A recombinant spike‐XBB.1.5 protein vaccine induces broad‐spectrum immune responses against XBB.1.5‐included Omicron variants of SARS‐CoV‐2

The XBB.1.5 subvariant has drawn great attention owing to its exceptionality in immune evasion and transmissibility. Therefore, it is essential to develop a universally protective coronavirus disease 2019 vaccine against various strains of Omicron, especially XBB.1.5. In this study, we evaluated and compared the immune responses induced by six different spike protein vaccines targeting the ancestral or various Omicron strains of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in mice. We found that spike‐wild‐type immunization induced high titers of neutralizing antibodies (NAbs) against ancestral SARS‐CoV‐2. However, its activity in neutralizing Omicron subvariants decreased sharply as the number of mutations in receptor‐binding domain (RBD) of these viruses increased. Spike‐BA.5, spike‐BF.7, and spike‐BQ.1.1 vaccines induced strong NAbs against BA.5, BF.7, BQ.1, and BQ.1.1 viruses but were poor in protecting against XBB and XBB.1.5, which have more RBD mutations. In sharp contrast, spike‐XBB.1.5 vaccination can activate strong and broadly protective immune responses against XBB.1.5 and other common subvariants of Omicron. By performing correlation analysis, we found that the NAbs titers were negatively correlated with the number of RBD mutations in the Omicron subvariants. Vaccines with more RBD mutations can effectively overcome the immune resistance caused by the accumulation of RBD mutations, making spike‐XBB.1.5 the most promising vaccine candidate against universal Omicron variants.

Heterologous vaccination with subunit protein vaccine induces a superior neutralizing capacity against BA.4/5‐included SARS‐CoV‐2 variants than homologous vaccination of mRNA vaccine

BA.4 and BA.5 (BA.4/5), the subvariants of Omicron, are more transmissible than BA.1 with more robust immune evasion capability because of its unique spike protein mutations. In light of such situation, the vaccination against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is in desperate need of the third booster. It has been reported that heterologous boosters might produce more effective immunity against wild‐type SARS‐CoV‐2 and the variants. Additionally, the third heterologous protein subunit booster should be considered potentially. In the present study, we prepared a Delta full‐length spike protein sequence‐based mRNA vaccine as the "priming” shot and developed a recombinant trimeric receptor‐binding domain (RBD) protein vaccine referred to as RBD–HR/trimer as a third heterologous booster. Compared to the homologous mRNA group, the heterologous group (RBD–HR/trimer vaccine primed with two mRNA vaccines) induced higher neutralizing antibody titers against BA.4/5‐included SARS‐CoV‐2 variants. In addition, heterologous vaccination exhibited stronger cellular immune response and long‐lasting memory response than the homologous mRNA vaccine. In conclusion, a third heterologous boosting with RBD–HR/trimer following two‐dose mRNA priming vaccination should be a superior strategy than a third homologous mRNA vaccine. The RBD–HR/trimer vaccine becomes an appropriate candidate for a booster immune injection. We prepared a Delta full‐length spike protein sequence‐based mRNA vaccine (Figure A, B) and developed a recombinant trimeric receptor‐binding domain (RBD) protein vaccine (Figure C). Later, the mRNA vaccine was injected as the "priming” shot, and the RBD–HR/trimer vaccine was used as a third heterologous booster (Figure D).

Trimeric protein vaccine based on Beta variant elicits robust immune response against BA.4/5-included SARS-CoV-2 Omicron variants.

The current Coronavirus Disease 2019 (COVID-19) pandemic, induced by newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants, posed great threats to global public health security. There is an urgent need to design effective next­generation vaccines against Omicron lineages. Here, we investigated the immunogenic capacity of the vaccine candidate based on the receptor binding domain (RBD). An RBDß-HR self-assembled trimer vaccine including RBD of Beta variant (containing K417, E484 and N501) and heptad repeat (HR) subunits was developed using an insect cell expression platform. Sera obtained from immunized mice effectively blocked RBD-human angiotensin-converting enzyme 2 (hACE2) binding for different viral variants, showing robust inhibitory activity. In addition, RBDß-HR/trimer vaccine durably exhibited high titers of specific binding antibodies and high levels of cross-protective neutralizing antibodies against newly emerging Omicron lineages, as well as other major variants including Alpha, Beta, and Delta. Consistently, the vaccine also promoted a broad and potent cellular immune response involving the participation of T follicular helper (Tfh) cells, germinal center (GC) B cells, activated T cells, effector memory T cells, and central memory T cells, which are critical facets of protective immunity. These results demonstrated that RBDß-HR/trimer vaccine candidates provided an attractive next-generation vaccine strategy against Omicron variants in the global effort to halt the spread of SARS-CoV-2.

Heterologous vaccination with subunit protein vaccine induces a superior neutralizing capacity against BA.4/5-included SARS-CoV-2 variants than homologous vaccination of mRNA vaccine.

