RS2 variant containing full-length S2 region. This modified RS2 induced higher levels of S2-specific antibodies and elicited slightly improved neutralization against Omicron subvariants. Additionally, the RBM-deleted RS2 was engineered to focus the immune response on a more conserved region of the RBD. While it displayed similar biophysical properties and immunogenicity to the wild-type RS2, further animal studies are needed to fully assess its potential. In the context of S2, most neutralizing antibodies target the S2 stem helix of the S2 subunit, a highly conserved epitope across all SARS-CoV-2 variants as well as other alpha and beta coronaviruses. In Chapter 10, we investigated the potential of this S2 stem helix as a key component for designing a broadly protective vaccine. Due to the small size of the S2 stem helix, we fused it with our previously reported stabilized RBD to develop both monomeric and trimeric, 30R and 31R immunogens. These immunogens were expressed in mammalian cells and extensively characterized. Although they exhibited reduced neutralization against SARSCoV- 2, mice immunized with 31R showed improved protection compared to those immunized with stabilized RBD against mouse-adapted SARS-CoV-2. Surprisingly, repeated heterologous immunization with spike and 30R induced robust neutralization against the XBB.1.5 Omicron pseudovirus and clade 3 pseudovirus (Lyra3). However, the precise reason for the observed broad neutralization—whether it is due to the priming antigen or the extended interval between priming and boosting—remains unclear and is under further investigation. Nonetheless, these findings support the notion that incorporating the S2 stem helix or the S2 subunit can significantly enhance both the immunogenicity and protective efficacy of vaccine candidates