key: cord-0310663-lxxl60nv
authors: Takanohashi, Asako; Alameh, Mohamad-Gabriel; Woidill, Sarah; Hacker, Julia; Davis, Benjamin; Helman, Guy; Gavazzi, Francesco; Adang, Laura; D’Aiello, Russell; Winters, Patrick; Cordova, Devon; Khandaker, Taibeen; Ni, Houping; Tam, Ying; Lin, Paulo; Weissman, Drew; Shults, Justine; Vanderver, Adeline
title: mRNA-based vaccines against SARS-CoV-2 do not stimulate interferon stimulatory gene expression in individuals affected by Aicardi Goutières Syndrome
date: 2022-05-19
journal: bioRxiv
DOI: 10.1101/2022.05.18.492546
sha: 78f8104abf6b6fbb43f75684b624a6496ac33f2b
doc_id: 310663
cord_uid: lxxl60nv

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses threats to individuals with rare disease, in part because so little is known about the impact of COVID-19 infection and vaccination safety in rare disease populations. Of particular concern, given the overlap in disease manifestations and interferon dysregulation, are a group of heritable autoinflammatory conditions called type I interferonopathies. The most common of these, Aicardi Goutières Syndrome (AGS), is caused by altered nucleic acid metabolism and sensing, resulting in additional concerns surrounding the use of mRNA vaccination approaches. To determine whether mRNA vaccines induce an interferon response in AGS, we applied mRNA SARS-CoV-2 vaccines to whole blood samples and assessed internalization and interferon signaling gene expression responses to the mRNA. In all cases (11 AGS and 11 control samples), interferon signatures did not significantly increase from baseline, regardless of baricitinib treatment status in the AGS subjects, and were even decreased, when using codon optimized SARS-CoV-2 di-proline modified spike sequence (S2P). Internalization of S2P in human dendritic cells was verified by Western Blot, and in control and AGS blood cells was verified by Luciferase activity. Although numbers of tested samples in this rare disease are small, based on these findings, we suggest that COVID vaccination is unlikely to directly stimulate the interferon signaling gene expression in AGS patients via response to mRNA internalization. The in vitro nature of this study cannot exclude an exaggerated interferon response to spike protein production at a systemic level in individuals with a primary heritable interferonopathy. In the context of continued SARS-CoV-2 spread in the community, we do not recommend withholding vaccination in this rare disease group. However, we recommend that vaccinations for AGS patients are provided in a controlled setting with appropriate observation and used with caution in individuals with prior vaccine associated adverse events.

Vaccination represents a safe and effective approach to prevent and control serious, and 28 sometimes deadly, infectious diseases [15] . The ongoing coronavirus disease 2019 29 (COVID-19) pandemic has required rapid development of vaccines to prevent disease, 30 reduce mortality, and curb the ongoing crisis. Approved vaccines have been shown to be 31 largely safe and well tolerated with demonstrated efficacy in both randomized clinical 32 trials (RCTs) and clinical practice [32] . More recently, as mRNA vaccines have been 33 approved for the pediatric population, individuals with rare disease remain a 34 marginalized group in vaccination efforts due to limited safety data. 35 Rare genetic disorders affecting the production of interferon (IFN) are known as 36 type I interferonopathies [9] . The type I interferonopathies include Aicardi Goutières 37 Syndrome (AGS), a neurologic disease with early infantile-onset and clinical features 38 caused by systemic inflammation [10, 11, 23, 24, 26] . This systemic autoinflammatory 39 state is believed to result from poor discrimination between endogenous and exogenous 40 nucleic acids, leading to constitutive upregulation of the IFN pathway [25] . Upregulation 41 of IFN pathways can be assessed by measuring the levels of interferon signaling gene 42 (ISGs) expression from whole blood [17, 27] . ISG signaling in blood samples has been 43 used in AGS clinical trials to understand therapeutic target response [1, 34] . 44 In viral infections, pathogen-derived nucleic acids (NAs) recognition by cell 45 membrane, endosomal, and intracellular sensors leads to the production of type I IFN 46 and subsequent ISG expression. IFN signaling upon SARS-CoV-2 infection is critical for 47 disease control [7] . IFN production results from endosomal recognition by toll-like double-stranded RNA fragments [31] . IFN inhibits viral replication while assisting in 53 stimulating the adaptive immune response. 54 mRNA-based vaccinations have been a rapid development in the clinical arsenal 55 against SARS-CoV-2 infection [28, 35] and other infectious diseases [3] . Translation of 56 mRNA vaccines and other mRNA based therapeutic modalities in clinical trials was 57 enabled by both the discovery that modified nucleosides, and proper purification, 58 improved translation and reduced the innate immune activating potential of in vitro 59 transcribed mRNA, with the parallel development of efficient non-viral delivery 60 systems [2, 20, 22, 36] . mRNA delivery has been enhanced by the addition of lipid 61 nanoparticles (LNPs) as a capsule for the modified mRNA [22] . BNT162b (Comirnaty, 62 Pfizer-BioNTech [35] ) and mRNA-1273 (Spikevax, Moderna [5] ) hereafter referred to by 63 their commercial names, are nucleoside modified mRNA encapsulated into LNPs 64 (mRNA-LNPs). mRNA-based vaccines are thought to provide a safer and more effective 65 form of immunity compared to other vaccine types [14] . Studies from the development 66 of nucleoside-modified mRNA demonstrated a lack of induction of proinflammatory 67 cytokines and type 1 interferons in dendritic cells treated in vitro or after in vivo 68 administration [16] . 69 

