key: cord-0750443-h7uopq0a authors: Shi, Juan; Zheng, Jian; Zhang, Xiujuan; Tai, Wanbo; Odle, Abby E; Perlman, Stanley; Du, Lanying title: RBD-mRNA vaccine induces broadly neutralizing antibodies against Omicron and multiple other variants and protects mice from SARS-CoV-2 challenge date: 2022-04-28 journal: Transl Res DOI: 10.1016/j.trsl.2022.04.007 sha: 2198b379831f8e4fb92854aa383681ae2bc48da2 doc_id: 750443 cord_uid: h7uopq0a Multiple SARS-CoV-2 variants are identified with higher rates of transmissibility or greater disease severity. Particularly, recent emergence of Omicron variant with rapid human-to-human transmission posts new challenges to the current prevention strategies. In this study, following vaccination with an mRNA vaccine encoding SARS-CoV-2 receptor-binding domain (RBD-mRNA), we detected serum antibodies that neutralized pseudoviruses expressing spike (S) protein harboring single or multiple mutations, as well as authentic SARS-CoV-2 variants, and evaluated its protection against SARS-CoV-2 infection. The vaccine induced durable antibodies that potently neutralized prototypic strain and B.1.1.7 lineage variant pseudoviruses containing N501Y or D614G mutations alone or in combination with a N439K mutation (B.1.258 lineage), with a L452R mutation (B.1.427 or B.1.429 lineage), or a L452R-E484Q double mutation (B.1.617.1 variant), although neutralizing activity against B.1.1.7 lineage variant containing 10 amino acid changes in the S protein was slightly reduced. The RBD-mRNA-induced antibodies exerted moderate neutralization against authentic B.1.617.2 and B.1.1.529 variants, and pseudotyped B.1.351 and P.1 lineage variants containing K417N/T, E484K, and N501Y mutations, the B.1.617.2 lineage variant harboring L452R, T478K, and P681R mutations, and the B.1.1.529 lineage variant containing 38 mutations in the S protein. Particularly, RBD-mRNA vaccine completely protected mice from challenge with a virulent mouse-adapted SARS-CoV-2 variant. Among these lineages, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 belong to Alpha, Beta, Gamma, Delta, and Omicron variants, respectively. Our observations reveal that RBD-mRNA vaccine is promising and highlights the need to design novel vaccines with improved neutralization against current and future pandemic SARS-CoV-2 variants. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), first reported in 2019, 1 causes the global pandemic of Coronavirus Disease 2019 . This pandemic has led to millions of deaths with devastating damages, significantly affecting the public health and global economy. The genome of SARS-CoV-2 encodes four structural proteins, including spike (S), nucleocapsid (N), membrane (M), and envelope (E); among them, the S protein plays an important role in virus infection and pathogenesis. 2, 3 Similar to other coronavirus S proteins, the S protein of SARS-CoV-2 consists of S1 and S2 subunits. 3 During SARS-CoV-2 infection, the virus first binds a cellular receptor, angiotensin converting enzyme 2 (ACE2), through the receptor-binding domain (RBD) in the S1 subunit, followed by virus-cell membrane fusion through the S2 subunit, mediating the virus entry into host cells. 4, 5 Therefore, the S protein, including the RBD, is a critical target for the development of SARS-CoV-2 vaccines and therapeutic antibodies. 4, 6, 7 SARS-CoV-2 has mutated frequently since its first emergence, and increasing numbers of variants have been identified with mutations in the S, M, N, and E structural proteins and several nonstructural proteins. Notably, mutation(s) in the S1 subunit of SARS-CoV-2, including RBD, generally have more significant effects than the non-S mutations. 8 (India) lineages have been efficiently transmitted to other regions and are therefore the variant strains of concern (VOCs) or variants of interest (VOIs). [10] [11] [12] [13] In addition, recent emergence of a new SARS-CoV-2 variant, B.1.1.529 (Omicron), which was first reported in South Africa and spread rapidly to other countries, [14] [15] [16] has posted new challenges to the current prevention strategies. All of these variant strains carry key mutations (substitutions, insertions, or deletions) in the S protein, particularly S1 subunit (N-terminal domain or RBD), including 69del, 70del, S371L, S373P, S375F, K417T/N, N440K, G446S, L452R, S477N, T478K, E484A, E484K, Q493R, Q498R, N501Y, D614G, P681H, and P681R, either alone or in combination (Table 1) [17] [18] [19] [20] [21] [22] The emergence of such mutant SARS-CoV-2 strains decreases the efficacy of current COVID-19 vaccines and therapeutic agents, potentially allowing the virus to evade vaccine-induced immune responses or antibody neutralization. [23] [24] [25] Therefore, development of vaccines with broad-spectrum activity against multiple SARS-CoV-2 strains is critically important to prevent infection by current mutant strains and future variants. In this study, we evaluated the broad-spectrum activity of antibodies induced by an mRNA vaccine encoding SARS-CoV-2 RBD (RBD-mRNA) to neutralize these SARS-CoV-2 variant strains, including B.1.1.529 (Omicron) and B.1.617.2 (Delta) variants. We also demonstrated that this vaccine induced complete protection of mice from challenge of a virulent mouse-adapted SARS-CoV-2. Mouse immunization and serum collection. 