key: cord-0849901-jzxvj0uq
authors: Chakraborty, Krishnendu; Chandrashekar, Abishek; Sidaway, Adam; Latta, Elizabeth; Yu, Jingyou; McMahan, Katherine; Giffin, Victoria; Manickam, Cordelia; Kroll, Kyle; Mosher, Matthew; Reeves, R. Keith; Gam, Rihab; Arthofer, Elisa; Choudhry, Modassir; Barouch, Dan H; Henley, Tom
title: A Novel CRISPR-Engineered, Stem Cell-Derived Cellular Vaccine
date: 2021-12-29
journal: bioRxiv
DOI: 10.1101/2021.12.28.474336
sha: 18c368412fd880a6d8e5185d93fcc977b40a0f50
doc_id: 849901
cord_uid: jzxvj0uq

COVID-19 has forced rapid clinical translation of novel vaccine technologies, principally mRNA vaccines, that have resulted in meaningful efficacy and adequate safety in response to the global pandemic. Notwithstanding this success, there remains an opportunity for innovation in vaccine technology to address current limitations and meet the challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) rationally designed to mimic the natural physiologic immunity induced post viral infection of host cells. Induced pluripotent stem cells were CRISPR engineered to delete MHC-I expression and simultaneously overexpress a NK Ligand adjuvant to increase rapid cellular apoptosis which was hypothesized to enhance viral antigen presentation in the resulting immune microenvironment leading to a protective immune response. Cells were further engineered to express the parental variant WA1/2020 SARS-CoV-2 spike protein as a representative viral antigen prior to irradiation and cryopreservation. The cellular vaccine was then used to immunize non-human primates in a standard 2-dose, IM injected prime + boost vaccination with 1e8 cells per 1 ml dose resulting in robust neutralizing antibody responses (1e3 nAb titers) with decreasing levels at 6 months duration. Similar titers generated in this established NHP model have translated into protective human neutralizing antibody levels in SARS-Cov-2 vaccinated individuals. Animals vaccinated with WA1/2020 spike antigens were subsequently challenged with 1.0 × 105 TCID50 infectious Delta (B.1.617.2) SARS-CoV-2 in a heterologous challenge which resulted in an approximately 3-log order decrease in viral RNA load in the lungs. These heterologous viral challenge results reflect the ongoing real-world experience of original variant WA1/2020 spike antigen vaccinated populations exposed to rapidly emerging variants like Delta and now Omicron. This cellular vaccine is designed to be a rapidly scalable cell line with a modular poly-antigenic payload to allow for practical, large-scale clinical manufacturing and use in an evolving viral variant environment. Human clinical translation of the UVC is being actively explored for this and potential future pandemics.

vaccines have established the capability of a rapid global vaccination program 1, 2, 3 . However, the 72 waning antibody responses seen with these emergency-use authorized vaccine technologies, and the 73 need for vaccine boosters, has highlighted the requirement for further improvements in vaccine 74 approaches to drive higher, longer-lasting protective immunity 4, 5, 6, 7, 8, 9 . The newly emerging viral 75 variants of SARS-CoV-2, and the evident reduced efficacy of the existing vaccines to protect against 76 transmissible and symptomatic infection of these variants, also highlights the need for vaccines that 77 can ideally deliver multiple variant antigens (polyvalency) and be rapidly manufactured at scale as 78 soon as new viral variants are discovered 10, 11, 12, 13 . 79 Theoretically, an ideal vaccine technology would have four core attributes, namely: hyper-80 immunity, self-adjuvancy, polyvalency and scalability. The first hyper-immunity is self-evident and 81 speaks to the requirement of generating a robust humoral neutralizing antibody, and ideally a 82 subsequent T cell amnestic response, such that protection remains durable. Self-adjuvancy, or 83 conversely the absence of the need for exogenous excipients to elicit a hyper-immune response may 84 prove to be a meaningful innovation in that the immune side-effects of current vaccines may be 85 mediated by the non-target antigen specific adjuncts 14 . Thirdly, polyvalency or the ability to protect 86 against multiple immunodominant epitopes, is a core feature of overlapping and orthogonal 87 mechanisms of protection and is a core principle or antibiotic and antiviral therapy in infection to 88 suppress underlying pathogenic genetic drift and mutation and acquired resistance 15, 16 . Lastly, 89 scalability or the ability to deliver preventative doses of vaccines in an economic, large scale and 90 clinically relevant fashion in both the developed and developing worlds, is a sine qua non of any human 91 vaccine.

