key: cord-0944417-0qznmhf1 authors: Yuan, Yaochang; Zhang, Xiantao; Chen, Ran; Li, Yuzhuang; Wu, Bolin; Li, Rong; Zou, Fan; Ma, Xiancai; Wang, Xuemei; Chen, Qier; Deng, Jieyi; Zhang, Yongli; Chen, Tao; Lin, Yingtong; Yan, Shumei; Zhang, Xu; Li, Congrong; Bu, Xiuqing; Peng, Yi; Ke, Changwen; Deng, Kai; Pan, Ting; He, Xin; Zhang, Yiwen; Zhang, Hui title: A Bivalent Nanoparticle Vaccine Exhibits Potent Cross-protection against the Variants of SARS-CoV-2 date: 2021-12-23 journal: Cell Rep DOI: 10.1016/j.celrep.2021.110256 sha: b8ee5a53c156a3413627c86d1cf88e5cc44735e7 doc_id: 944417 cord_uid: 0qznmhf1 The inoculation of vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is undergoing worldwide. However, the emergence of SARS-CoV-2 variants could confer immune evasion. Here we develop a bivalent nanoparticle vaccine, which displays the RBDs of both D614G and B.1.351 strains. With a prime-boost or a single-dose strategy, this vaccine elicits a robust neutralizing antibody and full protection against the infection of authentic D614G or B.1.351 strains in the human angiotensin-converting enzyme 2 transgene mice. Interestingly, after 8 months since the D614G-specific vaccine inoculated, a new boost with this bivalent vaccine potently elicits cross-neutralizing antibodies for SARS-CoV-2 variants in rhesus macaques. We suggest that the D614G/B.1.351-bivalent vaccine could be used as an initial single-dose or a sequential enforcement-dose to prevent SARS-CoV-2 and its variants. infection of authentic D614G and B.1.351 strains, the focus reduction neutralizing test 151 (FRNT) was conducted (Ma et al., 2020) . The nAbs in all nanoparticle vaccinated 152 mice strongly inhibited the replication of both authentic D614G and B.1.351 strains. weeks after boost vaccination, the splenocytes were obtained and intracellular 159 cytokine staining (ICCS) was conducted (Fig. S2A ). All the nanoparticle vaccinated 160 mice showed strong polyfunctional CD8 + T cell responses expressing 161 and Th1-biased CD4 + T cell responses expressing IFN-γ (Fig. S2B ). There was no 162 difference across different groups for Th2-biased IL-4 + -expressing CD4 + T cells (Fig. 163 S2B) . 164 To determine the stability of nanoparticle vaccines, we stored the 165 D614G/B.1.351_RBD-NP vaccine at −80, -20, 4 and 25°C for two weeks. profiles indicated that the protein remained highly stable after two weeks of storage at 167 all tested temperatures (Fig. S3A) , with no loss of immunogenicity in BALB/c mice 168 Moreover, the SARS-CoV-2 nucleocapsid (N) antigen was undetectable in the lung 193 tissue of all nanoparticle vaccinated hACE2 mice, but was detected in the control 194 mice ( Fig. 2F -I). Hematoxylin and eosin (HE) staining of lung tissue showed a 195 reduction of inflammation in immunized hACE2 mice compared to control mice In our previous study, we reported that a significant amount of nAbs had been 198 induced by our nanoparticle vaccines at four weeks post priming but before boost 199 vaccination, indicating that single-dose of nanoparticle vaccination strategy could be 200 enough to elicit sufficient nAbs against SARS-CoV-2 (Ma et al., 2020) . Herein, we 201 evaluate the immunogenicity and in vivo protection ability of a single-dose of the 202 bivalent D614G/B.1.351_RBD-NP vaccine (Fig. 3A) . Five hACE2 mice were 203 immunized with 10 μg of the D614G/B.1.351_RBD-NP vaccine, or an equimolar 204 amount of the D614G/B.1.351_RBD-monomer as a control. As the prime-boost 205 strategy of nanoparticle vaccine has been carefully evaluated using monomer as a 206 control in our previous study, here we also set the D614G/B.1.351_RBD-monomer 207 group as a control for the single-dose strategy (Ma et al., 2020) . Sera were collected 6 208 weeks after vaccination (Fig. 3A) . The RBD-specific IgG against D614G and B.1.351 209 strains was detectable in all nanoparticle immunized hACE2 mice (Fig. 3B ). With the 210 Finally, we assess the cross-protection of updated bivalent 248 D614G/B.1.351_RBD-NP vaccine as a third-dose on previously immunized rhesus 249 macaques (Wu et al., 2021) . Rhesus macaques were immunized with 50 μg of 250 D614G_RBD-NP vaccine on day 1 and 28, and the robustness of the nAbs against the 251 authentic D614G and B.1.351 strains were monitored over the course of more than 8 252 months ( Fig. 4A ) (Ma et