6-8-week-old BALB/c mice were intradermally (I.D.) immunized with LNP-encapsulated SARS-CoV-2 RBD-mRNA (10 μg/mouse), or empty LNPs (control), and boosted with the same immunogen at 4 weeks and 8 months, respectively. 28 Sera were collected at 10 days post-last dose, and detected for neutralizing antibodies against pseudotyped SARS-CoV-2 expressing S protein of prototypic strain or each of the mutant variants as described above. Mouse challenge study. 6-8-week-old BALB/c mice were I.D. immunized with SARS-CoV-2 RBD-mRNA or empty LNPs (control) as described above (for mouse immunization), and boosted with the same immunogen at 4 weeks. At 70 days post-2 nd immunization, the vaccinated mice were intranasally challenged with SARS2-N501YMA30 (a virulent mouse-adapted strain of SARS-CoV-2, 5×10 3 PFU/mouse, in 50 l DMEM), and observed for 2 weeks for survival and body weight changes. 27, 29, 30 Mouse sera collected at 10 days post-last immunization or before virus challenge were assessed for their activity to neutralize pseudotyped or authentic SARS-CoV-2 prototypic strain, B.1.1.529, or B.1.617.2 variant. We generated a series of mutant pseudoviruses (Table 1) The results revealed that SARS-CoV-2 RBD-mRNA induced long-term antibodies that potently neutralized infection by pseudovirus expressing S protein of SARS-CoV-2 (prototypic strain), with neutralizing antibody titer reaching ~1:44,000 ( Fig 1A) . These antibodies had similar or higher potency in neutralization of pseudoviruses containing single (N501Y or D614G) (Fig 1B, C) or combined (N501Y-D614G, N439K-D614G, 69-70del-N501Y-D614G, and 69-70del-N439K-D614G) mutations (Fig 1D-G) . By comparison, these antibodies had less neutralizing activity against pseudoviruses containing deletions of amino acids 69 and 70 (69-70del) (Fig 1H) or these deletions in combination with other single mutations (69-70del-N501Y and 69-70del-N439K) (Fig. 1I , J). Although neutralizing activity was slightly reduced against a B.1.1.7 lineage variant containing 10 amino acid changes in the S protein, the neutralizing antibody titer was still greater than 1:10,000 (Fig 1K) . In contrast, the control (empty LNPs) only induced a background level of 7 neutralizing antibodies against the aforementioned pseudoviruses (Fig 1) . These data indicate that SARS-CoV-2 RBD-mRNA vaccine elicited durable neutralizing antibodies against SARS-CoV-2 B.1.1.7 lineage variants harboring single or multiple mutations in the S protein, as well as combinations of these mutations with the B.1.258 lineage variant containing the N439K mutation at the RBD region. -2 B.1.427, B.1.429, B.1.617.1, B.1.351, Table 1 ). The results indicated that RBD-mRNA elicited durable and potent neutralizing antibodies against pseudovirus containing the L452R mutation in the RBD (B.1.427 or B.1.429 lineage), with the antibody titer (1:48,000) ( Fig 2B) higher than that against the prototypic pseudovirus (Fig 2A) . These antibodies neutralized pseudoviruses containing a single substitution (V483A, E484Q, or G485R), or L452R-E484Q double mutations (B.1.617.1 lineage) in the RBD (Fig 2C-F) , with slightly lower neutralizing antibody titers than those against the prototypic pseudovirus. These serum antibodies also neutralized pseudoviruses containing the E484K single mutation (Fig 2G) , or its combination with the K417N/T and N501Y mutations (Fig 2H, I) , albeit the neutralizing antibody titers were slightly lower than the titers of antibodies to neutralize other mutant pseudoviruses. Notably, K417N, particularly K417T, mutation potentially increased resistance to vaccine-induced neutralizing antibodies, with the neutralizing antibody titers against K417N-E484K-N501Y (B.1.351 lineage) and K417T-E484K-N501Y (P.1 lineage) variants were reduced to about 1:10,000 and 1:7,500, respectively (Fig 2H, I) . In addition to human strains, the vaccineinduced antibodies also neutralized a pseudovirus expressing S protein (F486L) of mink SARS-CoV-2, and the neutralizing titer was slightly reduced than the titer of antibodies to neutralize the SARS-CoV-2 prototypic strain (Fig 2J) . In contrast, sera from mice immunized with the control (empty LNPs) only had a background level of neutralizing antibodies against these pseudoviruses (Fig 2) . The