key: cord-0901951-e9iylmyp authors: Guirakhoo, Farshad; Kuo, Lucy; Peng, James; Huang, Juin-Hua; Kuo, Be-Shen; Lin, Feng; Liu, Yaw-Jen; Liu, Zhi; Wu, Grace; Ding, Shuang; Hou, Kou-Liang; Cheng, Jennifer; Yang, Vicky; Jiang, Hank; Wang, Jason; Chen, Tony; Xia, WeiGuo; Lin, Ed; Hung, Chung Ho; Chen, Hui-Jung; Shih, Zhonghao; Lin, Yi-Ling; Wang, Shixia; Ryan, Valorie; Schurter, Brandon T.; Hu, Mei Mei; Heppner, Gray; Malherbe, Delphine C.; Bukreyev, Alexander; Hellerstein, Michael; Monath, Thomas P.; Wang, Chang Yi title: A Novel SARS-CoV-2 Multitope Protein/Peptide Vaccine Candidate is Highly Immunogenic and Prevents Lung Infection in an AAV hACE2 Mouse Model and non-human primates date: 2021-10-07 journal: bioRxiv DOI: 10.1101/2020.11.30.399154 sha: 3f3184314302864c767f991f4b557a9d9e4eb383 doc_id: 901951 cord_uid: e9iylmyp A novel multitope protein-peptide vaccine against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and disease is described in this report. The initial development and characterization experiments are presented along with proof-of-concept studies for the vaccine candidate UB-612. UB-612 consists of eight components rationally designed for induction of potently neutralizing antibodies and broad T cell responses against SARS-CoV-2: the S1-RBD-sFc fusion protein, six synthetic peptides (one universal peptide and five SARS-CoV-2-derived peptides), a proprietary CpG TLR-9 agonist at low concentration as an excipient, and aluminum phosphate adjuvant. Through immunogenicity studies in Guinea pigs and rats, we optimized the design of protein/peptide immunogens and selected an adjuvant system, yielding a vaccine that provides excellent S1-RBD binding and high neutralizing antibody responses, robust cellular responses, and a Th1-oriented response at low doses. In challenge studies, UB- 612 vaccination reduced viral load and prevented development of disease in mouse and non-human primate challenge models. With a Phase 1 trial completed, a Phase 2 trial ongoing in Taiwan, and additional trials planned to support global authorizations, UB-612 is a highly promising and differentiated vaccine candidate for prevention of SARS-CoV-2 infection and COVID-19 disease. Author Summary SARS-CoV-2 virus, the causative agent of Coronavirus Disease 2019 (COVID-19), has spread globally since its origin in 2019, causing an unprecedented public health crisis that has resulted in greater than 4.7 million deaths worldwide. Many vaccines are under development to limit disease spread and reduce the number of cases, but additional candidates that promote a robust immune response are needed. Here, we describe a multitope protein-peptide vaccine platform that is unique among COVID-19 vaccines. The advantages of our approach are induction of both high levels of neutralizing antibodies as well as a Th/CTL response in the vaccinated host, which mimics the immune response that occurs after natural infection with SARS-CoV-2. We demonstrate that our vaccine is immunogenic and effective in preventing disease in several animal models, including AAV- hACE-2 transduced mice, and both rhesus and cynomolgus macaques. Importantly, no immunopathology was observed in the lungs of immunized animals, therefore showing that antibody-dependent enhancement (ADE) does not occur. Our study provides an additional, novel vaccine candidate for advancement in clinical trials to treat and prevent SARS-CoV-2 infection and COVID-19 disease. The UB-612 vaccine immunogen was designed to contain an S1-RBD-sFc fusion 150 protein plus five synthetic Th/CTL peptides for class I and II MHC molecules derived from 151 SARS-CoV-2 S2, M, and N proteins. To identify the best RBD immunogen to induce 152 neutralizing antibody responses, three S1-RBD-based protein antigen (sequences