Current mRNA, protein, and viral vector-based vaccines have certain limitations, such as their 93 requirement for excipient adjuvants to activate the recipient immune system, or to deliver the viral 94 antigenic payload 17, 18 . These include the artificial lipid nanoparticles delivering the mRNA, or MF59, 95 AS03, Alum, ISCOMATRIX, and Matrix-M chemical emulsions for example, or the adenoviral protein 96 antigens themselves that stimulate innate immune cell activation 18, 19, 20, 21, 22, 23, 24 . Adjuvants are 97 required to increase the effectiveness of vaccines and their use can cause side-effects including local 98 reactions (redness, swelling, and pain at the injection site) and systemic reactions (fever, chills, and with Thrombosis with Thrombocytopenia Syndrome (TTS) and Myocarditis, secondary to the current 101 26, 27 .

The size constraint of the adenoviral vector genome, and the limited length of stable mRNA 103 that can be produced and packaged into nanoparticles, restricts the number and size of nucleic acid-104 encoded antigens and epitopes that can be delivered in these vaccines 28 . Thus, these vaccines are 105 constrained in their ability to provide multiple immunodominant proteins to address emerging 106 pandemic variants, or to easily combine multiple pathogens into one vaccine. 

We first genetically engineered iPS cells to create an immunogenic phenotype by stable 146 integration of the SARS-CoV-2 full length spike antigen into the AAVS1 safe-harbor locus using 147 CRISPR/Cas9 gene editing (Fig. 1a) . We selected the original and well-characterized WA1/2020 variant 148 of SARS-CoV-2 and spike antigen sequence with mutation of the furin cleavage site and proline-149 stabilizing mutations that is identical to that in the current emergency-use authorized vaccines being 150 deployed globally to vaccinate against 36, 37 (Supplementary Fig. S1 ). By including the spike 151 transmembrane domain sequence in the gene encoding this antigen, we were able to detect high 152 levels of the viral spike on the cell surface of the engineered iPS cells (Fig. 1b) . Spike protein was also 153 readily observed in engineered cell lysates when measured by western blotting (Fig. 1c) . The yield of 154 antigen released upon lysis was quantified using a spike-specific ELISA assay and we observed an 155 abundant and dose-dependent release of protein from the cells, which would equate to approximately 156 ~20 micrograms of spike antigen protein delivered in a 1x10 8 cell vaccine dose of UVC (Fig. 1d ).

To ensure robust delivery of this immunodominant antigen to the recipient immune system, 158 we incorporate an apoptosis-inducing lethal irradiation step during vaccine manufacture by exposing 159 the UVC cells to a 10 Gy dose of gamma radiation prior to cryopreservation and vaccination. Thus, 160 when subjects are immunized with the UVC, we reasoned that the cells would undergo apoptosis and 161 release the SARS-CoV-2 spike antigen into the immune microenvironment via production of apoptotic 162 bodies (Fig. 1a) . In theory, these apoptotic bodies will be phagocytosed by innate immune cells and 163 antigen-presenting cells and presented to T and B lymphocytes to generate a spike antigen-specific 164 immune response.

