key: cord-0842756-an7nje0q
authors: Chaparian, Ryan R.; Harding, Alfred T.; Riebe, Kristina; Karlsson, Amelia; Sempowski, Gregory D.; Heaton, Nicholas S.; Heaton, Brook E.
title: Influenza viral particles harboring the SARS-CoV-2 spike RBD as a combination respiratory disease vaccine
date: 2021-04-30
journal: bioRxiv
DOI: 10.1101/2021.04.30.441968
sha: 0f33dbcf47f87fa5fd4ce9f91df3ab7c5e2e7bf1
doc_id: 842756
cord_uid: an7nje0q

Vaccines targeting SARS-CoV-2 have gained emergency FDA approval, however the breadth against emerging variants and the longevity of protection remains unknown. Post-immunization boosting may be required, perhaps on an annual basis if the virus becomes an endemic pathogen. Seasonal influenza virus vaccines are already developed every year, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed a recombinant influenza A virus (IAV) genetic platform that “reprograms” the virus to package an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicits neutralizing antibodies and provides protection from lethal challenge with both pathogens. This technology may allow for leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses.

Every year, sufficient influenza virus vaccines to vaccinate the global population are 47 produced, distributed, and administered 1 . These vaccines are widely accepted to be safe 48 and efficacious, however they must be constantly reformatted due to viral antigenic drift 2, 49 3, 4 . Additionally, a robust reverse genetics system for influenza viruses has been 50 developed, allowing for precise manipulation of the influenza viral genome 5 . Reverse 51 genetics has allowed for the introduction of non-influenza proteins and immune epitopes 52 into influenza viral strains 6, 7, 8 . Thus, at least in theory, leveraging existing influenza virus 53 vaccine production infrastructure to produce recombinant viral strains that express 54 immunogenic antigens from other pathogens of concern may be a practical approach to 55 generating cost-effective, easily implemented combination vaccines or boosters. 56 57 SARS-CoV-2 is a respiratory RNA virus that causes COVID-19 disease that is similar in 58 many respects to influenza-virus induced disease 9 . While a number of vaccines designed 59 to vaccinate immunologically naïve people and provide protection from COVID-19 are 60 either in use under emergency authorization from the FDA or in advanced stages of 61 development, these vaccines are for the most part: expensive, associated with higher-62 than-standard side effects, and difficult to produce/distribute 10, 11, 12, 13 . Further 63 complicating vaccination efforts is the emergence of variant or mutant strains of SARS-64

CoV-2 that have been associated with reduced protective vaccine efficacy 14 . Additionally, 65 protective immunity against human coronaviruses in general, is thought to be relatively 66 short lived 15, 16, 17, 18 . Thus, it is likely that a cost-effective, scalable, and safe vaccine to 67 periodically boost immunity against SARS-CoV-2 will be needed. 68

In order to develop a platform-based solution to regularly boost immunity against SARS-70

CoV-2 and associated variants, we have developed and tested a combination influenza 71

virus-based vaccine that can incorporate both IAV and SARS-CoV-2 antigens. By 72 generating a replication competent IAV that encodes, stably expresses, and packages a 73 key immunogenic domain of the SARS-CoV-2 S protein, we have generated a 74 combination vaccine that can be manufactured in the exact same way most influenza 75 TM-RBD HA viruses and analyzed their protein composition via western blot. We first 122 normalized loading based on the levels of the IAV matrix (M1) protein in the two samples. 123

The M1 protein forms the viral capsid and is not expected to be altered by changes to 124 glycoprotein composition 23 . As expected, we observed that SARS-CoV-2 RBD was 125 detectable in the recombinant RBD virus, but not in the WT virus (Figure 2A) . In order to 126 determine the influence of the incorporation of another protein in the viral envelope on the 127 native influenza viral surface proteins, we probed for expression of M2 as well as the HA 128 and NA proteins. While the levels of the M2 protein were unchanged, there was a slight 129 reduction in the amount of HA and an increase in the amount of NA packaged into nascent 130 virions compared to WT virus (Figure 2A) . Thus, this genetic approach can facilitate 131 packaging of a foreign protein onto a viral particle without dramatic effects on native viral 132 protein incorporation. 133