aa340-153 539) vaccine candidates were designed: S1-RBD-sFc (single chain Fc), S1-RBDa-sFc 154 (RBD domain modified to reduce a Cys-disulfide bond for better domain folding), and S1-155 RBD-Fc (double chain Fc) (structure of S1-RBD-sFc illustrated in Fig 1B) . These synthetic 156 genes were transfected into Chinese Hamster Ovary (CHO) cells for transient expression transferred into ACE2-ECD-sFc coated plates to test its inhibition activity. Although 195 the mean ID 50 inhibition values were not statistically significant (p ≥ 0.05), with the highest 196 ID 50 values observed for antibodies raised by S1-RBD-sFc (7251.5), followed by S1- Fc (3019.8) and S1- RBDa-sFc (1950.8) . This result indicates that all antigens elicited 198 antibodies capable of inhibiting hACE2 binding, with S1-RBD-sFc raising the most potent 199 responses. The function of anti-RBD antibodies was quantified both as inhibition of RBD 201 binding to hACE2 and as neutralization of live SARS-CoV-2. In the hACE2 binding 202 inhibition cell-based assay, HEK293 cells expressing hACE2 were treated with mixtures 203 of pooled GP sera and S1-protein (Fc tagged), then assayed by flow cytometry (FACS) 204 by staining cells with fluorescently labeled anti-human IgG Fc protein antibody. The GMT 205 ID 50 (the inhibitory dilutions at which 50% neutralization is attained) values were 1026 for 206 S1-RBD-sFc, 193 for S1-RBDa-sFc, and 325 for S1-RBD-Fc vaccine, again showing 207 functional activity for antibodies elicited by all candidates, still the highest activity was seen 208 for S1-RBD-sFc. Results are provided in Figs 2C, S1 and S2, respectively. To test 209 neutralizing capacity of the elicited antibodies, live virus cytopathic effect (CPE) 50% 210 reduction assay were adopted, using the anti-SARS-CoV-2 N protein antibody and 211 immunofluorescent visualization for neutralization titer (VNT 100 ) determination (Fig 2D and 212 Fig S2) . Sera from S1-RBD-sFc demonstrated superior activity, with neutralization titers 213 at 5 WPI 2-4-fold higher than those from the other two groups, protecting 50% of the cells 214 from viral infection at titers of 504-1,024 at 3 WPI and >32768 in pooled guinea pig sera 215 at 5 WPI (Fig S2) . In a separate experiment, we compared neutralizing titers in sera from GPs 217 vaccinated with S1-RBD-sFc with convalescent sera of COVID-19 patients, using the S1-Fig S2, demonstrated that GP immune sera diluted 1,000-fold (3 WPI) or 8,000-fold (5 220 WPI) exhibited comparable or higher inhibition of S1-RBD:ACE2 binding than by the 221 convalescent sera of 10 patients diluted at 20-fold, illustrating that the sera of GPs 222 contained ≥50-fold higher antibody titers than human convalescent sera. The initial immunogenicity assessment in GPs established the superior humoral Dawley rats, the immunogen doses and adjuvants were varied to allow selection of an 230 optimal adjuvant (Fig 3A) . S1-RBD-sFc was formulated with five Th/CTL peptides 231 selected from S2, M and N proteins of SARS-CoV-2 and our proprietary universal Th 232 peptide (UBITh®1a) [32] to generate the multitope protein-peptide vaccine candidate (Fig 233 1A ). We then combined the candidate vaccine with one of two different adjuvant systems: 234 ISA51/CpG3 or Adju-Phos®/CpG1. These vaccine-adjuvant combinations were most potent inhibitory activity was seen with the lowest dose of S1-RBD-sFc protein (10 244 µg) formulated with peptides and the Adju-Phos® adjuvant. In the replicating virus 245 neutralization assay against the Taiwanese SARS-CoV-2 isolate (representative of the 246 original Wuhan sequence), the Week 4 immune sera induced by UB-612 vaccine did not 247 show a significant dose-dependent effect in rats (Fig 3C) . The low doses of adjuvanted 248 protein,10 and 30 μg, could neutralize