key: cord-0823049-822h06el authors: Lee, I-Jung; Sun, Cheng-Pu; Wu, Ping-Yi; Lan, Yu-Hua; Wang, I-Hsuan; Liu, Wen-Chun; Tseng, Sheng-Che; Tsung, Szu-I; Chou, Yu-Chi; Kumari, Monika; Chang, Yu-Wei; Chen, Hui-Feng; Lin, Yin-Shiou; Chen, Tsung-Yen; Chiu, Chi-Wen; Hsieh, Chung-Hsuan; Chuang, Cheng-Ying; Lin, Chih-Chao; Cheng, Chao-Min; Lin, Hsiu-Ting; Chen, Wan-Yu; Chiang, Po-Cheng; Lee, Chong-Chou; Liao, James C.; Wu, Han-Chung; Tao, Mi-Hua title: Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants date: 2022-01-31 journal: bioRxiv DOI: 10.1101/2022.01.31.478406 sha: 6a1b2dbb26e2f0ae7c35970ee91cc581fd2cb4e4 doc_id: 823049 cord_uid: 822h06el The emerging SARS-CoV-2 variants of concern (VOC) harbor mutations associated with increasing transmission and immune escape, hence undermine the effectiveness of current COVID-19 vaccines. In late November of 2021, the Omicron (B.1.1.529) variant was identified in South Africa and rapidly spread across the globe. It was shown to exhibit significant resistance to neutralization by serum not only from convalescent patients, but also from individuals receiving currently used COVID-19 vaccines with multiple booster shots. Therefore, there is an urgent need to develop next generation vaccines against VOCs like Omicron. In this study, we develop a panel of mRNA-LNP-based vaccines using the receptor binding domain (RBD) of Omicron and Delta variants, which are dominant in the current wave of COVID-19. In addition to the Omicron- and Delta-specific vaccines, the panel also includes a “Hybrid” vaccine that uses the RBD containing all 16 point-mutations shown in Omicron and Delta RBD, as well as a bivalent vaccine composed of both Omicron and Delta RBD-LNP in half dose. Interestingly, both Omicron-specific and Hybrid RBD-LNP elicited extremely high titer of neutralizing antibody against Omicron itself, but few to none neutralizing antibody against other SARS-CoV-2 variants. The bivalent RBD-LNP, on the other hand, generated antibody with broadly neutralizing activity against the wild-type virus and all variants. Surprisingly, similar cross-protection was also shown by the Delta-specific RBD-LNP. Taken together, our data demonstrated that Omicron-specific mRNA vaccine can induce potent neutralizing antibody response against Omicron, but the inclusion of epitopes from other variants may be required for eliciting cross-protection. This study would lay a foundation for rational development of the next generation vaccines against SARS-CoV-2 VOCs. However, the pandemic is still far from over due to the constant emergence of new SARS-CoV-2 variants 51 of concern (VOC) 2 . Among the earlier identified VOCs, B.1.351 (Beta) exhibited the greatest immune 52 escape against convalescent sera obtained from COVID-19 patients or vaccinated individuals 3,4 . The 53 B.1.617.2 (Delta) variant that emerged in early December, 2020, quickly outpaced all other circulating 54 isolates and showed a significant reduction in vaccine effectiveness. Importantly, acquisition of favorable 55 mutations in Delta strain enhances transmissibility among individuals and leads to more severe outcomes 56 5,6 . In late November 2021, the B.1.1.529 (Omicron) variant was first discovered and rapidly spread 57 globally. This variant contains novel genomic sequence changes different from any of the previously 58 defined ancestral or VOC isolates of SARS-CoV-2, including 37 mutations in the spike protein, 15 of 59 which are located in the RBD 7 . Recent studies have shown that an increase in the number and complexity 60 of spike mutations leads to inability of therapeutic monoclonal antibodies against Omicron strain 8 . 61 Furthermore, constellation mutations render Omicron more antigenically distant from ancestral viruses or 62 other VOCs, leading to reduced antibody neutralizing activity from vaccination or natural infection 7,9 . 63 Although the disease symptoms induced by Omicron variant are milder than that of Delta 10 , higher 64 transmission rates may inevitably lead to an increase in case numbers and pose a threat on the public 65 health and economics of society. Therefore, there is an urgent need to develop a new generation of 66 vaccines to prevent from VOCs pandemic. 67 In this study, we developed monovalent receptor-binding domain (RBD)-based mRNA vaccines 69 targeting on two currently major predominant VOCs, Omicron and Delta. We also tested the concept of 70 bivalent vaccines containing both Delta and Omicron RBD, and a Hybrid vaccine, which combined the 71 mutation sites of Delta and Omicron in single RBD construct since multivalent vaccines containing 72 various SARS-CoV-2 VOC antigens are recommended to effectively control the spread of SARS-CoV-2 73 variants according to the recommendations of the WHO Technical Advisory Group on COVID-19 samples from the Omicron vaccinated mice can effectively neutralize Omicron, but not the wild-type 76 (D614G), or other VOCs (Beta and Delta) of SARS-CoV-2. In contrast, the Omicron/Delta bivalent 77 mRNA vaccine elicited broadly cross-reactive neutralizing antibodies, effectively neutralizing Omicron 78 and other VOCs. Taken together, our data demonstrate that a new generation of multivalent COVID-19 79 mRNA vaccine is a viable approach to prevent infection from ancestral or VOCs of SARS-CoV-2. 