al., 2020) . By the measurement with the FRNT50 assay 253 J o u r n a l P r e -p r o o f against the D614G authentic viruses, the nAb titers of immunized rhesus macaque 254 sera were elicited at 14 days after the priming by the D614G_RBD-NP vaccine, 255 peaked following another 14 days after the first boosting. The nAb titers since then 256 yet remained a significant amount but eventually waned, during a course of over 8-257 months. Interestingly, the immunized sera showed consistently higher nAb titers 258 against the D614G strain than the B.1.351 strain (Fig. 4B) . protection not only occurs in the initial prime-boost strategy but also in the initial 295 single-dose strategy. Therefore, this bivalent nanoparticle vaccine could be used for 296 initial prime-boost or single-dose vaccination for those who never received any 297 COVID-19 vaccine inoculation. In addition, we proposed a third-dose (second booster) 298 strategy, which is particularly important given the ever accelerating rollout of global 299 vaccination (Kreier, 2021) . Notably, in the single-dose strategy, the robust immune 300 response against B.1.351 viruses depend on a at least 5 g-dose in our experiment 301 settings. This result suggest that a certain amount of vaccine is required for initiating 302 the immune response. In rhesus macaques receiving initial prime-boost inoculation of 303 the D614G-specific nanoparticle vaccine, the nAbs for the D614G and viral variants 304 eventually decreased. A third-dose inoculation with a bivalent nanoparticle vaccine 305 significantly boosted the nAb titers against the D614G and B.1.351 strains at almost 306 the same level. Notably, the deterioration rate of the D614G neutralizing antibody 307 titer is slower after the third-dose, compared to that after the second-dose, suggesting 308 the involvement of a stronger memory immune response. Importantly, the third-dose 309 J o u r n a l P r e -p r o o f of immunization with bivalent nanoparticle vaccine also potently elicited nAbs 310 against almost all the variants we tested, albeit a slight deficiency for B.1.617.1. 311 A recent work indicates that nanoparticle vaccine based upon early strain is 312 enough to fully protect the animal from infection of viral variants including B.1.351 313 (Saunders et al., 2021) . Although our data support this claim, we have indicated that, 314 after initial prime-boost with nanoparticle vaccine targeting D614G, the nAb titers for 315 the D614G strain will eventually decrease, and the decrease of nAb titers for B.1.351 316 is even much significant. Therefore, it is worrisome for the long-persistence of 317 immunity against viral variants after nanoparticle vaccine targeting the early strain. 318 Besides, given that the SARS-CoV-2 variants with E484K/Q mutant eventually 319 dominate in many regions of the world, and the bivalent vaccine exerts a potent 320 capability to boost the nAb titers against viral variants, we suggest that it is necessary 321 to develop the bivalent nanoparticle vaccine targeting both early strain and B.1.351 322 strain, which could be sufficient for current COVID-19 pandemic under the threat of 323 484K/Q mutants. Given that SARS-CoV-2 exhibits a significant capability to evolve 324 after an almost two year epidemic, the vaccine for SARS-CoV-2 variants could be 325 routinely developed. 326 To deal with the pandemic caused by SARS-CoV-2 variants, in addition to 327 updating the design of immunogen, the inoculation strategy should also be optimized 328 (Huang et al., 2021b; Powell et al., 2021; Yahalom-Ronen et al., 2020) . As the 329 bivalent nanoparticle vaccine exerts potent protection efficiency in hACE2 mice with 330 J o u r n a l P r e -p r o o f a single-dose and is stable at ambient temperature, resistant to freeze-thaw with 331 minimal loss in immunogenicity, the long-distance distribution of this bivalent 332 nanoparticle vaccine could become quite easy, especially for these countries where • All data supporting the findings of this study are available within the paper or from 484 the corresponding author upon request. 485 • This paper does not report original code. 486 • Any additional information required to reanalyze the data reported in this paper is 487 available from the lead contact upon request. for RBD-specific antibodies (Liu et al., 2020) . 