viral infection at VNT 50 of >10,240 dilution. The rat immune sera at Week 6 (i.e. 4 weeks after the 2 nd immunization) from each 250 vaccinated dose group were assayed two ways: first, in comparison with a set of 251 convalescent sera of COVID-19 patients for titers in S1-RBD:ACE2 binding inhibition 252 ELISA, expressed in blocking level of μg/mL; and second, through a SARS-CoV-2 CPE 253 assay in Vero-E6 cells, expressed as VNT 50 . As shown in Fig 3D, all doses of the vaccine 254 formulations elicited neutralizing titers in rats that were significantly higher than those in 255 convalescent patients by S1-RBD:ACE2 binding ELISA and higher (but not achieving 256 statistical significance due to variation in patient data and low number of animals) by 257 VNT 50 . To assess the Th1/Th2 response, vaccinated rats were evaluated using ELISpot. Rats were dosed at Weeks 0 and 2 with 30 µg or 100 µg of UB-612 vaccine. Splenocytes 260 were then collected at Week 4 and restimulated in vitro with the Th/CTL peptide pool plus 261 S1-RBD or with the Th/CTL peptide pool alone. High levels of IFN-γ and IL-2 secretion 262 was observed in splenocytes after the stimulations with Th/CTL peptide pool plus S1-RBD 263 or with the Th/CTL peptide pool alone, while only minor amounts of IL-4 were seen (Figs 264 4A and 4B). The individual peptide stimulations also demonstrated that the rat 265 splenocytes also produced high levels of IFN- and IL-2 (Th1) responses but very low 266 levels of IL-4 (Th2) against S2 peptides (p5752, p5753 and p5755) (Fig 4S-A formulated with Adju-Phos®. S1-RBD-specific antibody titers were evaluated at Weeks 0, 275 3 and 4. After 2 doses of vaccine, S1-RBD specific antibody responses were detected in 276 all three dose groups with significant dose dependent response pattern (Fig 5B) , p < 0.05 277 between 3 and 9 µg groups and p < 0.005 between 3 and 30 µg groups. The mice were 278 infected with adeno-associated virus (AAV) expressing hACE2 at 4 WPI and challenged 279 2 weeks later with 10 6 TCID 50 of SARS-CoV-2 by the intranasal (IN) route (Fig 5A) . Efficacy of the vaccine was measured using lung viral loads and body weight Immunogenicity and challenge studies in rhesus and 295 cynomolgus macaques Based on an established model using rhesus macaques (RM) [37, 38] , an 297 immunization study of UB-612 by IM injection was initiated with RM (N = 4/group) receiving 298 0, 10, 30 or 100 μg of UB-612 at 0 and 4 weeks in the first NHP study (Study 1) (Fig 6A) . IgG binding antibody to S1-RBD was increased over baseline in all animals, with titers 300 reaching around 3 logs at 5 and 7 weeks (Fig 6B) . Strong neutralizing antibody responses 301 were induced, with highest titers observed at the 30 μg dose (Fig 6C) . In ELISpot antigen-302 specific IFN-γ-secreting T cells were elicited in a dose-dependent manner (Fig 6D) , with 303 highest responses at the 100 μg dose level. To test the response to boosting, the 3 rd 304 immunization was given at Day 70 (6 weeks after the 2 nd immunization). One week after 305 the 3 rd dose, S1-specific IgG titers were significantly boosted (~5-fold) at all three dose 306 levels (Fig 7A) . Neutralizing antibody responses against the Wuhan strain also increased 307 in a live virus CPE assay one week after the 3 rd dose, with the greatest increase (5~10-308 fold) seen for the 100 μg dose level (Fig 7B) . We also measured neutralization in a 309 pseudovirus assay expressing the Spike proteins from the Wuhan strain and 5 variants of had increased neutralization activity against all five VOC (Fig 7C) . In a live virus assay, 313 sera taken one week after the 3 rd dose (Day 77) demonstrated potent neutralization or D614G strains (Fig 7D and 7E ). Animals were challenged on Day 77 with SARS-CoVthat high