AGS is a disorder of disrupted nucleic acid metabolism that may be treated with 70 immunosuppressive medications [13, 33, 34] and Moderna vaccines except for the 3' and 5' UTRs. mRNA was stored at -20°C until 116 use.

Production of empty LNPs and mRNA-LNP vaccines 118 mRNA loaded LNPs, or vaccines, were formulated using a total lipid concentration of 119 40mM as previously described [3] . The ethanolic lipid mixture comprising ionizable 120 cationic lipid, phosphatidylcholine, cholesterol and polyethylene glycol-lipid was rapidly 121 mixed with an aqueous solution containing cellulose-purified UTP or N1-mψ in vitro 122 transcribed mRNAs to generate the unmodified mRNA-LNP vaccine and the nucleoside 123 modified mRNA-LNP resembling the clinical vaccine Comirnaty, respectively [21] . The 124 LNP formulation used in this study is proprietary to Acuitas Therapeutics; the 125 proprietary lipid and LNP composition are described in US patent US10,221,127.

Empty particles were prepared as described above without the addition of mRNA in the 127 aqueous phase. RNA-loaded and empty particles were characterized and subsequently 128 stored at -80°C at an RNA concentration of 1 µg/µL (in the case of loaded particles) 129 and total lipid concentration of 30 µg/µL-1 (both loaded and empty particles). Membranes were revealed using the Amersham ECL prime reagent (GE healthcare) and 165 imaged on an Amersham A600 system. Membranes were stripped and re-blotted in the 166 presence of an anti-GAPH antibody as a loading control. NanoString nCounter® analysis approaches have been previously described for the 186 measurement of ISG expression [17] . After hybridization at 65°C for 16h, nCounter 187 "Elements" sample runs were immediately processed using the "High Sensitivity" We next evaluated the impact of treatment on change from no treatment (Table 3 ) 230 while also allowing the change from no treatment to vary between AGS and non-AGS 231 samples. We accomplished this by fitting QLS regression models on change from no 232 treatment with the following variables: an indicator variable for each of the 4 vaccine 233 types (with untreated as the reference category); an indicator variable for control 234 (versus AGS); baseline ISG (no treatment); and a control by treatment type interaction 235 term. Naked unmodified and naked modified could not be included in this analysis 236 because there was only one AGS sample for these vaccine types. Due to the limited 237 sample size, we included the interaction terms one at a time, in separate models for 238 each treatment type. To account for the correlation among the multiple ISG scores per 239 subject, we fit the exchangeable and unstructured correlation matrices. We evaluated 240 the fit of the exchangeable and robust structures using several criteria and selected the 241 structure with the best fit. We then fit the model for the best fitting correlation 242 structure. After fitting the model, we used the lincom procedure in Stata 17 to estimate 243 the difference in ISG scores (with 95% confidence intervals [CI] and p-value for test that 244 the difference is zero) between treatment types. Figure 1C ). Detection of the protein using western blot or flow 261 cytometry in whole blood was difficult to achieve. In order to demonstrate that these 262 LNPs are able to enter cells and express their cargo following their addition into blood, 263 we used a luciferase reporter mRNA for improved sensitivity. Blood from AGS patients 264 and control subjects was transfected with either Luc mRNA-LNP or empty for 6 hours, 265 and PBMCs were purified, lysed and subjected to the luciferase assay. Luciferase 