above data suggest that neutralizing antibodies induced by RBD-mRNA vaccine (Fig 3A) , albeit the neutralizing antibody titer was ~2 times lower than that against the pseudotyped SARS-CoV-2 prototypic strain ( Fig 3B) . Notably, the RBD-mRNA vaccine-induced antibodies also neutralized pseudotyped B.1.1.529 variant, although its neutralizing antibody titer was ~6 and 3 times lower than the neutralizing antibody titers against the pseudotyped prototypic strain and B.1.617.2 variant, respectively (Fig 3C) . These antibodies were able to neutralize authentic SARS-CoV-2 prototypic, B.1.617.2 and B.1.1.529 variant strains, respectively, with a trend of neutralizing antibody titers similar to that against pseudotyped SARS-CoV-2 (Fig 3D-F) . Sera collected before SARS-CoV-2 challenge also neutralized pseudotyped B.1.617.2 variant, with a reduced neutralizing antibody titer (~3.6 times lower) than that against the prototypic pseudovirus (Fig 4A-B) . In contrast, sera of mice injected with the control (empty LNPs) only elicited a background level of neutralizing antibodies against these pseudotyped and authentic SARS-CoV-2 viruses (Fig 3 and 4A-B) . Data from the challenge studies revealed that 100% of RBD-mRNA-immunized mice survived SARS2-N501YMA30 infection (Fig 4C) , and body weight continuously increased during 14 days after virus infection ( Fig 4D) . In contrast, the control mice receiving empty LNPs significantly lost weight, and 60% of them died at day 8, after virus challenge (Fig 4C-D) . The above data indicate that antibodies induced by RBD-mRNA vaccine were able to neutralize B.1.617.2 and B.1.1.529 lineage variants. Importantly, these neutralizing antibodies were sufficient to protect immunized mice against infection of a virulent mouse-adapted SARS-CoV-2. 49 Overall, the neutralizing antibody titers induced by RBD vaccines are generally lower against these SARS-CoV-2 variants than against the prototypic virus strain. 29, [47] [48] [49] In this study, we found that RBD-mRNA vaccine, which contains the RBD sequence of the prototypic SARS-CoV-2, induced durable neutralizing antibodies that broadly neutralized B. 50 Of note, since SARS2-N501YMA30 contains several mutations found in the Omicron strain, our results suggest that the RBD-mRNA vaccine will have some efficacy against this newly emergent virus strain. Further studies are needed to design novel and effective universal vaccines with improved broadspectrum neutralizing activity and protective efficacy against multiple current pandemic SARS-CoV-2 variants and future variant virus strains which have pandemic potential. In particular, vaccines with strong efficiency against Omicron and other SARS-CoV-2 variants which present high transmissibility and/or infectivity would be a high priority for development. While a 3 rd dose of prototypic BNT162b2 or mRNA-1273 mRNA vaccine may induce moderate neutralizing 11 antibodies against Omicron variant, 51-53 other strategies, such as T-cell-inducing vaccines based on the conserved viral sequences, will have potential to enhance the potency against not only the prototypic strain but also Omicron and other SARS-CoV-2 variants. A pneumonia outbreak associated with a new coronavirus of probable bat origin Subunit vaccines against emerging pathogenic human coronaviruses Neutralizing antibodies for the prevention and treatment of COVID-19 Therapeutic antibodies and fusion inhibitors targeting the spike protein of SARS-CoV-2 Structural basis of receptor recognition by SARS-CoV-2 SARS-CoV-2 spike protein: a key target for eliciting persistent neutralizing antibodies Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2 Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies Estimation of secondary household attack rates for emergent spike L452R SARS-CoV-2 variants detected by genomic surveillance at a community-based testing site in San Francisco Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England The emergence of novel SARS-CoV-2 variant P.1 in Amazonas (Brazil) was temporally associated with a change in the age and sex profile of COVID-19 mortality: A population based ecological study Household transmission of COVID-19 cases associated with SARS-CoV-2 delta variant (B.1.617.2): national case-control study International risk of SARS-CoV-2 Omicron variant importations originating in South Africa SARS-CoV-2 B.1.1.529 (Omicron) variant transmission within households -Four Introduction and rapid spread of SARS-CoV-2 Omicron variant and dynamics of BA.1 and BA.1.1 sublineages CMMID COVID-19 Working Group, et al. Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7 Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant Community transmission and viral load kinetics of the SARS-CoV-2 delta (B.1.617.2) variant in vaccinated and unvaccinated individuals in the UK: a prospective, longitudinal, cohort study Severity of disease among adults hospitalized with laboratory-confirmed COVID-19 before and during the period of SARS-CoV-2 B.1.617.2 (Delta) predominance -COVID-NET, 14 States Epidemiological characterisation of the first 785 SARS-CoV-2 Omicron variant cases in Denmark Investigation of a SARS-CoV-2 B.1.1.529 (Omicron) variant cluster -Nebraska SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Identification of SARS-CoV RBD-targeting monoclonal antibodies with cross-reactive or neutralizing activity against SARS-CoV-2 The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2 Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19 SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion Transmission of Omicron (B.1.1.529) -SARS-CoV-2 variant of concern in a designated quarantine hotel for travelers: a challenge of elimination strategy of COVID-19 BNT162b2 mRNA COVID-19 vaccine: first approval The Advisory Committee on immunization practices' interim recommendation for use of Moderna COVID-19 vaccine -United States Adverse events of active and placebo groups in SARS-CoV-2 vaccine randomized trials: A systematic review Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants Delta virus in serum of mRNA-vaccinated patients receiving Tumor Necrosis Factor-alpha inhibitors BNT162b2 and mRNA-1273 COVID-19 vaccine effectiveness against the SARS-CoV-2 Delta variant in Qatar SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing antibodies elicited by ancestral spike vaccines Potency of BNT162b2 and mRNA-1273 vaccineinduced neutralizing antibodies against severe acute respiratory syndrome-CoV-2 variants of concern: A systematic review of in vitro studies Serum neutralizing activity of mRNA-1273 against SARS-CoV-2 variants Daily briefing: Omicron was already spreading in Europe Probable transmission of SARS-CoV-2 Omicron variant in Quarantine Hotel Omicron is supercharging the COVID vaccine booster debate SARS-CoV-2 receptor binding domain displayed on HBsAg virus-like particles elicits protective immunity in macaques Induction of broadly cross-reactive antibodies by displaying receptor binding domains of SARS-CoV-2 on virus-like particles Kappa-RBD produced by glycoengineered Pichia pastoris elicited high neutralizing antibody titers against pseudoviruses of SARS-CoV-2 variants Organization Health Organization. Tracking SARS-CoV-2 variants Third BNT162b2 vaccination neutralization of SARS-CoV-2 Omicron infection mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant Vaccine booster efficiently inhibits entry of SARS-CoV-2 omicron variant SARS-CoV-2 RBD-mRNA induced neutralizing antibodies against SARS-CoV-2 B.1.351, P.1, B.1.617.1, B.1.427, and B.1.429 lineage variants. Same mouse sera described in Figure 1 were tested for neutralizing antibodies against pseudoviruses expressing SARS-CoV-2 prototypic (wildtype) S protein control (A) E484K (G), and F486L (J), dual mutations of L452R and E484Q (F), or the combinations of E484K and N501Y with K417N/T (H, I) in the S1 subunit of SARS-CoV-2 S protein. NT50 was calculated against each pseudovirus infection in hACE2-293T cells, and the data are presented as mean ± s.e.m of four wells/dilution of pooled sera Experiments were repeated twice, yielding similar results SARS-CoV-2 RBD-mRNA induced neutralizing antibodies against SARS-CoV-2 2 and B.1.1.529 lineage variants. BALB/c mice were immunized with SARS-CoV-2 Sera were collected at 10 days post-2 nd immunization, and tested for neutralizing antibodies against the following SARS-CoV-2: pseudotyped SARS-CoV-2 expressing S protein of B.1.617.2 lineage variant containing L452R, T478K, and P681R mutations in the S1 subunit (A), prototypic (wildtype) virus strain (B), and B.1.1.529 lineage variant containing 38 mutations in the full-length S (C), as well as authentic SARS-CoV-2 B.1.617.2 variant (D), prototypic (wild-type) virus strain (E), and B.1.1.529 variant (F). NT50 was calculated against each pseudovirus or live virus infection, and the data are presented as mean ± s.e.m of 5 mouse sera SARS-CoV-2 RBD-mRNA protected mice against a virulent SARS-CoV-2 challenge BALB/c mice were immunized and boosted with SARS-CoV-2 RBD-mRNA (e.g., RBD) or empty LNPs (control) (as described in Figure 3), and challenged with a virulent mouse-adapted SARS-CoV-2 variant lineage variant (A) and prototypic (wild-type) strain (B). NT50 was calculated against each pseudovirus infection. Experiments were repeated twice, yielding similar results. Mouse survival (C) and body weight changes (D) were observed for 14 days after challenge. The data (in A, B, and D) are presented as mean ± s.e.m This study was supported by NIH grants (R01AI139092, R01AI137472, and R01AI157975). We