titers of neutralizing antibody titers were achieved against live virus Wuhan strain 325 on Day 50 (3 weeks after the 2 nd immunization for both the 30 μg and 100 μg doses of 326 UB-612) (Fig 8B) and also against the B.1.617.2 Delta variant (Fig 8C) , though a drop in 327 titer of 2-fold for the 30 μg dose and 1.5-fold for the 100 μg dose was seen for Delta as 328 Initially, we developed three RBD-sFc fusion protein vaccine candidates, which 346 were then down-selected to a single candidate through immunogenicity tests in GPs, showing robust S1-RBD binding antibody responses and functional activity, including 348 neutralization of live SARS-CoV-2 and inhibition of s1:hACE2 binding. Of the three 349 candidates tested, S1-RBD-sFc (S1-RBD fused to a single-chain Fc) gave the strongest 350 responses in all measurements. S1-RBD-sFc was slightly more immunogenic than the 351 other two constructs in S1-RBD binding antibody assays, but the strength of the S1-RBD- Further confirmation of the immunogenicity of this vaccine candidate was obtained 359 by comparison of neutralizing titers in sera from S1-RBD-sFc-immunized GPs with titers 360 in convalescent sera from COVID-19 patients. The results demonstrated that highly diluted 361 GP immune sera (e.g. 1:1000 after one dose or 1:8000 after two doses) exhibited 362 comparable or higher inhibition of S1-RBD:ACE2 binding than convalescent sera of 10 derived from S2, M and N structural proteins of SARS-CoV-2, to generate the final unadjuvanted vaccine candidate, which was studied in Sprague Dawley rats to compare 369 two adjuvant combinations (ISA 51VG/CpG3 and Adju-Phos®/CpG1). In the initial rat study, which tested the two vaccine-adjuvant combinations at a 371 dose range of 10 to 300 μg per injection, our results indicated that vaccines formulated 372 with both adjuvant systems elicited similar BAb titers across all doses, indicating excellent 373 immunogenicity of the vaccine even for low quantities of the primary protein immunogen. As in the earlier GP studies, however, functional antibody assays demonstrated clear 375 differences between candidate vaccine formulations. These tests showed the equivalency 376 in immunogenicity between the two adjuvant combinations while confirming excellent 377 immunogenicity at low doses. We chose Adju-Phos®/CpG1 as the adjuvant in our final 378 vaccine formulation due to its long safety record and ability to improve immune responses. showed excellent neutralizing immunogenicity. All doses of protein elicited neutralizing 381 titers significantly higher than those in convalescent patients, as determined through an 382 S1-RBD:ACE2 binding ELISA, Additionally, titers were higher (but not achieving statistical 383 significance due to the spread in patient data and the low number of animals) by VNT 50 . A Th1-oriented immune response against SARS-CoV-2 is potentially important to 385 avoid ADE or VAERD, as demonstrated by studies with SARS and MERS coronaviruses 386 as well as a commercial formalin inactivated RSV vaccine (inducing a Th2-biased 387 response), which led to the death of several vaccinated children who were later exposed 388 to live RSV [9, 41, 42] . Therefore, the FDA has recommended that any vaccines for COVID- (e.g., lack of ADE or VAERD) and efficacy of vaccine candidates including UB-612. We 402 infected mice with AAV-expressing hACE2 at 4 WPI and challenged 2 weeks later with In addition, we tested UB-612 in rhesus and cynomolgus macaque models. While 407 SARS-CoV-2 does not cause a lethal COVID-19-like disease in monkeys, the virus can 408 cause infection and illness. In these animals, disease is generally mild, self-limiting and 409 resolves within 2 weeks [48] [49] [50] . In an initial study, rhesus macaques received three 410 vaccinations