key: cord-0817198-kvh5q7i2 authors: Jia, Liqiu; Zhou, Yang; Li, Shaoshuai; Zhang, Yifan; Yan, Dongmei; Wang, Wanhai; Zhang, Wenhong; Wan, Yanmin; Qiu, Chao title: Omicron booster in ancestral strain vaccinated mice augments protective immunities against both the Delta and Omicron variants date: 2022-02-23 journal: bioRxiv DOI: 10.1101/2022.02.19.481110 sha: 31995ef13cf7ac1fee09a45feb434da028eba85b doc_id: 817198 cord_uid: kvh5q7i2 A booster vaccination is called for constraining the evolving epidemic of SARS-CoV-2. However, the necessity of a new COVID-19 vaccine is currently unclear. To compare the effect of an Omicron-matched S DNA vaccine and an ancestral S DNA vaccine in boosting cross-reactive immunities, we firstly immunized mice with two-dose of a DNA vaccine encoding the spike protein of the ancestral Wuhan strain. Then the mice were boosted with DNA vaccines encoding spike proteins of either the Wuhan strain or the Omicron variant. Specific antibody and T cell responses were measured at 4 weeks post boost. Our data showed that the Omicron-matched vaccine efficiently boosted RBD binding antibody and neutralizing antibody responses against both the Delta and the Omicron variants. Of note, antibody responses against the Omicron variant elicited by the Omicron-matched vaccine were much stronger than those induced by the ancestral S DNA vaccine. Meanwhile, CD8+ T cell responses against both the ancestral Wuhan strain and the Omicron strain also tended to be higher in mice boosted by the Omicron-matched vaccine than those in mice boosted with the ancestral S DNA vaccine, albeit no significant difference was observed. Our findings suggest that an Omicron-matched vaccine is preferred for boosting cross-reactive immunities. The highly mutated SARS-CoV-2 Omicron (B.1.1.529) variant has been shown to substantially evade the neutralizing antibody responses elicited by current vaccines and early pandemic Alpha, Beta, Gamma, or Delta variant (1) (2) (3) (4) (5) (6) (7) (8) (9) . A third dose of either homologous or heterologous COVID-19 vaccine was reported to enhance neutralizing antibody responses against the Omicron variant (3, 4, (10) (11) (12) (13) (14) (15) . However, the magnitudes of neutralizing activities towards Omicron after the booster dose were still far lower compared to earlier variants of concern (3, 5, 11, (16) (17) (18) (19) . Therefore, more effective vaccines or vaccination strategies are urgent in need to control the evolving pandemic of SARS-CoV-2 (20) (21) (22) . Recently preprinted studies demonstrated that Omicron infection of previously vaccinated individuals could boost broadly neutralizing antibodies against different SARS-CoV-2 variants (23, 24) , suggesting that the spike protein of the Omicron variant might serve as a good candidate antigen for a new COVID vaccine. Meanwhile, contradictory findings suggest that Omicron-matched vaccination shows no superiority in protection compared to immunization with current vaccines (25) (26) (27) (28) , which exaggerates concern about the effect of original antigenic sin caused by exposures to ancestral SARS-CoV-2 variants (29) . To investigate the effect of using Omicron-matched spike protein as a booster antigen, we firstly immunized mice with two-dose of a DNA vaccine encoding the spike protein of the ancestral Wuhan strain. Then the mice were boosted with DNA vaccines encoding spike proteins of either the Wuhan strain or the Omicron variant. Our data showed that the Omicron S DNA vaccine boosted cross-reactive antibody and T cell responses more efficiently than the ancestral S DNA booster vaccine. All experiments and methods were performed in accordance with relevant guidelines and regulations. Mice experiments were reviewed and approved by the Research Ethics Review Committee of Shanghai Public Health Clinical Center. The full-length s genes of the SARS-CoV-2 Wuhan and Omicron strain were optimized according to the preference of human codon usage and synthesized by Genewiz (Genewiz Biotech Co., Ltd., Suchow, China). The codon optimized spike genes were subcloned into the pJW4303 eukaryotic expression vector (Kindly gifted by Dr. shan Lu's laboratory at the University of Massachusetts). The sequence of insertion was confirmed by Sanger sequencing (Sangon Biotech Co., Ltd., Shanghai, China). The recombinant plasmids for mouse vaccination were prepared using an EndoFree Plasmid Purification Kit (Cat# 12391, Qiagen, Hilden, USA). Female