507 508 HEK293T, CHO-K1 and Vero E6 cells were obtained from ATCC. These adherent 510 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented 511 with 1% penicillin-streptomycin (ThermoFisher) and 10% FBS (ThermoFisher). 512 HEK293T expressing hACE2 (hACE2/HEK293T) was constructed in home. All cells 513 were cultured in the sterile incubator at 37℃ and 5% CO2. All cells have been 514 confirmed to be mycoplasma free. 515 SARS-CoV-2 strains, named as hCoV-19/CHN/SYSU-IHV/2020 (D614G) 516 (Accession ID on GISAID: EPI_ISL_444969) and 19nCoV-CDC-Tan-GDPCC 517 (B.1.351) were propagated in Vero E6 cells as published before (Huang et al., 2021a; 518 Ma et al., 2020) . The D614G strain was isolated from the sputum of a female 519 COVID-19 patient who was infected by a UK traveler in April 2020 by us, and the 520 Transgenic hACE2 mice (C57BL/6) were purchased from GemPharmatech Co, Ltd. 529 The generation procedure was described as published before (Zhang et al., 2021d) . Protein expression and purification 540 The RBD nanoparticle vaccine was constructed as described previously (Ma et al., 541 2020) . To further improve the conjugation efficiency of the NP vaccine, we screened 542 and modified a variety of natural CnaB2 proteins with isopeptide bonds from various 543 bacterial strains. Additionally, we optimized the conjugation efficiency by generating 544 several mutants based upon the structural prediction. Finally, we identified a potent 545 combination of GvTagOpti(Gv) and SdCatcher(Sd) from Gardnerella vaginalis and 546 Streptococcus dysgalactiae respectively, which significantly enhanced the assemble 547 efficiency and the production of the SARS-CoV-2 NP vaccine . 548 The Sd-Ferritin was expressed and purified from Escherichia coli (E.coli). Briefly, the 549 Sd was genetically fused at the N terminus of Ferritin (Sd-Ferritin), DNA sequences 550 of Sd-Ferritin were cloned to the pET28a vector. The construct was transformed into 551 BL21 (Takara). A single clone was amplified in LB with kanamycin while shaking at 552 37°C. The isopropyl b-D-1 thiogalactopyranoside (IPTG) (Takara) was added to the 553 bacterial solution to induce protein expression. Eighteen hours after induction, the 554 bacteria expressing the protein were harvested and lysed by sonication. After 555 and National Study 788 Group for Vaccine against the B.1.1.7 and B.1.351 Variants Adjuvanting a subunit COVID-19 vaccine to induce protective immunity. 794 Nature Two-component spike nanoparticle vaccine 798 protects macaques from SARS-CoV-2 infection Convergent 802 evolution of SARS-CoV-2 spike mutations, L452R, E484Q and P681R, in 803 the second wave of COVID-19 in Maharashtra SARS-CoV-2 806 variants show resistance to neutralization by many monoclonal and 807 serum-derived polyclonal antibodies Infection and vaccine-induced neutralizing antibody responses to the 812 SARS-CoV-2 B. 1.617. 1 variant. bioRxiv Emergence of SARS-CoV-2 B.1.1.7 Lineage -United 816 Multiple SARS-CoV-2 variants escape 821 neutralization by vaccine-induced humoral immunity Comprehensive mapping of 824 mutations in the SARS-CoV-2 receptor-binding domain that affect 825 recognition by polyclonal human plasma antibodies Emergence in late 2020 of multiple lineages of 830 SARS-CoV-2 Spike protein variants affecting amino acid position 677. 831 medRxiv SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing 835 Neutralization of SARS-CoV-2 VOC 501Y.V2 by human antisera 838 elicited by both inactivated BBIBP-CorV and recombinant dimeric RBD 839 ZF2001 vaccines A single-dose mRNA 842 vaccine provides a long-term protection for hACE2 transgenic mice 843 from SARS-CoV-2 Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein 847 Nanoparticle Vaccine Unprecedented achievement': who received the 850 first billion COVID vaccinations? Nature Possible 852 link between higher transmissibility of B. 1.617 and B. 1.1. 