at three dose levels. All vaccinated animals developed high titers of S1-RBD 411 binding antibodies that were also potently neutralizing. In a second study, cynomolgus demonstrated that S1-RBD-Fc exists in two major isoforms, S1-RBD-sFc1 and S1-RBD- The immune sera from rats (n = 3 for each dose group) were collected at weeks 0, 2, 3, and 4 for assessment of antigenic activities. Vaccination and challenge procedure in AAV6/CB-hACE2 mice 514 A total of 12 male BALB/C at 8-10 weeks of age were purchased from BioLASCO 515 Taiwan Co., Ltd. After a 3-day acclimation, animals were randomly assigned to 4 groups. All procedures on animals were performed in accordance with the regulations and and PM) during the study periods for clinical signs which included, but were not limited to 553 mortality, morbidity, feces, emesis, and changes in water and food intake. Animals were 554 bled at regular intervals for the immunogenicity studies described below. The first NHP study was conducted at JOINN Laboratories (Beijing) in Rhesus The macaques were challenged at 11 days after the 3 rd immunization (Day 81) with SARS- CoV-2 (10 6 TCID50) intratracheally. The viral loads were determined by viral RNA 563 copies/gram of lung tissue at 7 days after challenge (Day 88). The second NHP study was conducted in cynomolgus macaques (3-6 years old). At Biomere animals were divided into three groups (5/group) and injected intramuscularly Titer Calculation Program was used to calculate the relative titer. The anti-S1-RBD 591 antibody level was expressed as log 10 of an end point dilution for a test sample (SoftMax 592 Pro 6.5, Quadratic fitting curve, Cut-off value 0.5). 594 96-well ELISA plates were coated with 2 µg/mL ACE2-ECD-Fc antigen (100 595 mL/well in coating buffer, 0.1M sodium carbonate, pH 9.6) and incubated overnight ( The RT-PCR assay for the sgmRNA utilizes primers and a probe specifically designed to 679 amplify and bind to a region of the N gene messenger RNA from SARS-CoV-2 (Primers: (A) Guinea pig study design: animals were immunized with S1-RBD-sFC, S1-RBDa-sFC, or S1-RBD-Fc (n = 5 each group) at weeks 0 and 3 via intramuscular route. Immune sera were collected at 0, 3, and 5 weeks post initial immunization (WPI). Anti-S1 binding antibodies were detected by ELISA and neutralizing antibody titers were detected via CPE assay against wild Week 3 GP-1 5 S1-RBD-sFc* 200 g 100 g GP-2 5 S1-RBDa-sFc** 200 g 100 g GP-3 5 S1-RBD-Fc*** 200 g 100 g * S1-RBD-sFc: RBD (aa340-539) -single chain Fc fusion protein; ** S1-RBDa-sFc: RBD (aa340-539, Cys-mutation) -single chain Fc fusion protein; *** S1-RBD-Fc: RBD (aa340-539) -double chain Fc fusion protein. Weeks Post Immunization S1-specific IgG titers S1-RBD-sFc S1-RBDa-sFc S1-RBD-Fc * p < 0.01 S1-RBD-sFc S1-RBDa-sFc S1-RBD-Fc Pre-immune (B) S1-specific antibody temporal responses. Results shown as group geometric mean (GMT) ± standard error (SE). Immunization time points are shown with black arrows. * indicates that the statistical significance compared S1-RBD-sFC with S1-RBDa-sFC or S1-RBD-Fc immunization groups. (C) Neutralization and inhibitory dilution ID50 titers in S1 protein binding to ACE2 on ELISA by guinea pig sera collected at 2 weeks after the 2nd immunization (5 WPI). Cells were stained with human anti-SARS-CoV-2 N protein antibody and detected with antihuman IgG-488 (green). The nuclei were counterstained with DAPI (4',6-diamidino-2phenylindole) (blue). (A) Rat immunization study design. Immunogenicity of UB-612 adjuvanted with ISA51/CpG3 or Adju-Phos(R)/CpG1. Sprague Dawley rats were immunized at weeks 0 and 2 with UB-612 vaccine (at a dose range of 10-300 μg of S1-RBD-sF, formulated with synthetic designer peptides and adjuvants). A novel coronavirus outbreak Anti-spike IgG causes severe 