In addition to creating a mechanism for delivery of immunogenic antigens via apoptotic 166 bodies, the irradiation of the UVC can be considered a safety feature as it renders the cells unable to proliferation or persist in vivo upon vaccination. In support of this, we observed a robust elevation in 

Rapid growth kinetics of engineered UVC 197 198 Prior to irradiation and cryopreservation of the UVC ready for immunization, we evaluated the growth 199 kinetics of the cells to confirm the capacity for rapid, scalable proliferation that would be needed for 200 a vaccine technology to address the needs of a pandemic. IPS cells are known to have a relatively short doubling times in the range of 18-20 hours 45, 46 , and we observed similar kinetics with an average 202 exponential growth of >50-fold over a 7-day culture period (Fig. 1i) 

To evaluate the immunogenicity of the UVC and the vaccine's ability to engender a humoral immune 254 response, we immunized cynomolgus macaques and followed the production of neutralizing and 255 spike-specific antibodies over a 10-week period, and a duration follow up at 6 months. We immunized 256 9 macaques, aged 6-12 years old, with either 1x10 7 UVC (n=3) or 1x10 8 UVC (n=3) expressing the 257 WA1/2020 SARS-COV-2 spike antigen, and sham controls (n=3). Macaques received a prime dose 258 immunization by the intramuscular route without adjuvant at week 0, followed by a boost dose 259 immunization (same cell number as prime dose) at week 6 ( Fig. 3a) . Neutralizing antibody responses 260 were assessed using a pseudovirus neutralization assay 49, 50, 51, 52 , and we observed neutralizing 261 antibodies in all UVC vaccinated macaques at week 2 that further increased by week 4 (Fig. 3b) . The 262 higher dose of 1x10 8 UVC resulted in the most robust titers of neutralizing antibodies at all timepoints 263 tested. Following UVC boost dose immunization at week 6, neutralizing antibody titers elevated 264 further, reaching close to 1x10 3 titers with the higher 1x10 8 cell dose. Six months after the initial UVC 265 immunization, neutralizing antibody showed a durable response, and levels in macaques immunized 266 with the 1x10 8 UVC dose remained elevated beyond that seen with the initial prime UVC dose. We 267 also observed robust spike-specific and receptor-binding-domain (RBD)-specific antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) in vaccinated macaques ( Fig. 3c and d) .

These antibody responses and durability at 6 months in the 1x10 8 dose, were like those seen with the 270 neutralizing antibody titers, and thus collectively demonstrating that the UVC vaccine can engender a 271 robust humoral response against the SARS-CoV-2 spike antigen with demonstrable durability. At 6 272 months after immunization, detectible levels of neutralizing antibodies against Beta and Delta were 273 also observed, albeit lower than seen with the immunizing antigen variant WA1/2020 spike, 274 suggesting humoral immunity is also generated against SARS-CoV-2 variants (Fig. 3e) . This prompted 275 us to assess the protective immunity of the humoral immune response generated by UVC 

While neutralizing antibody titers specific for the WA1/2020 variant spike was high in both 295 macaque immunization studies ( Fig. 3 and supplementary Fig. S2 ), the titers specific for other SARS-296 CoV-2 variants (Beta and Delta) was lower, which is to be expected given the divergence in antigen 297 protein sequence. Thus, the partial protection seen in animals immunized with WA1/2020 spike UVC 298 and challenged with B.1.617.2 (Delta) SARS-CoV-2, is also expected given the heterologous nature of 299 the challenge. We would predict a more robust and complete reduction in virus from animals 300 immunized with UVC and challenged with SARS-CoV-2 in which the antigen and variant are matched.

Collectively these data demonstrate that a prime and boost dose of 1x10 8 WA1/2020 Spike expressing 303 UVC promote a robust antigen-specific antibody response with levels of neutralizing antibodies and 304 durability similar to the current approved COVID-19 vaccines 49, 54, 55 , and this can lead to partial 305 protective immunity in a heterologous WA1/2020 versus Delta SARS-CoV-2 virus challenge. 

Cell pellets were harvested and lysed in 20 µl Cell Extraction Buffer (Invitrogen) containing protease 520 inhibitors (Sigma) on ice for 30 minutes, with 3 brief vortexing every 10 minutes. Samples were 521 centrifuged at 13,000 rpm for 10 minutes at 4°C to pellet insoluble contents. S1 Spike protein was 522 detected using a Covid-19 S-protein ELISA kit (Abcam) specific to S1RBD. Samples were diluted to a 523 range determined to be within the working range of the ELISA kit used and the assay procedure was 

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