A variant-proof SARS-CoV-2 vaccine targeting HR1 domain in S2 subunit of spike protein

The ongoing COVID-19 pandemic, fueled by the emergence of SARS-CoV-2 variants of concern (VOCs), particularly Omicron sublineages, poses significant challenges. These variants exhibit strong immune evasion and high transmission rates, weakening the efficacy of existing vaccines and antibody therapies. The development of variant-proof, or even pan-sarbecovirus, vaccines is crucial. SARS-CoV-2 entry into host cells is a primary target for vaccine development. The spike (S) protein, a key component of the virus, facilitates this entry. The S1 subunit binds to the host receptor angiotensin-converting enzyme 2 (ACE2), while the S2 subunit drives membrane fusion, releasing the viral genome into the host cell. The S1 subunit, especially its receptor-binding domain (RBD) and N-terminal domain (NTD), is a major target for vaccine design, eliciting neutralizing antibodies (nAbs). However, the high mutation rate in S1, driven by selective pressure from the host immune system, compromises the effectiveness of existing vaccines against emerging variants, leading to breakthrough infections. In contrast, the S2 subunit, more conserved among coronaviruses, induces fewer nAbs and has been less explored for vaccine development. The S2 subunit comprises two highly conserved domains: heptad repeat 1 (HR1) and heptad repeat 2 (HR2). According to the membrane fusion model for class I enveloped viruses, including coronaviruses, HR1 and HR2 are crucial for the fusion process. These domains are transiently exposed in the fusion intermediate conformation. Antibodies targeting this intermediate conformation could disrupt the fusion process, demonstrating broad-spectrum antiviral activity. However, this fusion intermediate is highly unstable, posing challenges for vaccine design. Previous attempts to create immunogens mimicking the fusion intermediate conformation in HIV-1 and influenza viruses resulted in weak nAb responses. This study aims to overcome these challenges by designing a novel recombinant protein, HR121, that effectively mimics the HR1 trimeric inner core conformation of the S2 subunit fusion intermediate. The goal is to develop a variant-proof vaccine that elicits potent, broadly neutralizing antibodies against diverse SARS-CoV-2 variants.

Existing literature highlights the challenges posed by SARS-CoV-2 variants and the need for variant-proof vaccines. Many studies focus on the S1 subunit, particularly the RBD, as the primary target for vaccine design. The success of vaccines targeting RBD demonstrates the efficacy of this approach. However, the high mutation rate in S1 necessitates the exploration of alternative targets. Previous studies have explored using the conserved HR1 and HR2 domains in the S2 subunit, though attempts to create stable immunogens mimicking the transient fusion intermediate conformation have been largely unsuccessful. Studies on HIV-1 and influenza have shown that mimicking the fusion intermediate is difficult and often results in weak neutralizing antibody responses. The development of vaccines targeting conserved regions, such as the S2 subunit, holds promise in overcoming the limitations of existing vaccines. Some research has explored using RBD-HR fusion proteins as vaccine candidates. This research provides a foundation for the current study's approach to target the conserved HR1 domain in the S2 subunit while addressing the challenges of the transient nature of the fusion intermediate.

The study involved designing and characterizing a recombinant protein, HR121, derived from the SARS-CoV-2 S2 subunit. HR121 consists of HR1-linker1-HR2-linker2-HR1 sequences, designed to mimic the HR1 trimeric inner core of the fusion intermediate conformation. The HR121 protein was expressed and purified from *E. coli*. Its structure was determined using X-ray crystallography, revealing a dimeric, asymmetric 6-HB conformation. Functional characterization was performed using GST pull-down assays and surface plasmon resonance (SPR), confirming the selective binding of HR121 to HR2, demonstrating its functional analogy to the fusion intermediate. Immunogenicity was evaluated by immunizing rabbits, BALB/c mice, hACE2 transgenic mice, Syrian golden hamsters, and rhesus macaques with HR121 formulated with different adjuvants (Freund's adjuvant and aluminum adjuvant). Antibody responses were assessed using ELISA, measuring both binding antibodies (bAbs) and neutralizing antibodies (nAbs). Neutralization assays used both VSV pseudotyped SARS-CoV-2 and authentic SARS-CoV-2 viruses, evaluating the breadth of neutralization against various variants, including Omicron sublineages. In vivo protection studies challenged the immunized animals with SARS-CoV-2 or Omicron BA.2, assessing viral loads in lung tissues via qPCR and evaluating histopathological changes via H&E staining and immunohistochemistry. ELISpot assays measured cellular immune responses (IFNγ production). Statistical analyses included one-way ANOVA, two-tailed Mann-Whitney test, and Wilcoxon test, as appropriate.