743 acute lung injury by skewing macrophage responses during acute SARS-CoV 744 infection Two Different Antibody-Dependent Enhancement (ADE) Risks for vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-763 precipitated respiratory syncytial virus vaccine A 766 double-inactivated severe acute respiratory syndrome coronavirus vaccine 767 provides incomplete protection in mice and induces increased eosinophilic 768 proinflammatory pulmonary response upon challenge Anti-spike IgG causes severe 771 acute lung injury by skewing macrophage responses during acute SARS-CoV 772 infection An HIV 774 Envelope gp120-Fc Fusion Protein Elicits Effector Antibody Responses in Rhesus 775 Immunization With Fc-Based Recombinant Epstein-Barr Virus gp350 Elicits 779 Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model Methods for the study of 895 irritation and toxicity of substances applied topically to the skin and mucous 896 membranes A Simple method of estimating fifty per cent endpoints components are mixed with CpG1 and Adju-Phos adjuvant to constitute the UB-612 vaccine drug product Light blue shading indicates RBD of SARS-CoV-2 and no shading indicates the sFc fragment of an IgG1. The substitution of His297 for Asn297 (EU-index numbering) in single chain Fc, His282 in S1-RBD-sFc, is indicated by underline. S1-RBD-sFc protein contains 431 amino acid residues hACE2 binding ability of S1-RBD-sFc, as determined via ELISA. by ELISA. The rat groups in the left or right panels received vaccines with ISA51/CpG3 or Adju-Phos(R)/CpG1 as adjuvant Samples taken 4 WPI from rats immunized at weeks 0 and 2 with UB-612 vaccine adjuvanted with ISA51/CpG3 or Adju-Phos/CpG1. Potent neutralization of live SARS-CoV-2 by rat immune sera. Neutralization titers expressed as VNT50 hACE2 binding inhibiting (left) and neutralizing antibody (right) titers of sera from UB-612 with Adju-Phos(R)/CpG1 vaccinated rats are higher than titers in convalescent COVID-19 patients (HCS). * p ≦ 0.05, ** p ≦ 0.01 and NS (not significant) (Kruskal-Wallis ANOVA Week 4 and challenged with 10 6 PFU TCID50 of SARS-CoV-2 virus (hCoV-19/Taiwan/4/2020) by intratracheal infection at Week 6. The lung viral load and pathology were detected at 5 days after infection Immunization and challenge schedule S1-RBD-specific antibody titers at Weeks 0, 3 and 4 were measured with significant dose dependent response trend. * p < 0.05 between 3 and 9 µg groups 005 between 3 and 30 µg groups SARS-CoV-2 viral load RNA in lung were determined by RT-PCR. Significant difference is indicated between the saline and 30 µg groups Lung pathological scores on Day 5 after challenge. Significant difference is indicated between the saline and 30 µg groups Stained sections of mouse lung tissues from different vaccination groups of mice challenged with live virus. The vaccine dose: Low dose: 3 g; Middle dose: 9 g, or High dose: 30 g UB-612 vaccine; and Saline as negative control. (Study 2). The macaques received Saline, UB-612 30 μg or UB-612 100 μg on Day 0 and Day 28 The immunization and challenge study design in rhesus macaques Wuhan strain was used for challenge on Day 55 with a total of 1.0 × 105 TCID50 of SARS-CoV-2 divided equally between intranasal Neutralizing antibody responses against wild type Wuhan (WA) strain at different time points Neutralizing antibody tiers against WA and Delta variant on Day 50 (3 weeks after the second immunization) Viral loads were detected in (D) BAL, (E) nasal swabs and (F) rectal swabs through sgmRNA RT-PCR after challenge with SARS-CoV-2 Wuhan strain. The black curves represent viral loads of the individual animals, and the red curves are the median viral load of each group We thank Dr. Qian Gao of Sinovac for providing CPE neutralization titrations free of 736 charge.