We also wanted to assess the protein packaging under non-denaturing conditions by 135 performing enzyme-linked immunosorbent assays (ELISAs) with fully intact virions. 136

Confirming our western blot results, we observed a statistically significant but minor 137 reduction of HA in the recombinant TM-RBD HA virus compared to unmodified WT virus 138 ( Figure 2B) . We further probed for the SARS-CoV-2 RBD using antibodies that recognize 139 either linear or structural epitopes. A non-conformation specific antibody raised against 140 the RBD yielded strong signal only against the RBD virus ( Figure 2C ). To test if the RBD-141 TM fusion protein was folding correctly, we utilized DH1041 and DH1044 which are 142 conformation specific human monoclonal antibodies that bind to the SARS-CoV-2 RBD 143 and also display neutralizing abilities 24 . Both of these antibodies specifically bound the 144 TM-RBD HA virus and not the parental WT IAV (Figure 2D-E) . Taken together, these 145 results indicate that the recombinant TM-RBD HA virus packages properly folded SARS-146

CoV-2 RBD protein while maintaining the packaging of the other IAV envelope proteins 147 HA, NA, and M2. 148

The H1N1/SARS-CoV-2 combination vaccine elicits IAV and SARS-CoV-2 neutralizing 150 antibodies 151 Our next step was to define if the recombinant IAV/SARS-CoV-2 virus elicited the desired 152 immune responses after vaccination. We therefore took naïve mice and primed them 153 with a live vaccination of either WT or the combination IAV/SARS-CoV-2 viral vaccines. 154

Three weeks later, the animals were boosted with an IM injection of the inactivated 155 vaccine preps or BSA as a negative control. Two weeks after the boost, peripheral blood 156 was collected and serum reactivity against the IAV HA protein or the SARS-CoV-2 RBD 157 protein, as well as the neutralizing viral titers against IAV and SARS-CoV-2, were 158 determined ( Figure 3A) . 159

We observed high serum IgG reactivity against the IAV HA protein in both experimental 161 vaccine groups but not the BSA control ( Figure 3B) . Further, there was no significant 162 differences in the reactivity against HA between the two viral vaccine groups, despite 163 there presumably being slightly less HA in the IAV/SARS-CoV-2 combination vaccine 164 preparation ( Figure 3C ). For both experimental vaccine groups, the high antibody 165 binding reactivity was correlated with high antibody neutralizing activity against authentic, 166 infectious H1N1 IAV ( Figure 3D ). The average 50% plaque reduction neutralization titer 167 (PRNT50) was calculated to be 1:3236 for WT and 1:2767 for TM-RBD HA, however those 168 values were not significantly different from each other ( Figure 3E) . The development and production of novel vaccines is frequently labor intensive, costly, 204 and difficult to scale upon high demand. The existing infrastructure for producing influenza 205 vaccines is highly optimized and capable of delivering more than a billion doses per 206

year 28 . In order to potentially leverage influenza vaccine infrastructure for the production 207 Despite the data presented in this study, there remain a number of questions that need 228 to be answered. Perhaps most obvious question is if the S protein RBD is the most 229 appropriate antigen relative to either the full S protein or other coronaviral proteins. While 230 clearly vaccine responses against the RBD are sufficient to mediate some protection, it 231

will be important to determine if boosting only RBD directed responses is ultimately an 232 efficacious strategy in people. Further, our genetic manipulation of the virus was 233 associated with a reduction in viral yield. Additional studies will be required to understand 234 how decreased yield would affect the eventual vaccine cost or timelines for 235 manufacturing. Finally, we have only utilized one genetic background and corresponding 236 set of viral glycoproteins for these studies. While PR8 is a standard background for 237 vaccine development, it will also be important to understand if other H1 (or indeed, H3 or 238 influenza B virus) HA proteins are amenable to our manipulations to segment length and 239 composition such that a membrane anchored RBD domain can be functionally encoded. 240 241