BA.4 and BA.5 (BA.4/5), the subvariants of Omicron, are more transmissible than BA.1 with more robust immune evasion capability because of its unique spike protein mutations. In light of such situation, the vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in desperate need of the third booster. It has been reported that heterologous boosters might produce more effective immunity against wild-type SARS-CoV-2 and the variants. Additionally, the third heterologous protein subunit booster should be considered potentially. In the present study, we prepared a Delta full-length spike protein sequence-based mRNA vaccine as the "priming" shot and developed a recombinant trimeric receptor-binding domain (RBD) protein vaccine referred to as RBD-HR/trimer as a third heterologous booster. Compared to the homologous mRNA group, the heterologous group (RBD-HR/trimer vaccine primed with two mRNA vaccines) induced higher neutralizing antibody titers against BA.4/5-included SARS-CoV-2 variants. In addition, heterologous vaccination exhibited stronger cellular immune response and long-lasting memory response than the homologous mRNA vaccine. In conclusion, a third heterologous boosting with RBD-HR/trimer following two-dose mRNA priming vaccination should be a superior strategy than a third homologous mRNA vaccine. The RBD-HR/trimer vaccine becomes an appropriate candidate for a booster immune injection.

A self-assembled trimeric protein vaccine induces protective immunity against Omicron variant.

The recently emerged Omicron (B.1.1.529) variant has rapidly surpassed Delta to become the predominant circulating SARS-CoV-2 variant, given the higher transmissibility rate and immune escape ability, resulting in breakthrough infections in vaccinated individuals. A new generation of SARS-CoV-2 vaccines targeting the Omicron variant are urgently needed. Here, we developed a subunit vaccine named RBD-HR/trimer by directly linking the sequence of RBD derived from the Delta variant (containing L452R and T478K) and HR1 and HR2 in SARS-CoV-2 S2 subunit in a tandem manner, which can self-assemble into a trimer. In multiple animal models, vaccination of RBD-HR/trimer formulated with MF59-like oil-in-water adjuvant elicited sustained humoral immune response with high levels of broad-spectrum neutralizing antibodies against Omicron variants, also inducing a strong T cell immune response in vivo. In addition, our RBD-HR/trimer vaccine showed a strong boosting effect against Omicron variants after two doses of mRNA vaccines, featuring its capacity to be used in a prime-boost regimen. In mice and non-human primates, RBD-HR/trimer vaccination could confer a complete protection against live virus challenge of Omicron and Delta variants. The results qualified RBD-HR/trimer vaccine as a promising next-generation vaccine candidate for prevention of SARS-CoV-2, which deserved further evaluation in clinical trials.

Histones released by NETosis enhance the infectivity of SARS-CoV-2 by bridging the spike protein subunit 2 and sialic acid on host cells.

Neutrophil extracellular traps (NETs) can capture and kill viruses, such as influenza viruses, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV), thus contributing to host defense. Contrary to our expectation, we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2, as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model. The histone H3 or H4 selectively binds to subunit 2 of the spike (S) protein, as shown by a biochemical binding assay, surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids. Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein. Moreover, histones enhance cell-cell fusion. Finally, treatment with an inhibitor of NETosis, histone H3 or H4, or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model. These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.

S19W, T27W, and N330Y mutations in ACE2 enhance SARS-CoV-2 S-RBD binding toward both wild-type and antibody-resistant viruses and its molecular basis.

SARS-CoV-2 recognizes, via its spike receptor-binding domain (S-RBD), human angiotensin-converting enzyme 2 (ACE2) to initiate infection. Ecto-domain protein of ACE2 can therefore function as a decoy. Here we show that mutations of S19W, T27W, and N330Y in ACE2 could individually enhance SARS-CoV-2 S-RBD binding. Y330 could be synergistically combined with either W19 or W27, whereas W19 and W27 are mutually unbeneficial. The structures of SARS-CoV-2 S-RBD bound to the ACE2 mutants reveal that the enhanced binding is mainly contributed by the van der Waals interactions mediated by the aromatic side-chains from W19, W27, and Y330. While Y330 and W19/W27 are distantly located and devoid of any steric interference, W19 and W27 are shown to orient their side-chains toward each other and to cause steric conflicts, explaining their incompatibility. Finally, using pseudotyped SARS-CoV-2 viruses, we demonstrate that these residue substitutions are associated with dramatically improved entry-inhibition efficacy toward both wild-type and antibody-resistant viruses. Taken together, our biochemical and structural data have delineated the basis for the elevated S-RBD binding associated with S19W, T27W, and N330Y mutations in ACE2, paving the way for potential application of these mutants in clinical treatment of COVID-19.

A bivalent recombinant vaccine targeting the S1 protein induces neutralizing antibodies against both SARS-CoV-2 variants and wild-type of the virus.

The emerging variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in pandemic call for the urgent development of universal corona virus disease 2019 (COVID-19) vaccines which could be effective for both wild-type SARS-CoV-2 and mutant strains. In the current study, we formulated protein subunit vaccines with AS03 adjuvant and recombinant proteins of S1 subunit of SARS-CoV-2 (S1-WT) and S1 variant (K417N, E484K, N501Y, and D614G) subunit (S1-Mut), and immunized transgenic mice that express human angiotensin-converting enzyme 2 (hACE2). The S1 protein-specific antibody production and the neutralization capability for SARS-CoV-2 and B.1.351 variant were measured after immunization in mice. The results revealed that the S1-Mut antigens were more effective in inhibiting the receptor-binding domain and ACE2 binding in B.1.351 variant than in wild-type SARS-CoV-2. Furthermore, the development of a bivalent vaccine exhibited the ideal neutralization properties against wild-type and B.1.351 variant, as well as other variants. Our findings may provide a rationale for the development of a bivalent recombinant vaccine targeting the S1 protein that can induce the neutralizing antibodies against both SARS-CoV-2 variants and wild-type of the virus and may be of importance to explore the potential clinical use of bivalent recombinant vaccine in the future.

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells1,2. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.