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SPECIFIC AIMS

Adeno-associated virus (AAV) vector has been successfully applied to target the liver in clinical trials with hemophilia patients1,2. These trials have suggested that the AAV capsid specific cytotoxic T lymphocytes (CTLs) eliminate AAV vector targeted liver cells, following AAV2 or AAV8 transduction, and result in therapeutic failure. Our studies and others have demonstrated that both classical antigen presentation and cross-presentation pathways are involved in mounting an AAV capsid specific CTL response3-7. In clinical trials, ion exchange chromatography has been used to purify AAV vectors. Unlike CsCl purification approach, the chromatographic method cannot currently separate genome-containing AAV capsids (full particles) from empty particles. The contamination of empty virions potentially increases the AAV capsid antigen load in transduced cells and it has been demonstrated that empty virion contamination in vector preparations induces liver damage, which potentially enhances capsid antigen presentation from full virion transduction8,9. Although we have observed a lower capsid antigen presentation from AAV empty virion infection compared to full particles in vitro10, our in vivo preliminary result demonstrated that AAV empty capsids still elicit capsid antigen presentation. In this proposal we will investigate the kinetics of capsid antigen presentation from empty virions and the effect of empty particles on antigen presentation from full particle transduction (Aim 1a and 1b). We have demonstrated that AAV capsid cross-presentation is dependent on virion endosomal escape and proteasome-mediated capsid degradation in AAV transduced cells in vitro10. However, the mechanistic insights of the work were largely elucidated in vitro, and over a limited time period (24 to 48 hrs), and therefore it remains unclear which aspects of our discoveries translate in vivo regarding long-term antigen processing and presentation. The mechanism of capsid antigen presentation from empty virions and full particles in vivo will be performed using mouse models deficient in the genes responsible for classical class-I antigen presentation (TAP -/- mice) or classical class-II antigen presentation (Cat S -/- mice) (Aim 1c). Our data have shown that capsid antigen presentation is dose-dependent and requires capsid ubiquitination for proteasome mediated degradation11,12. To decrease antigen presentation on AAV transduced cells for avoiding capsid specific CTL-mediated elimination, it has been proposed to modify the AAV capsid surface in order to enhance AAV transduction while lowering the effective dose, or to escape capsid ubiquitination13,14. It is unclear whether the enhancement of liver transduction with AAV mutants or a decrease in capsid ubiquitination influences capsid antigen presentation in vivo (Aim 2a). Proteasome inhibitors have been shown to enhance AAV transduction and inhibit antigen presentation12,15. However, our further in vitro study demonstrated varying effects of the proteasome inhibitor on capsid antigen cross-presentation in a dose related manner. A high dose of the proteasome inhibitor bortezomib blocks capsid antigen presentation, while a lower dose of bortezomib increases capsid antigen presentation without enhanced transduction. We hypothesize that proteasome inhibitor treatment will change the profile of AAV antigen presentation in vivo and the combination of AAV mutants and proteasome inhibitors will further increase AAV transduction while inhibiting capsid antigen presentation (Aim 2b and 2c). It is well-known that the transduction of AAV vectors in mouse models does not always translate into the human host. To address this, a mouse model xenografted with human hepatocytes has been used to develop AAV vectors for human liver targeting gene therapy16. In this proposal we will explore the directed evolution approach combined with a rational design strategy to isolate AAV vectors with human hepatocyte specific tropism and the ability to evade a capsid specific CTL response in humanized mice (Aim 3). Elucidation of AAV empty capsid antigen presentation in vivo and the development of an AAV vector with enhanced human liver transduction and CTL immune-evasion, will allow us to design safer and more effective strategies that address the current clinical complications for human liver gene therapy using AAV. To address these issues, we will execute the following specific aims: 1. Study the effect of AAV empty particles on AAV capsid antigen cross-presentation in vivo.

a. The kinetics and dose-response of AAV capsid antigen presentation from AAV empty virions in vivo. b. The effect of empty particles on capsid antigen presentation from full-particle AAV transduction in vivo. c. AAV capsid antigen presentation in TAP-/- and in Cat S-/- mice.

