key: cord-0287216-p3af4pmx authors: Davenport, Bennett J.; Catala, Alexis; Weston, Stuart M.; Johnson, Robert M.; Ardunay, Jeremy; Hammond, Holly L.; Dillen, Carly; Frieman, Matthew B.; Catalano, Carlos E.; Morrison, Thomas E. title: Phage-like particle vaccines are highly immunogenic and protect against pathogenic coronavirus infection and disease date: 2021-11-09 journal: bioRxiv DOI: 10.1101/2021.11.08.467648 sha: 97e803caa3b8cd739abc36b9dffa0ebd0bc068ce doc_id: 287216 cord_uid: p3af4pmx The response by vaccine developers to the COVID-19 pandemic has been extraordinary with effective vaccines authorized for emergency use in the U.S. within one year of the appearance of the first COVID-19 cases. However, the emergence of SARS-CoV-2 variants and obstacles with the global rollout of new vaccines highlight the need for platforms that are amenable to rapid tuning and stable formulation to facilitate the logistics of vaccine delivery worldwide. We developed a “designer nanoparticle” platform using phage-like particles (PLPs) derived from bacteriophage lambda for multivalent display of antigens in rigorously defined ratios. Here, we engineered PLPs that display the receptor binding domain (RBD) protein from SARS-CoV-2 and MERS-CoV, alone (RBDSARS-PLPs, RBDMERS-PLPs) and in combination (hCoV-RBD PLPs). Functionalized particles possess physiochemical properties compatible with pharmaceutical standards and retain antigenicity. Following primary immunization, BALB/c mice immunized with RBDSARS- or RBDMERS-PLPs display serum RBD-specific IgG endpoint and live virus neutralization titers that, in the case of SARS-CoV-2, were comparable to those detected in convalescent plasma from infected patients. Further, these antibody levels remain elevated up to 6 months post-prime. In dose response studies, immunization with as little as one microgram of RBDSARS-PLPs elicited robust neutralizing antibody responses. Finally, animals immunized with RBDSARS-PLPs, RBDMERS-PLPs, and hCoV-RBD PLPs were protected against SARS-CoV-2 and/or MERS-CoV lung infection and disease. Collectively, these data suggest that the designer PLP system provides a platform for facile and rapid generation of single and multi-target vaccines. 7 reaction mixtures by agarose gel electrophoresis (AGE) demonstrated that particles can be 144 decorated in a defined manner with either and both gpD-RBD constructs (Fig 3A-C) . Decorated particles were purified by SEC (Fig 3D) and characterized by multiple 146 approaches. Electron microscopy (EM) confirmed that decorated PLPs retain icosahedral 147 symmetry and are well dispersed. Close inspection of the micrographs revealed that particles 148 decorated with RBD densities greater than 20% are characterized by surface projections whose 149 number increases with increasing surface decoration (Fig 3E) ; we attribute these projections to 150 RBD proteins extending from the particle surface. Physiochemical characterization of PLP 151 preparations by dynamic and electrophoretic light scattering analyses reveal that the mean 152 hydrodynamic diameter of particles decorated with RBDs also increases as a function of RBD 153 surface density and have an overall surface charge that ranges from -12 to -29 mV ( Table 1) . The 154 polydispersity index (PDI) of the decorated particles ranges from (0.156 ± 0.05) to (0.285 ± 0.09). In sum, these data indicate that these particle preparations have a high degree of homogeneity 156 and are acceptable by pharmaceutical standards. titers comparable to those detected in convalescent samples from SARS-CoV-2 recovered specific IgG out to day 174 post-prime (Fig 4B) , indicating that these particles elicit durable immunized with WT PLPs at all time points evaluated (Fig 4B) . Furthermore, IgG subclass 176 analysis at 14 days post-prime and boost revealed that 60% RBDSARS-PLPs elicit high levels of mice immunized with WT PLPs did not display neutralizing activity (Fig S1) , whereas serum from 181 mice immunized with 60% RBDSARS-PLPs 14 days post-prime neutralized SARS-CoV-2 infection 182 comparable to the neutralizing activity detected in convalescent samples from SARS-CoV-2 183 recovered patients (Fig 4E-F) . Neutralizing activity was enhanced 4.7-fold following the booster 184 immunization (P < 0.05), and high