7 853 variants of SARS-CoV-2 and increased structural stability of its 854 spike protein and hACE2 affinity Functional assessment 856 of cell entry and receptor usage for SARS-CoV-2 and other lineage B 857 betacoronaviruses SARS-CoV-2 501Y.V2 variants 861 lack higher infectivity but do have immune escape The challenge of emerging 864 SARS-CoV-2 mutants to vaccine development Differential efficiencies to 868 neutralize the novel mutants B.1.1.7 and 501Y.V2 by collected sera 869 convalescent COVID-19 patients and RBD nanoparticle Recovered COVID-19 patients 874 with recurrent viral RNA exhibit lower levels of anti-RBD antibodies Nanoparticle Vaccines Based on 878 the Receptor Binding Domain (RBD) and Heptad Repeat Elicit Robust Protective Immune Responses Responses Elicited by SARS-CoV-2 501Y.V2 (B.1.351) Sensitivity of infectious SARS-CoV-2 B.1.1.7 and 889 B.1.351 variants to neutralizing antibodies The variant gambit: COVID-19's next move A Single 896 Immunization with Spike-Functionalized Ferritin Vaccines Elicits 897 Neutralizing Antibody Responses against SARS-CoV-2 in Mice CoV-2 B. 1.1. 7 and B. 1.351 Spike variants bind human ACE2 with 901 increased affinity Spike E484K mutation in the first 905 SARS-CoV-2 reinfection case confirmed in Brazil Neutralizing antibody vaccine for pandemic and pre-emergent 909 coronaviruses Neutralization of SARS-CoV-2 912 1.429 and B.1.351 Efficacy of NVX-CoV2373 Covid-19 Vaccine against the B.1.351 916 Variant Vaccine-induced immunity provides more robust 920 heterotypic immunity than natural infection to emerging SARS-CoV-2 921 variants of concern Potent 924 neutralizing nanobodies resist convergent circulating variants of 925 SARS-CoV-2 by targeting novel and conserved epitopes. bioRxiv Emergence and rapid spread of a new severe acute respiratory 930 syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple 931 spike mutations in South Africa Elicitation of Potent Neutralizing Antibody Responses by Designed 935 Protein Nanoparticle Vaccines for SARS-CoV-2 Structure, Function, and Antigenicity of the 939 SARS-CoV-2 Spike Glycoprotein A novel 942 coronavirus outbreak of global health concern Antibody 946 resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 Spike mutations in SARS-CoV-2 950 variants confer resistance to antibody neutralization. bioRxiv Dual-targeting 954 nanoparticle vaccine elicits a therapeutic antibody response against 955 chronic hepatitis B Variant SARS-CoV-2 959 mRNA vaccines confer broad neutralization as primary or booster 960 series in mice A single dose of recombinant VSV-G-spike vaccine provides 964 protection against SARS-CoV-2 challenge The 968 interferon-stimulated exosomal hACE2 potently inhibits SARS-CoV-2 969 replication through competitively blocking the virus entry Emergence of a Novel SARS-CoV-2 Variant 974 Improvement of a SARS-CoV-2 977 vaccine by enhancing the conjugation efficiency of the immunogen to 978 self-assembled nanoparticles The ORF8 protein of SARS-982 CoV-2 mediates immune evasion through down-regulating MHC-Iota Amino acids and RagD 986 potentiate mTORC1 activation in CD8(+) T cells to confer antitumor 987 immunity Evidence of escape of SARS-CoV-2 variant B.1.351 from 991 natural and vaccine-induced sera A pneumonia 995 centrifugation, the supernatants were loaded onto a Sepharose 6 FF (GE Healthcare) 556 size exclusion column that was pre-equilibrated with 20 mM Tris 50 mM NaCl buffer 557 (pH 7.5) and eluted with the same buffer at a rate of 10 ml min −1 . The total volume of 558 the column (Vt) was 53 ml, and the elution volume (Ve) of Sd-ferritin NP was 26 ml. 559The concentration of Sd-Ferritin was determined by BCA assay. The bacterial 560 endotoxins in nanoparticles were quantified by the Tachypleus amebocyte lysate test 561 (≤10 EU/dose). Coomassie blue staining, size-exclusion chromatography (SEC), and 562 transmission electron microscopy (TEM) were executed to confirm the purity and 563 homogeneity (Wang et al., 2020b) . 564The D614G_RBD and B.1.351_RBD were expressed and purified from Chinese 565 hamster ovary K (CHO-K1) cells. Briefly, The Gv coding sequence was fused at the 566 N-terminus of the D614G_RBD or B.1.351_RBD sequence. DNA sequences of SP-567Gv-D614G_RBD and SP-Gv-B.1.351_RBD were codon-optimized for mammalian 568 cell expression and cloned into vector plasmid pLVX. The constructed pLVX, 569 lentiviral packaging plasmids psPAX2, and pLP/VSVG were transfected into 570 HEK293T cells cultured in DMEM conditioned medium. Lentiviruses released in the 571 supernatant were collected 60 h after transfection and then infected anchorage-572 dependent CHO-K1 cells cultured in F12K medium. The supernatant was removed 8 573 h later and changed with new a F12K medium for another day of culture. The F12K 