2. Investigate AAV capsid antigen presentation following administration of AAV mutants and/or proteasome inhibitors for enhanced liver transduction in vivo. a. Capsid antigen presentation from AAV mutants with enhanced liver transduction in mice. b. The effect of proteasome inhibitors (high vs low dose) on natural AAV capsid antigen presentation in

vivo. c. The effect of a combination of AAV mutants with proteasome inhibitors on antigen presentation in vivo.

3. Isolate AAV chimeric capsids with human hepatocyte tropism and the capacity for CTL evasion. a. Verify AAV human liver transduction efficiency in xenograft mice. b. Characterization of AAV mutants recovered from human liver xenografted mice. c. Investigation of capsid CTL evasion from humanized AAV mutants.

Contact PD/PI: Li, Chengwen

Specific Aims Page 33

Contact PD/PI: Li, Chengwen

LITERATURE CITED

1 Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12, 342-347, doi:10.1038/nm1358 (2006).

2 Nathwani, A. C. et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365, 2357-2365, doi:10.1056/NEJMoa1108046 (2011).

3 Li, C. et al. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo. J Virol 81, 7540-7547, doi:10.1128/JVI.00529-07 (2007).

4 Chen, J., Wu, Q., Yang, P., Hsu, H. C. & Mountz, J. D. Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy. Molecular therapy : the journal of the American Society of Gene Therapy 13, 260-269, doi:10.1016/j.ymthe.2005.10.006 (2006).

5 Wang, L., Figueredo, J., Calcedo, R., Lin, J. & Wilson, J. M. Cross-presentation of adeno-associated virus serotype 2 capsids activates cytotoxic T cells but does not render hepatocytes effective cytolytic targets. Human gene therapy 18, 185-194, doi:10.1089/hum.2007.001 (2007).

6 Sabatino, D. E. et al. Identification of mouse AAV capsid-specific CD8+ T cell epitopes. Molecular therapy : the journal of the American Society of Gene Therapy 12, 1023-1033, doi:10.1016/j.ymthe.2005.09.009 (2005).

7 Li, H. et al. Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes. Molecular therapy : the journal of the American Society of Gene Therapy 15, 792-800, doi:10.1038/sj.mt.6300090 (2007).

8 Wright, J. F. AAV empty capsids: for better or for worse? Molecular therapy : the journal of the American Society of Gene Therapy 22, 1-2, doi:10.1038/mt.2013.268 (2014).

9 Gao, K. et al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Molecular Therapy — Methods & Clinical Development 1, 1-8 (2014).

11 He, Y. et al. Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical. Human gene therapy 24, 545-553, doi:10.1089/hum.2013.065 (2013).

12 Li, C. et al. Adeno-associated virus capsid antigen presentation is dependent on endosomal escape. J Clin Invest 123, 1390-1401, doi:10.1172/JCI66611 (2013).

13 Shen, S. et al. Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency. J Biol Chem 288, 28814-28823, doi:10.1074/jbc.M113.482380 (2013).

14 Zhong, L. et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci U S A 105, 7827-7832, doi:10.1073/pnas.0802866105 (2008).

15 Mitchell, A. M. & Samulski, R. J. Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors. J Virol, doi:10.1128/JVI.01826-13 (2013).

16 Lisowski, L. et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506, 382-386, doi:10.1038/nature12875 (2014).

17 Inaba, K. & Inaba, M. Antigen recognition and presentation by dendritic cells. Int J Hematol 81, 181- 187, doi:10.1532/IJH97.04200 (2005).

18 Gao, G. et al. Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo. Human gene therapy 11, 2079-2091, doi:10.1089/104303400750001390 (2000).