In conclusion, the influenza-based, multi-valent vaccines may represent a generalizable 242 approach to reduce the time and manufacturing requirements for development of novel 243

vaccines. Since the current influenza vaccine is composed of three or four distinct 244 strains 34 , this approach could be even more highly multiplexed than eliciting responses 245 against two pathogens. While there remain questions to be answered and technical 246 challenges to overcome, "reprogramming" influenza viruses may be an attractive 247 approach to produce and package antigens that are difficult to purify or are poorly 248

immunogenic on their own. Continued work on this and other generalizable vaccine 249 platforms will not only help with the current response to the COVID-19 pandemic but will 250 help poise us for rapid response during future epidemic/pandemic outbreaks. 

by IDT. Importantly, we also encoded the neuraminidase (NA) transmembrane domain 286 (amino acids 1-40 of the NA open reading frame) 5' to the RBD to allow it to be 287 incorporated into the viral particle, and a FLAG tag 3' to the RBD to aid in detection as 288 needed. Once this was done, the codon optimized RBD was PCR amplified and cloned 289 into the bicistronic pDZ rescue plasmid system for A/Puerto Rico/8/1934 using the 290 NEBuider HiFi DNA assembly kit (NEB). Specifically, the SARS-CoV-2 RBD-TM construct 291 was cloned into the previously reported mNeon-HA construct, wherein the RBD-TM 292 sequence replaced the mNeon reporter allowing expression of the transgene 5' to the HA 293 22 . Successful cloning was then confirmed by Sanger sequencing. Viral rescue was then 294 performed by transfecting the modified RBD-TM HA plasmid alongside the 7 plasmids 295 encoding the other PR8 segments into 293Ts using the Mirus TransIT-LT1 reagent. 296

chicken eggs (Charles River) at 37 °C for three days. 298 299

Influenza virus titering was performed as previously reported 22 . Briefly, approximately 500 301 PFU of each plaque-purified stock was injected in 10-day old embryonated hen eggs 302 purchased from Charles River Laboratories, Inc. and incubated for 72 hours at 37 °C. The 303 allantoic fluid was then harvested from and titer determined via plaque assay on MDCK 304

MDCKs with 500 µL of the diluted sample for 1 hour at 37 °C. After incubation, the viral 306 suspension was aspirated, agar overlay was applied, and cells were incubated at 37 °C 307 for 48-72 hours depending on plaque size. Plaque assays were then fixed by adding 2mL 308 of 4% paraformaldehyde solution and incubating overnight at room temperature in a fume 309 hood. The next day paraformaldehyde was aspirated, and cells were washed prior to 310 performing antigen staining to detect A/Puerto Rico/8/1934 HA protein as described 311 above. For viral growth curves and endpoint titer assays, eggs were injected with 10,000 312 pfu/egg and eggs were collected for plaque assay at 24, 48, and 72 hours post infection. 313

Hemagglutination assays were performed by serially diluting virus 1: 2 in PBS in a 96 well 314 plate then adding chicken blood to each well. Plates were incubated 1 hour then each 315 well was scored as positive or negative. SARS-CoV-2 stocks were grown on Vero E6 316 cells in virus infection media (MEM+Earl's Salts, penicillin/streptomycin, 2% FBS, 1mM 317 Na Pyruvate, 1x MEM NEAA) for 72 hours. Stocks were frozen at -80 °C and were titered 318

Vero E6 cells growing in 6-well, poly-L-lysine treated, plates for 1 hour. Inoculum was 320 then removed and an agarose overlay was added. Cells were incubated at 37 °C and 321 5% CO2 for 72 hrs then were stained with 0.05% neutral red in PBS for 3 hours. 322 323