levels of SARS-CoV-2 neutralizing antibodies were maintained 185 out to day 174 post-prime (Fig 4E and Fig S1) . Vaccination with RBDSARS-PLPs protects from virulent SARS-CoV-2 challenge. We 187 evaluated the protective activity of particles decorated with gpD-RBDSARS using a recently 188 developed mouse-adapted strain of SARS-CoV-2 (SARS-CoV-2 MA10), which productively 189 replicates in the mouse lung and results in clinical manifestations of disease consistent with 190 severe COVID-19 in humans (55). Six-week-old BALB/c mice were immunized i.m. with WT PLPs 191 or 60% RBDSARS-PLPs. At 184 days post-prime, mice were challenged intranasally with euthanized, and lungs were collected for viral burden analysis and histopathology. While high levels of infectious virus were detected in the lungs of mice immunized with WT PLPs, infectious 197 virus was undetectable in the lungs of mice immunized with 60% RBDSARS-PLPs, as determined 198 by plaque assay (Fig 5B) . Moreover, greatly reduced levels of genomic (Fig 5C) and subgenomic ( Fig 5D) viral RNA were detected in the lungs of mice vaccinated with 60% RBDSARS-PLPs, as 200 compared to those immunized with WT PLPs. Collectively, these data indicate that i.m. immunization with RBDSARS-PLPs elicits durable protection against SARS-CoV-2 lung infection. The effect of immunization with RBDSARS-PLPs on lung inflammation and disease also was 203 assessed by analyzing lung tissue for histopathological changes. Mice immunized with WT PLPs 204 and challenged with SARS-CoV-2 MA10 had an abundant accumulation of immune cells in 205 perivascular and alveolar locations, vascular congestion, and interstitial edema (Fig 5E) . Conversely, immunization with 60% RBDSARS-PLPs resulted in a marked reduction of lung-207 associated histopathological changes (Fig 5E) , indicative of protection against SARS-CoV-2 208 induced lung inflammation and injury. CoV-2 challenge. We next evaluated the immunogenicity and protective efficacy of de-escalating 211 doses of particles decorated with gpD-RBDSARS. Six-week-old BALB/c mice were immunized by 212 i.m. injection with 2.5, 1.0, or 0.25 μg of 60% RBDSARS-PLPs and received a booster dose of the 213 same amount three weeks later. Control mice received two i.m. injections of 2.5 μg of WT PLPs, 214 following the same immunization schedule. Serum samples were collected on days 14, 35 (14 evaluated by ELISA. At 14 days, little response was observed in mice immunized with a single 217 injection of 0.25 μg of 60% RBDSARS-PLPs, and no response was detected in control mice (Fig 218 6A ). In contrast, mice immunized with 2.5 or 1.0 μg of 60% RBDSARS-PLPs had detectable levels 219 of RBDSARS-specific IgG which were comparable to mice immunized with a 10 μg dose (compare 220 Figs 4B and 6A), and to RBDSARS-specific IgG detected in convalescent samples from SARS-IgG responses were elevated in mice that received all doses of 60% RBDSARS-PLPs, although immunized with all doses maintained high levels of RBDSARS-specific IgG up to 84 days post-prime 225 (Fig 6A) . We also evaluated serum from this dose de-escalation study for the capacity to neutralize 227 SARS-CoV-2 infection (Fig 6B and Fig S2) . Serum from mice immunized with WT PLPs did not 228 display neutralizing activity (Fig S2) . While day 14 post-prime serum from mice immunized with 229 all doses of 60% RBDSARS-PLPs displayed little to no neutralizing activity, day 35 (14 days post-230 boost) serum from mice immunized with 2.5 or 1.0 μg of 60% RBDSARS-PLPs displayed potent 231 neutralizing activity that was maintained up to 84 days post-prime (Fig 6B) . Despite detectable 232 levels of RBDSARS-specific IgG, immunization with 0.25 μg of 60% RBDSARS PLPs did not result in 233 neutralizing activity at any of the time points evaluated (Fig 6B) . Mice immunized with de-escalating doses of 60% RBDSARS PLPs were challenged with 235 10 4 PFU of SARS-CoV-2 MA10 at day 90 post-prime. Compared to control mice, mice immunized 236 with all doses (including 0.25 μg) of 60% RBDSARS PLPs were protected from weight loss 237 associated with SARS-CoV-2 infection (Fig 6C) . At 4 dpi, lungs were collected and assessed for 238 viral burden. Immunization with 2.5 or 1.0 μg of 60% RBDSARS-PLPs resulted in potent protection 239 against viral infection (Fig 6D) and reduced levels of viral genomic and subgenomic RNA (Fig 240 6E ,F) in the lungs. Furthermore, histopathological analysis of lung tissues of mice immunized at 241 these doses showed minimal perivascular and alveolar infiltrates at 4 dpi, whereas those of 242 control mice were characterized by extensive inflammation (Fig 6G) . Mice immunized with 0.25 243 μg of 60% RBDSARS PLPs had levels of infectious virus and viral RNA in the lungs similar to those 244 in control mice (Fig 6D-F) , consistent with an inability to neutralize SARS-CoV-2 infection (Fig for protection against SARS-CoV-2 infection and suggest that additional adaptive immune 248 response elicited by vaccination with RBDSARS-PLPs may contribute to protection against severe 249 disease characterized by more extensive weight loss, such as the generation of memory T cells. Vaccination with mosaic hCoV-PLPs protects from virulent SARS-CoV-2 and MERS-251 CoV challenge. As described above, PLPs can be decorated with multiple hCoV RBDs at varying 252 surface densities to generate mosaic PLPs. Thus, we evaluated the immunogenicity and 253 protective efficacy of particles simultaneously decorated with gpD-RBDSARS and gpD-RBDMERS in 254 comparison with their mono-RBD decorated counterparts (Fig 7) . Six-week-old BALB/c mice were (Fig 7A, 7B ). In addition, immunization with 20% hCoV-RBD-PLPs induced potent neutralizing 263 antibody responses against both SARS-CoV-2 ( Fig 7C) and MERS-CoV (Fig 7D) . We note, 264 however, that neutralizing titers against MERS-CoV were somewhat lower in mice immunized 265 with the bi-valent vaccine compare with the mono-valent MERS vaccine (Fig 7D) . Next, these mice were challenged with 10 4 PFU of SARS-CoV-2 MA10 or 10 5 PFU MERS- CoV at day 102 post-prime. Five days prior to MERS-CoV challenge, mice were i.n. inoculated 268 with Ad-hDPP4 to deliver the MERS-CoV cell entry receptor to lung cells (56, 57) . Compared with 269 control mice, mice immunized with either RBDSARS-PLPs or hCoV-PLPs were protected from 270 weight loss (Fig 7E) and lung viral burden (Fig 7F) associated with SARS-CoV-2 infection. In this study, we designed and constructed lambda PLPs decorated with the RBDs from used in the enzyme immunoassays binds to a conserved epitope in the RBD that is only 330 accessible when the S protein is in conformation competent to bind the hACE2 receptor (79, 80). Additionally, RBD-specific IgG subclass responses were assessed; however, further 332 characterization of the potential role of these in vaccine-mediated protection was not determined. Considering the implications these may have to the development of safe vaccines (e.g., clinical 334 syndromes associated with vaccine-enhanced disease) (81), additional studies are needed to 335 examine the possible impacts to cellular effector function and evaluate mucosal and cellular 336 immunity in addition to immunity against variants of SARS-CoV-2 and MERS-CoV. In summary, the versatility and robustness of the lambda system presented provides a SARS-CoV-2 genomic or subgenomic RNA copies were measured by qPCR using the primer and 521 probe combinations listed in Table S1 (Integrated DNA Technologies). To quantify SARS-CoV-2 522 genomic RNA, we extrapolated viral RNA levels from a standard curve generated from known 523 FFU of SARS-CoV-2 from which RNA was isolated and cDNA generated as previously described were overlaid with 1% (w/v) methylcellulose in MEM plus 2% FBS and incubated at 37°C. At 3 538 dpi, overlays were removed, and plates were fixed with 4% PFA for 20 minutes at room 539 temperature. After removal of PFA, plates were stained with 0.05% (w/v) crystal violet in 20% 540 methanol (10-20 min). Crystal violet was removed, and plates were rinsed with water or PBS. Plaques were counted manually to determine infectious virus titer. Histopathology. Mice were euthanized at 4 days following SARS-CoV-2 MA10 or MERS- CoV challenge. The lungs were removed and fixed with 10% formalin. 5-micron sections were 544 stained with H&E for histological examination. Slides were examined in a blinded fashion for total 545 inflammation, periarteriolar, and peribronchiolar inflammation and epithelial cell denuding. 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Kim for providing the Balb/c