19 Lock, M., Alvira, M. R. & Wilson, J. M. Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography. Hum Gene Ther Methods 23, 56-64 (2012).

20 Qu, G. et al. Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography. J Virol Methods 140, 183-192, doi:10.1016/j.jviromet.2006.11.019 (2007).

21 Urabe, M. et al. Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion- exchange chromatography potentiates transgene expression. Molecular therapy : the journal of the American Society of Gene Therapy 13, 823-828, doi:10.1016/j.ymthe.2005.11.024 (2006).

References Cited Page 51

SARS-CoV-2 Omicron VOC Transmission in Danish

Households

Frederik Plesner Lyngse1,2,3,∗, Laust Hvas Mortensen4,5, Matthew J. Denwood6,

Lasse Engbo Christiansen7, Camilla Holten Møller3, Robert Leo Skov3,

Katja Spiess3, Anders Fomsgaard3, Maria Magdalena Lassaunière3,

Morten Rasmussen3, Marc Stegger8, Claus Nielsen3,

Raphael Niklaus Sieber8, Arieh Sierra Cohen3, Frederik Trier Møller3,

Maria Overvad3, Kåre Mølbak3, Tyra Grove Krause3, Carsten Thure Kirkeby6

December 22, 2021

∗Correspondence to Frederik Plesner Lyngse, [email protected] Affiliations: 1Department of Economics & Center for Economic Behaviour and Inequality, University of Copenhagen, Copenhagen, Denmark; 2Danish Ministry of Health, Copenhagen, Denmark; 3Statens Serum Institut, Copenhagen, Denmark; 4Statistics Denmark; 5Department of Public Health, Faculty of Health and Medical Sciences, University of Copenhagen; 6Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. 7Department of Applied Mathematics and Computer Science, Dynamical Systems, Technical University of Denmark, Kgs. Lyngby, Denmark.; 8Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Copenhagen, Denmark

1

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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

1 Abstract

The Omicron variant of concern (VOC) is a rapidly spreading variant of SARS-CoV-2

that is likely to overtake the previously dominant Delta VOC in many countries by the

end of 2021.

We estimated the transmission dynamics following the spread of Omicron VOC within

Danish households during December 2021. We used data from Danish registers to estimate

the household secondary attack rate (SAR).

Among 11,937 households (2,225 with the Omicron VOC), we identified 6,397 secondary

infections during a 1-7 day follow-up period. The SAR was 31% and 21% in households

with the Omicron and Delta VOC, respectively. We found an increased transmission

for unvaccinated individuals, and a reduced transmission for booster-vaccinated individ-

uals, compared to fully vaccinated individuals. Comparing households infected with the

Omicron to Delta VOC, we found an 1.17 (95%-CI: 0.99-1.38) times higher SAR for un-

vaccinated, 2.61 times (95%-CI: 2.34-2.90) higher for fully vaccinated and 3.66 (95%-CI:

2.65-5.05) times higher for booster-vaccinated individuals, demonstrating strong evidence

of immune evasiveness of the Omicron VOC.

Our findings confirm that the rapid spread of the Omicron VOC primarily can be ascribed

to the immune evasiveness rather than an inherent increase in the basic transmissibil-

ity.

2

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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 27, 2021. ; https://doi.org/10.1101/2021.12.27.21268278doi: medRxiv preprint

2 Introduction

The SARS-CoV-2 variant B.1.1.529, which is referred to as the Omicron variant of concern

(VOC), has overtaken the Delta VOC in South Africa and has spread rapidly to at least 28

countries countries in Europe (7), Asia, the Middle East and South America (9; 17). The

Omicron VOC has been reported to be three to six times as infectious as previous variants

(4), with a short doubling time (11), including early estimates from countries with a high

vaccination coverage indicating doubling times of 1.8 days (UK), 1.6 days (Denmark), 2.4

days (Scotland) and 2.0 days (United States) (26). Transmission of the Omicron VOC

has been high among individuals being fully vaccinated against SARS-CoV-2 infection as

well as among individuals with a history of COVID-19 infection (19).