HR121, a recombinant protein designed to mimic the HR1 domain in the SARS-CoV-2 S2 subunit fusion intermediate, induced potent cross-neutralizing antibodies in various animal models. In rabbits, HR121 elicited high titers of nAbs with broad neutralization against a panel of SARS-CoV-2 variants, including Omicron sublineages (NT50s ranging from 1.8 x 10³ to 3.6 x 10⁴). The neutralizing activity was mainly attributed to the inhibition of HR2 binding to the HR1 trimer, based on competitive ELISA results. In BALB/c mice, HR121 induced prolonged humoral and cellular immune responses, with sustained antibody titers and HR1-specific IFNγ production three months post-immunization. HR121 vaccination provided near-full protection against SARS-CoV-2 infection in hACE2 transgenic mice and long-term protection in Syrian golden hamsters, with significantly reduced viral loads and minimal lung pathology. In rhesus macaques, HR121 elicited potent and homogeneous nAbs against a wide range of SARS-CoV-2 variants, including Omicron sublineages, leading to nearly full protection against SARS-CoV-2 infection. The study also showed that HR121, formulated with either Freund's or aluminum adjuvant, induced similar levels of protection against Omicron BA.2 infection in hamsters. The protective efficacy observed in these animal models correlates strongly with the high titers of neutralizing antibodies elicited by the HR121 vaccine.

The findings demonstrate that HR121, targeting the conserved HR1 domain in the S2 subunit, is a promising candidate for a variant-proof SARS-CoV-2 vaccine. Unlike existing vaccines that primarily target the highly mutable S1 subunit, HR121 focuses on a conserved region crucial for viral entry. The high titers of broadly neutralizing antibodies induced by HR121 in multiple animal models translate into robust protection against both ancestral and variant strains, including Omicron. The success of HR121 in eliciting neutralizing antibodies, in contrast to previous attempts with fusion intermediate mimetics from other viruses, might be attributed to factors like differences in antigenic epitopes, steric hindrance in the fusion intermediates, and full-length HR1 and HR2 sequences. The results highlight the importance of targeting the fusion mechanism for vaccine development. This approach offers a potential solution to the challenge of continuous SARS-CoV-2 evolution, as it targets a conserved region less prone to mutation. The observed lack of antibody-dependent enhancement of infection further strengthens the potential of HR121 as a safe and effective vaccine.

This study demonstrates the potential of HR121 as a promising variant-proof SARS-CoV-2 vaccine. HR121 successfully mimics the HR1 domain's fusion intermediate conformation, inducing potent and broadly neutralizing antibodies across various animal models, leading to significant protection against infection. The conserved nature of the target region suggests the potential for long-lasting immunity against currently circulating and future SARS-CoV-2 variants. Future studies should focus on further optimizing the HR121 vaccine, including preclinical and clinical trials, and exploring its potential application in other coronavirus vaccine platforms.

The study has several limitations. The structure of the HR121/HR2 complex and HR121/antibody complex remains unsolved, hindering a precise identification of the neutralizing epitopes. The relatively small sample sizes in some animal studies could limit the generalizability of the findings. The lack of measurement for anamnestic nAb titers post-infection, due to near-full protection in the studies, should be addressed in future studies. While humoral immunity was extensively investigated, the role of cellular immune responses, especially CD4+ and CD8+ T cells, needs more thorough investigation.