Purification of influenza viral particles was performed prior to use in vaccination and 325 ELISA experiments. First, viral stocks were grown in 10-day old embryonated hen eggs 326 as described above. Then allantoic fluid was collected and dialyzed overnight using the 327 Spectra-Por Float-a-lyzer G2 10 mL, 100 kDA MWCO tubes according to manufacturer's 328 instructions (Millipore Sigma cat. no. Z727253-12EA). After samples were dialyzed to 329 remove larger impurities, the allantoic fluid was collected and virus samples concentrated 330 by ultracentrifugation using a 30% sucrose cushion for 1 hour at 25,700 RPM using the 331 Sorvall TH-641 swinging bucket rotor. Virus samples were then resuspended in PBS and 332 pooled prior to being fixed in 0.02% formalin for 30 minutes at room temperature. Samples 333 were then once again dialyzed overnight to remove formalin using Slide-A-Lyzer casettes 334 (ThermoScientific cat. no. PI66370) before being stored at 4 °C until use. 

Protein extracts were quantified and normalized via Bradford assay. SDS-PAGE was 370 performed using 4-20% polyacrylamide gels (BioRad) electrophoresed at 120V for 60 371 minutes. Proteins were transferred to 0.45 µm nitrocellulose membranes at 90V for 60 372 minutes at 4 °C and blocked using PBST + 5% milk for a minimum for 1 hour at room 373 temperature. For cellular lysates, 20 µg of total protein was loaded per sample. To 374 normalize viral protein extracts, 0.5 µg of PR8 and PR8-TM-RBD HA were initially loaded 375 and analyzed via western blot. Viral protein extracts were probed for M1 and normalized 376 via densitometry (ImageJ). After normalization to M1, 0.5 µg PR8 and 1.32 µg PR8-TM-377 RBD HA were loaded for subsequent western blot analyses. The following antibodies 378

were used for protein detection; PY102 (HA, 1 µg/mL), 4A5 (NA, 0.45 µg/mL), anti-matrix 379 protein [E10] (M1 and M2, 1:1,000, Kerafast cat. no. EMS009), and anti-SARS-CoV-2 380 spike protein (RBD) polyclonal antibody (RBD, 1:1,000, Invitrogen cat. no. PA5-114451) . 381

All primary antibodies were diluted in PBST + 5% milk and applied to membranes for ≥16 382 hours at 4 °C. Anti-mouse (1:20,000, Thermo cat. no. A16072) and anti-rabbit (1:10,000, 383

Thermo cat. no. A16104) secondary antibodies were diluted in PBST + 5% milk and 384 applied to membranes for 60 minutes at room temperature. Membranes were developed 385 using Clarity ECL or Clarity ECL MAX. 386 387

Proteins/viruses were immobilized to 96-well plates using carbonate coating buffer (30 389 mM Na2CO3, 70 mM NaHCO3, pH 9.5) for ≥16 hours at 4 °C. For protein samples, 50-390 100 µL of sample at 10 µg/mL was added to wells. For viral samples, 1 x 10 6 PFUs were 391 added to wells in a volume of 50-100 µL. All samples were diluted using PBS + 3% BSA. Vaccine doses for the boost were prepared using the purified inactivated virus described 429 above. Virus samples were then diluted 1:1 with the adjuvant Addavax (Invivogen cat. no. 430

vac-adx-10) to a final concentration of 100 µg/mL. After preparation of doses, mice were 431 anesthetized as described above and then administered a 100 µL injection 432 intramuscularly into the left leg. Mice were then monitored the next day for side effects 433 and then housed for the indicated period of time before collection or viral challenge. If 434 serum was collected, mice were administered a lethal dose of the ketamine-xylazine (200 435 µL) and then blood was harvested and serum collected using Sarstedt Z-Gel tubes 436 according to manufacturer's instruction (Sarstedt cat. no. 41.1378.005) . Serum was then 437 stored at -20 °C until use. 438 439 Plaque reduction neutralization assays 440 MDCK and Vero cells were used for influenza and SARS-CoV-2 plaque reduction assays, 441 respectively. A master mix of virus was diluted to the indicated concentration (~40-80 442 PFU/mL) and aliquoted prior to being mixed with antibody dilutions. Following a 45-minute 443 incubation at room temperature with antibody, the media was aspirated from cells and 444 500 µL of the virus-antibody mixture was added to each well of cells. For each experiment 445 a no antibody control was included to accurately record how much virus had been used 446 to infect cells. Cells were incubated with the virus-antibody mixture at 37 °C for 1 hour, 447 rocking the samples every 15 minutes to ensure cells were completely covered by the 448 solution. After this period, the solution was aspirated, and an agar overlay was applied. 449