A current concern worldwide is that the Omicron VOC is able to evade immunity induced

by the currently used vaccines, and a preliminary meta-analysis of neutralization studies

indicated that the vaccine effectiveness is reduced to around 40% against symptoms and

to 80% against severe disease, but that the effect for booster vaccinations is at 86% and

98%, respectively (12). These results are supported by laboratory studies establishing a

markedly reduced elimination of the Omicron VOC by neutralizing antibodies, indicating

that the vaccination effectiveness with Pfizer-Biontech against infection is only at 35%

for the Omicron VOC (5). This was corroborated by another in vitro study reporting an

8.4-fold reduction in neutralization for the Omicron VOC vs. the PV-D614G reference

strain, whereas there was only a 1.6-fold reduction in neutralization for the Delta VOC

(27). Therefore, the advantage of the Omicron VOC seems to be a combination of high

transmissibility and increased immune evading abilities.

Studies on the transmission of the Omicron VOC are yet sparse and a critical prerequisite

for effective control of this variant worldwide (3). In particular, it is important to clarify

whether the growth advantage can be ascribed to immune evasiveness, i.e., a higher pro-

portion of vaccinated or previously infected individuals being susceptible to infection, an

increased inherent transmissibility for this variant, or both.

3

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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 27, 2021. ; https://doi.org/10.1101/2021.12.27.21268278doi: medRxiv preprint

The aim of the present study is to investigate the household transmission of the Omicron

VOC. Specifically, we address the following questions: 1) Is the secondary attack rate

higher for the Omicron VOC than for the Delta VOC? 2) Does the Omicron VOC show a

higher immune evasiveness relative to the Delta VOC? 3) Is booster vaccination effective

for reducing transmission?

3 Data and Methods

3.1 Study design and participants

Since July 2021, the Delta VOC has been the dominant variant in Denmark. The

first Danish case infected with the Omicron VOC was detected on 22nd November 2021

(Danish Covid-19 Genome Consortium, DCC), and community transmission was deter-

mined to be present by late November 2021. On 8th December, Danish authorities dis-

continued intensive contact tracing of close contacts for cases specifically infected with the

Omicron VOC. We therefore started the study period on 9th December 2021 when cases

of both variants were treated approximately equally, thus reducing bias from intensified

contact tracing and active case finding of the Omicron VOC that was implemented shortly

after it’s discovery in Denmark (24). The end of the inclusion period for primary cases

was set at 12th December to balance the inclusion of enough cases for proper estimation

and early dissemination of the results. Potential secondary cases were followed up to 7

days after, i.e., until 19th December 2021 to allow for test results to be obtained. We

obtained the last test results on 21st December.

We used Danish register data for this study. All individuals in Denmark have a unique

identification number, enabling cross linking between administrative registers. Using this,

we obtained individual level information on home address, data on all antigen and RT-

PCR tests for SARS-CoV-2 from the Danish Microbiology Database (MiBa; (20)), and

records in the Danish Vaccination Register (13).

4

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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 27, 2021. ; https://doi.org/10.1101/2021.12.27.21268278doi: medRxiv preprint

Households were defined based on residential addresses. We included households with

2-6 members to exclude care facilities and other places, where many individuals share

the same address. If two individuals tested positive on the same day, we excluded these

households from the data, to ensure proper identification of the primary case within each

household.

We defined a primary case as the first individual within a household to test positive

with an RT-PCR test within the study period. We followed all tests of other household

members in the study period. A positive secondary case was defined by either a positive

RT-PCR test or a positive antigen test (10). Almost all samples that tested positive with

RT-PCR were tested with Variant PCR to determine the VOC (21) (see Appendix section

6.2). Based on the variant PCR test result of the primary case, we classified households

into households with either the Omicron or the Delta VOC. The Delta VOC has been

the dominating variant in Denmark since early July 2021, accounting for approximately

100% of all positive RT-PCR samples August-November 2021 (23).