AGS and related heritable interferonopathies have unregulated IFN production, which 306 can worsen in the context of infection or other immune triggers [1] . In addition, several 307 AGS related genes are involved in the detection (sensing) and the regulation of 308 endogenous nucleic acids [11, 12, 23, 24, 26] . This has led to concern that nucleic acid 309 based vaccines such as the FDA approved Comirnaty [35] and Spikevax [5] 310 mRNA-based vaccines could result in further dysregulation of IFN signaling and has led 311 to hesitancy around vaccine adoption in this rare disease population.

Our experiments do not suggest an increase in IFN signaling, as measured by the 313 calculation of an ISG score, after internalization of the clinical S2P in a LNP (Table 1 , 314 Figure 1 ). Although unmodified mRNA vaccines increase ISGs in both control and AGS 315 samples, the internalization of the nucleoside modified mRNA vaccine representing the 316 clinical vaccine significantly decreases ISGs relative to untreated samples in both control 317 and AGS samples. It should be noted that the SARS-CoV-2 spike protein itself has 318 been shown in vitro to result in decreased production of type 1 IFN [30] , but that 319 SARS-CoV-2 overall has a complex interaction with IFNs, likely associated with tissue 320 specificity and disease severity [18, 29, 31] . It is promising that the expression of RNA 

The elevated ISGs seen on in vitro treatment of AGS patient cells with unmodified 327 mRNA vaccines is of particular concern. The HERALD phase 2b/3 clinical trial using a 328 chemically unmodified mRNA vaccine, named CVnCoV, demonstrated only 53% 329 efficacy against SARS-CoV-2 compared to the 95% and 94% efficacy of the 330 Comirnaty [35] and Spikevax [5] vaccines, respectively [19] . Importantly, in this trial the 331 frequency of adverse events was more frequently reported in CVnCoV recipient 332 population than in a placebo group in healthy participants despite a reduced dose (12µg) 333 being used to immunize participants [19] . We observed elevated ISGs and elevation in 334 11/18 RNA sensing genes upon administration of an unmodified mRNA LNP vaccine in both 335 AGS and control patient cells, demonstrating the immunogenicity of this formulation. 336 We suggest that the use of mRNA-based vaccinations overall in AGS patients should be 337 limited to nucleoside modified based formulations and the development of other 338 unmodified mRNA LNP vaccine formulations should take this into account.

It is important to note that the safety and effectiveness of COVID-19 vaccines in 340 those with AGS has not been studied. It is however unlikely that such a clinical trial 341 will occur in a rare disease population. Because of limited information, our guidance is 342 based on this limited in vitro data. Although a systemic autoinflammatory response to 343 the spike protein is likely and may increase ISG signaling, it is likely that this will not 344 be as great as the autoinflammatory response seen in the context of an actual infection. 345 Thus, in the context of continued COVID-19 infections in the community, however, we 346 are cautiously recommending that AGS affected individuals consider being vaccinated report that DW is named on patents that describe the use of nucleoside-modified 369 mRNA as a platform to deliver therapeutic proteins and vaccines. DW and MGA are 370 also names on patents describing the use of lipids nanoparticles, and lipid compositions 371 for nucleic acid delivery and vaccination. We have disclosed those interests fully to the 372 University of Pennsylvania, and we have in place an approved plan for managing any 373 potential conflicts arising from licensing of our patents. PW, DC, and TK are 374 volunteers of the AGSAA whose patient registry development was supported by Biogen. 375

The data that support the findings of this study are available on request from the 377 corresponding author. The data are not publicly available due to privacy or ethical 378 restrictions. 379 12/18

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