For influenza plaque reduction assays, staining and plaque counting was performed as 450 described above in the titering section. SARS-CoV-2 plaque reduction assays were 451 evaluated by first staining plaques with .05% Neutral Red solution for 3 hours at 37 °C 452 (Sigma Aldrich cat. no. N2889-100mL). Neutral red was then aspirated from the wells and 453 plaques were counted. The percent reduction in plaques was calculated in reference to 454 a no sera control. The PRNT50 value was calculated by using a non-linear regression 455 model, [Agonist] vs. response-Find ECanything with f constrained to 50. The reciprocal 456 50% neutralization titer was calculated by averaging the greatest dilution of mouse sera 457 that had a >50% reduction in plaques compared to a no sera control for each sera sample 458 in a vaccination group. indicates the stock of virus used for the experiment. Statistical analyses were performed 596 using unpaired t-tests. For all panels, P-values denoted with asterisks correspond to the 597 following values: * < 0.05, *** < 0.001, and ns = not significant. 598 599 600 601

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Figure 2. The TM-RBD-HA virus incorporates appropriately folded RBD without 605 disrupting incorporation of the IAV viral envelope proteins. (A) Western blot analysis 606 of WT and TM-RBD HA viruses. Samples were normalized via M1 protein signal using 607 pixel densitometry. (B) (left) ELISAs using the PY102 anti-HA antibody against whole 608 virus particles and (right) area under the curve analysis

ELISAs using a SARS-CoV-2 neutralizing 611 antibody (DH1041, binds a structural epitope on the RBD), against whole virus particles 612 and (right) area under the curve analysis. (E) Same analysis as in (D) using a different 613 conformation-specific SARS-CoV-2 neutralizing antibody, DH1044. Statistical analyses 614 were performed using unpaired t-tests

Experimental design displaying 621 vaccination strategy, sample collection, and downstream assays. (B) ELISAs using serum 622 from vaccinated mice against purified soluble HA protein. (C) Area under the curve 623 analysis for (B). (D) IAV plaque reduction neutralization tests (PRNT) with serum from 624 vaccinated mice. (E) Reciprocal 50% neutralization titer using IAV plaque neutralization 625 data. (F) ELISAs using serum from vaccinated mice against purified soluble SARS-CoV-626 2 RBD protein. (G) Area under the curve analysis for (B). (H) SARS-CoV-2 plaque 627 reduction neutralization tests (PRNT) with serum from vaccinated mice. (I) Reciprocal 628 50% neutralization titer using SARS-CoV-2 plaque neutralization data. Statistical 629 analyses were performed using unpaired t-tests

Mice vaccinated with TM-RBD HA virus are protected from lethal disease 635 after challenge with IAV and SARS-CoV-2. (A) Diagram illustrating vaccination strategy 636 and lethal challenges using IAV or SARS-CoV-2. (B) Bodyweight measurements of mice 637 vaccinated with TM-RBD HA virus, WT IAV virus, or BSA and subsequently challenged 638 with a lethal dose of A/Puerto Rico/8/1934 virus

Bodyweight measurements of mice vaccinated with TM-RBD HA virus or WT IAV virus 640 and subsequently challenged with a lethal dose of USA-WA1/2020 SARS-CoV-2 virus. 641 (E) Survival of mice from (D)