We classified individuals by vaccination status into three groups: i) unvaccinated; ii)

fully vaccinated (defined by the vaccine used, Comirnaty (Pfizer/BioNTech): 7 days after

second dose; Vaxzevria (AstraZeneca): 15 days after second dose; Spikevax (Moderna):

14 days after second dose; Janssen (Johnson & Johnson): 14 days after vaccination, and

14 days after the second dose for cross vaccinated individuals) or 14 days after previous

infection; or iii) booster-vaccinated (defined by 7 days after the booster vaccination, (16;

2)). Partially vaccinated individuals were regarded as unvaccinated in this study. By 22nd

December 2021, of all vaccinated individuals, 85% were vaccinated with Comirnaty, 14%

with Spikevax, 1% with Janssen, and approximately 0% with AstraZeneca (22).

3.2 Statistical analyses

We defined the secondary attack rate (SAR) within households as the proportion of po-

tential secondary cases within the same household that tested positive between 1-7 days

following the positive test of the primary case within the household (15). Adjusted odds

5

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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 27, 2021. ; https://doi.org/10.1101/2021.12.27.21268278doi: medRxiv preprint

ratios (OR) were estimated from multivariable logistic regression models fit to the binary

outcome of test status of each potential secondary case, with the primary explanatory

variable reflecting the strain type in the household (Omicron vs. Delta VOC) and po-

tentially confounding variables of age and sex of the primary case, age and sex of the

potential secondary case, and household size (2-6 individuals). To test if vaccine sta-

tus conferred differential protection against the Omicron and Delta VOC, we included

an interaction term between vaccination status of primary and potential secondary cases

and the variant. Standard errors were adjusted to control for clustering at the household

level.

We have conducted a number of supplementary analyses to support the main analysis.

We re-analysed each strata of the data separately using another set of logistic regression

models (see appendix 7.4). To examine the potential mediating role of viral load of

primary cases infected with the Omicron VOC relative to the Delta VOC, we plotted

the distributions of Ct values for each variant (appendix Figure 3). We also examined

the extent to which the Ct value of the primary case could explain the difference in

transmission between the variants (see appendix Table 8). Our study relies on Variant

PCR testing to determine if each primary case was Delta or Omicron. We estimated the

intra-household correlation of variants, i.e., the probability that the positive secondary

case was infected with the same variant as the primary case. To investigate if there

was bias in the selection of samples for Variant PCR, we investigated the probability of

sampling for Variant PCR by sample Ct value and age. Moreover, we tested the robustness

of potential secondary cases being tested and testing positive by only using RT-PCR tests,

which are more sensitive.

3.3 Ethical statement

This study was conducted using data from national registers only. According to Dan-

ish law, ethics approval is not needed for this type of research. All data management

and analyses were carried out on the Danish Health Data Authority’s restricted research

servers with project number FSEID-00004942. The study only contains aggregated results

6

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is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted December 27, 2021. ; https://doi.org/10.1101/2021.12.27.21268278doi: medRxiv preprint

and no personal data. The study is, therefore, not covered by the European General Data

Protection Regulation (GDPR).

3.4 Data availability

The data used in this study are available under restricted access due to Danish data

protection legislation. The data are available for research upon reasonable request to The

Danish Health Data Authority and Statens Serum Institut and within the framework of

the Danish data protection legislation and any required permission from Authorities. We

performed no data collection or sequencing specifically for this study.

4 Results

A total of 2,225 primary cases with the Omicron VOC and 9,712 primary cases with the

Delta VOC were included (Table 1). The SAR was 31% in households with the Omicron

VOC and 21% in households with the Delta VOC. Generally, the estimated SAR was

higher for the Omicron VOC than for the Delta VOC, for all age groups. Unvaccinated </

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