80 81 To cope with the emergence of new variants, 4 different mRNA vaccines were designed to encode 84 the SARS-CoV-2 spike receptor-binding domain (RBD) region of wild-type (WT, Wuhan strain), Delta, 85 Omicron, and Omicron with additional L452R mutation (named Hybrid), respectively. The RNA 86 constructs with mutation sites were summarized in figure 1A . The in vitro transcription reaction was used 87 to synthesize mRNA and the fragment analysis was conducted to analyze RNA integrity. Four synthesized 88 RNA had expected length (around 1000nt) and showed great integrity with 93% or 94% of intact RNA 89 and only limited amounts of degraded transcripts ( Fig 1B and Sup 1). The mRNA was then transfected to 90 293T cells for RBD expression examination. Two days post transfection, supernatants were collected and 91 cocultured with 293T cells that stably expressed human angiotensin-converting enzyme 2 (293T-hACE2) 92 to conduct binding assay. The bound RBD was then detected by polyclonal anti-RBD antibodies. All WT, 93 Delta, Omicron, and Hybrid RBD mRNA efficiently expressed RBD with around 99% of cells stained 94 positive in each construct. Also, we assessed the ability of expressed RBD to bind mouse ACE2 as 95 previous study had shown that RBD of Omicron variant gained the ability to bind mouse ACE2 7 . In this 96 case, 3T3 cells that stably expressed mouse ACE2 were used. In contrast to WT and Delta RBD, which 97 showed no binding capacity against mouse ACE2, RBD from Omicron and Hybrid mRNA transfected showed that the average size of these LNP ranged between 86 nm and 99 nm with a narrow distribution 103 (pdI around 0.121 to 0.147). The zeta potential was about 6 -9 mV (Sup 2). We also examined the RBD 104 expression capacity of these RBD-LNP by transfection into 293T cells. Two days post transfection, 105 supernatants were collected and cocultured with 293T-hACE2 cells to conduct binding assay. All 5 106 RBD-LNP vaccine efficiently expressed RBD with around 99% of cells stained positive in each group 107 ( Fig 1D) . Recently, several studies analyzing the sera from vaccinated or convalescent subjects revealed that 135 the major antigenic shift of Omicron variant lead to immune evasion 7,11-13 . By using vaccinated mouse 136 model which provided an identical genetic background and immune profile, we fairly assessed the 137 neutralizing antibody response induced by various RBD mRNA-LNP vaccines. Our data showed that WT 138 vaccine can induce high neutralization titers against D614G, Beta, and Delta variants (Fig 2A) , but only 139 caused a marginal effect (7.8% of D614G) to Omicron variant (Fig 2A) . The loss of WT 140 vaccine-mediated immunity against Omicron may be due to the loss of epitopes critical for neutralizing 141 antibody recognition since it has been reported that mutations on Omicron variant such as K417N, G446S, 142 E484A, and Q493R impaired a large panel of monoclonal antibodies under commercial development 143 7,8,14,15 . In addition, a cross-variant protection of WT vaccine against Omicron still existed (Fig 2A) , 144 indicating that certain conserved epitopes shared by WT and Omicron may confer neutralization effect, 145 despite low immunogenic and limited protection to Omicron. However, we do not know whether 146 vaccines based on ancestral SARS-CoV-2 strain. To answer this question, immunoanalysis by using mice received heterologous WT/Omicron prime-boost vaccination is needed. 149 To efficiently prevent Omicron pandemic, we generated an Omicron-specific vaccine which can 151 elicit extremely high neutralizing antibody titers against Omicron itself but failed to neutralize other 152 SARS-CoV-2 variants (Fig 2C) . However, at present, Delta is still another dominant variant associated 153 with more severe illness, making up 28% of all cases as of 22nd Jan (COVID-19 Weekly Epidemiological 154 Update, 75th edition). To simultaneously prevent spread of Delta and Omicron, we designed a bivalent 155 vaccine which contained both RBD-LNPs in half dose. Our data showed that combinatorial vaccination 156 generated broadly neutralizing activity (Fig 2E) . Although the neutralizing activity elicited by bivalent 157 vaccine was lower than that generated by the full dose Omicron vaccine (Fig 2C) or Hybrid vaccine (Fig 158 2D ), perhaps because of Omicron RBD dose halved, bivalent vaccine is still a potent strategy to increase 159 the breadth and potency of vaccine. In the future, different Delta/Omicron RBD-LNP ratios can be tested 160 for improvement of vaccine effectiveness. To our surprise, monovalent Delta RBD-LNP also showed 161 cross-strains immunity against Omicron (Fig 2B) . An Updated Review of SARS-CoV-2 Vaccines and the Importance of 274 Effective Vaccination Programs in Pandemic Times Evolution of viruses and the 277 emergence of SARS-CoV-2 variants Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma. 279 Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies. 281 Effectiveness of Covid-19 Vaccines against the B.1.617.2 (Delta) Variant. N 283 SARS-CoV-2 Delta VOC in Scotland: demographics, risk of hospital admission, 285 and vaccine effectiveness Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic 287 shift An infectious SARS-CoV-2 B.1.1.529 Omicron virus escapes 289 neutralization by therapeutic monoclonal antibodies SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 292 elicited neutralization and requires ACE2 for infection. medRxiv Reduced neutralisation of SARS-CoV-2 omicron B.1.1.529 variant by 295 post-immunisation serum Activity of convalescent and vaccine serum against SARS-CoV-2 Omicron Antibodies elicited by SARS-CoV-2 infection or mRNA vaccines have 299 reduced neutralizing activity against Beta and Omicron pseudoviruses Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from 304 neutralizing antibody responses Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies Limited cross-variant immunity after infection with the SARS-CoV-2 308 Omicron variant without vaccination. medRxiv The authors declare no conflict of interest. 271