C57BL/6J mice, 6-8-week-old, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in the SPF animal facility of Shanghai Public Health Clinical Center. The schedule of vaccination is shown in Figure 1 . Briefly, 20μg of the S_Wuhan DNA vaccine was injected intramuscularly into each mouse at Week 0 and Week 2. Subsequently, the mice were boosted with 50μg of either the S_Omicron or the S_Wuhan DNA vaccine at Week 6. The control group was boosted with PBS. Four weeks after the final vaccination, the mice were euthanized. Peripheral blood and splenocytes were collected for assays of S protein specific immune responses. VSV-backboned SARS-CoV-2 pseudo-viruses were prepared according to a reported method (30) . The neutralization assay was conducted by following the previously described procedure (30, 31) . Briefly, 100μl of serially diluted mice sera were added into 96-well cell culture plates. Then, 50μl of pseudoviruses with a titer of 13000 TCID50/ml were added into each well and the plates were incubated at 37°C for 1 hour. Next, Vero cells were added into each well (2×10 4 cells/well) and the plates were incubated at 37°C in a humidified incubator with 5% CO2. 24 hours later, luminescence detection reagent (Bright-Glo™ Luciferase Assay System, Promega, USA) was added to each well following the manufacturer`s instruction. The luminescence was measured using a luminescence microplate reader (GloMax® Navigator Microplate Luminometer, Promega, USA) within 5 minutes. The Reed-Muench method was used to calculate the virus neutralization titer. Antibody neutralization titers were presented as 50% maximal inhibitory concentration (IC50). All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons between two groups were conducted by the method of t-test. P<0.05 was considered as statistically significant. In comparison with CD4 + T cell responses, specific CD8 + T cell responses were significantly improved by the booster doses of both the S_Wuhan and the S_Omicron DNA vaccines compared with the control group ( Figure 3D -3F, 4C and 4D). It is worth noting that the booster shot of the S_Omicron DNA vaccine elicited the highest IFN-γ + CD8 + , TNF-a + CD8 + , IL-2 -TNF-a -IFN-γ + CD8 + and IL-2 -TNF-a + IFN-γ + CD8 + T cell responses against spike proteins of both the ancestral and the Omicron variants among the three groups. However, no significant differences of CD8 + T cell responses were observed between the groups boosted with the S_Omicron and the S_Wuhan DNA vaccines. Extensive mutations in the spike protein render the Omicron variant more likely to escape current vaccines than ancestral SARS-CoV-2 variants (32, 33) . A third dose can reduce the risk of hospitalization due to Omicron, however, the effectiveness may diminish quickly (34) . In addition to humoral responses, cross-reactive T cell responses have also been observed in previously infected or vaccinated people (19, (35) (36) (37) , which hold the potential to protect against severe disease with Omicron. Here we show that the Omicron-matched vaccine is more efficient in boosting crossreactive T cell responses. As a recent study demonstrates that the Omicron variant might escape T cell immunity in some individuals with prior infection or vaccination (38) , a booster vaccination that can improve T cell cross-reactivity will very likely help to protect against emerging SARS-CoV-2 variants. We note two major limitations in our study. First, we were not able to conduct a live-virus challenge experiment, therefore, we do not know whether the observed improvements of humoral and cellular immunities can be translated into superior protection. Second, the results were generated using a mouse model, which might not completely mimic the characteristics of human immune responses. The Omicron variant challenge studies in animals and vaccination in humans will be required for corroboration. The authors declare that they have no relevant conflicts of interest. Parametric t-test was used to analyze the data in B. Non-parametric t-test was used to analyze the data in A, C and D. Statistical analyses were performed using the method of parametric t-test. Next, a gating of FSC vs SSC was used to find the lymphocyte population. Then, CD3 + T cells were found via a gating of SSC vs CD3-PE-Cy7, which were further divided into CD3 + CD4 + and CD3 + CD8 + T cells via gating on CD4 vs CD8. 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