574 medium was then replaced with CHO S4 medium, which was used for cell suspension 575 and expansion to a density of 3×10 6 cells/mL. Seven days later, supernatants were 576 J o u r n a l P r e -p r o o f collected and centrifuged to discard cellular debris. The cleared supernatants were 577 passed through the KR2i TangentialFlow Filtration system equipped with filters 578 (Repligen) with 10-kDa and 100-kDa molecular weight cutoffs (MWCO) to obtain 579 the 10-100kDa molecular weight proteins. The concentrates were purified by AKTA 580 pure system using Capto SP Impres (GE Healthcare) columns running phosphate-581 buffered (PH= 7) and eluted with the 150 mM NaCl phosphate-buffered (PH= 7) 582 buffer at a rate of 5 ml min −1 . The purified proteins were concentrated and buffer-583 replaced with conventional Tris buffer. The concentrations of D614G_RBD and 584 B.1.351_RBD were determined by BCA assay. Coomassie blue staining was executed 585 to confirm the purity. 586The purified Gv-D614G_RBD and Gv-B.1.351_RBD were irreversibly covalently 587 retention of retaining 11 ml to 14 ml. The elution of nanoparticles was concentrated, 608 and the concentration was measured by BCA assay. Coomassie blue staining, size-609 exclusion chromatography (SEC), and transmission electron microscopy (TEM) were 610 executed to confirm the purity and homogeneity (Ma et al., 2020) . were immunized with equal moles of ferritin, which were the same as the 630 D614G/B.1.351_RBD-NP group. All the mice were vaccinated with the above 631 vaccines in a prime/boost manner, which vaccinated mice at week 0 and week 4. 632Serum was collected every two weeks. Mice were euthanized at week 6. 633 published before (Zhang et al., 2021d) . 705 706 The method was consistent as described previously (Li et al., 2021c; Ma et al., 2020) . with PBS solution containing 1 % BSA. 50 μl of the diluted secondary antibody was 727 added to each well and incubated at 37 °C for 1 h, followed by washing with PBS/T 728 three times. 50 μl TrueBlue (KPL) was added to each well and set for 5 mins shaking 729 at room temperature. Plates were washed with ddH2O, and the liquid was eradicated, 730 followed by spot counting using with ImmunoSpot Microanalyzer. The reduction 731 rates of the serial dilution assay were analyzed by GraphPad Prism 8.0 using non-732 linear regression to measure the FRNT50 titer (Zhang et al., 2021b) . 733 734 Briefly, HEK293T cells were co-transfected with packaging plasmid psPAX2, 736 luciferase-expressing lentivirus plasmid, and respective variant spike protein-737 expressing plasmid using polyethyleneimine (PEI, Sigma). Forty-eight hours after 738 transfection, culture supernatants were collected, clarified of cells before stored in -739 and infectivity was measured by detection of luminescence, details would be shown 741 as follows. All the convalescent serum and immunized animal serum were serially 742 diluted and incubated with pre-titrated amounts of SARS-CoV-2 pseudotyped virus at 743 37℃ for 1 h. Then the serum/virus mixture was then added into wells containing 744 J o u r n a l P r e -p r o o f 1×10 4 hACE2 cells and incubated at 37 ℃ in 5% CO2 for 48 h. Cells were then lysed 745 with lysis buffer (Promega), and the lysate was the measure for luciferase activity by 746 detecting relative luminescence units (RLU) in a luminometer (Promega). 747Neutralizing antibody titers of serum against the pseudotyped virus were analyzed 748 using GraphPad Prism 8.0 software (Zhang et al., 2021d) . or Microsoft Excel. Triplicate, sextuplicate, and other replicative data were presented 775 as mean ± SD. Value of p < 0.05 was considered to be statistically significant and 776represented as an asterisk (*). Value of p < 0.01 was supposed to be more statistically 777 significant and described as double asterisks (**). Value of p < 0.001 was considered 778 the most statistically significant and represented as triple asterisks (***). Value of p < 779 0.0001 was supposed to be extremely statistically substantial and described as 780 quadruple asterisks (****). For comparison between two treatments, a Student's t-test 781 was used. For comparison between each group with the mean of every other group 782 within a dataset containing more than two groups, one-way ANOVA with Tukey's 783 multiple comparisons test was used.