key: cord-0283008-knnwycjg
authors: Ballmann, Mónika Z.; Raus, Svjetlana; Engelhart, Ruben; Kaján, Győző L.; van der Zalm, Chantal; Papp, Tibor; John, Lijo; Khan, Selina; Custers, Jerome; Bakker, Wilfried A.M.; van der Schaar, Hilde M.; Arnberg, Niklas; Lemckert, Angelique A.C.; Havenga, Menzo; Baker, Andrew H.
title: Human AdV-20-42-42, a promising novel adenoviral vector for gene therapy and vaccine product development
date: 2021-03-12
journal: bioRxiv
DOI: 10.1101/2021.03.11.435055
sha: 21e2bbead13a831f8e2d0932407f0a92755238ef
doc_id: 283008
cord_uid: knnwycjg

Pre-existing immune responses towards adenoviral vector limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest to vectorize novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton-base which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In addition, the vector did not sequester in the liver upon intravenous administration in rodents. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon vaccination. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop safe and efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are currently under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that pre-existing immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrates that we have successfully vectorized a novel adenovirus serotype with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

seroprevalence. The cell transduction data and antigen-specific immune responses induced in 56 vivo demonstrate that this vector is highly promising for the development of gene therapy and 57 vaccine products. 59 Adenoviral vectors have been studied for decades as they hold great promise as tools to develop 60 safe and effective gene therapy and vaccine products. As such, there are dozens of therapeutic 61 applications being pursued utilizing adenoviral vectors. As it has been amply demonstrated that acknowledged serotypes, many hybrid adenoviruses have been discovered and as these so-76 called chimeras can be neutralized by parental serum they do not qualify as distinct serotypes. 77 Most likely such hybrids originate from homologous recombination events between two or 78 more viruses replicating simultaneously in a host cell during co-infection (5) (6) (7) (8) . 79 In the present study we describe the generation of a novel replication-incompetent 80 vector based on a natural hybrid that we named HAdV-20-42-42. The recombinant HAdV-20-81 42-42 vector was used for characterization of its seroprevalence, receptor usage, and tropism. 82 In addition, we explored the utility of the vector as a potential tool to develop gene therapy and 83 vaccine products. The data obtained and described here warrant further studies into the 84 utilization of the HAdV-20-42-42 vector to develop vaccines and cardiovascular intervention 85 strategies.

Identification of HAdV-20-42-42, a natural chimera 88 In order to identify possible new vector candidates, e.g. new and/or rare human adenovirus 89 types, 281 human adenovirus strains isolated from patients in Sweden between 1978 and 2010 90 were screened previously (9). From these samples, the hexon, the penton base, and the 91 polymerase genes were amplified and sequenced to allow for identification of new adenovirus 92 types or possible recombinants. Strains with interesting genotypes were propagated on A549 93 cells, after which the complete viral genome was sequenced using Next-Generation Sequencing 94 and annotated based on HAdV reference strains. Phylogenetic analyses were conducted based 95 on the complete genome sequence and amino acid translations of the hexon, penton base, and 96 the fiber knob.

During the screening process, strain 212 was pinpointed and analyzed further. The 98 complete genome sequence of this strain was 35,187 bp long with a GC-content of 57.0%. A 99 typical HAdV-D genome layout was observed with 37 protein coding sequences and two virus-100 associated RNAs ( Figure 1A ), pointing to a recombination event of two types that resulted in 101 a hybrid. The strain is closest related to HAdV-42 (species Human mastadenovirus D) in most 102 phylogenetic analyses except for the penton base, which has the highest sequence identity with 103 HAdV-20 ( Figure 1B) . Thus, the genomic composition of strain 212 was determined as HAdV-104 20-42-42 concerning the sequence of the penton base, the hexon, and the fiber knob.

HAdV-20-42-42 shows low seroprevalence in human subjects 107 High levels of pre-existing anti-vector humoral immunity in vaccine target populations may 108 hamper potential use of a novel adenoviral vector as an efficacious vaccine platform, such as 109 found for HAdV-5-based vectors (10-12). We investigated the levels of pre-existing 110 neutralizing antibodies against HAdV-20-42-42 using a panel of serum samples (n=103) taken 111 from a cohort of healthy >50-year-old US citizens. In line with previous findings (13), ~60 % 112 of the serum samples exhibited high levels of neutralizing activity (effective at dilutions 113 >1 : 200) against HAdV-5 (Fig. 2) . For on the other hand only 114 ~15 % of serum samples neutralized viral activity at dilutions >200 (Fig. 2) . These data indicate 115 that the seroprevalence of HAdV-20-42-42 in this cohort was low with antibody levels 116 comparable to that of the rare serotype HAdV-35 (species HAdV-B). In order to study the therapeutic applicability of HAdV-20-42-42, we first generated 120 replication-incompetent vectors expressing reporter genes β-galactosidase (LacZ), luciferase 121 (Luc) or Enhanced Green Fluorescent Protein (EGFP). To do so, engineered HAdV-20-42-42 122 genomic DNA sequences were cloned into three plasmids, called the adaptor plasmid, the 123 intermediate plasmid, and the right-end plasmid, with overlapping regions to allow for 124 homologous recombination events (see Figure 3A ). To produce replication-incompetent 125 reporter viruses, the E1 region of HAdV-20-42-42 was replaced with an expression cassette 126 and a reporter gene in the adaptor plasmid. In the right-end plasmid, the E3 region was deleted 127 to create capacity for insertion of larger transgenes. To enhance the growth in a standard 128 producer cell line (i.e. HAdV-5 E1-complementing HEK293 cells), the native E4 ORF6/7 129 region was exchanged with that of . these findings indicate that the novel adenoviral vector HAdV-20-42-42 is able to bind to both 159 CAR and CD46 receptors, primarily the C2 isoform. As these receptors are present in many 

In the control group of animals without CL pretreatment, HAdV-5-Luc was mainly 191 distributed in liver and spleen at the levels of ~2.5 × 10 5 genome copy numbers per 100 ng total 192 DNA, while in the group pre-treated with CL the liver and spleen distribution was higher, closer 193 to ~5 × 10 5 genome copy numbers (Fig. 6 ). HAdV-20-42-42 on the other hand appeared to 194 have only a spleen tropism, since luciferase activity was not detected in other organs. As 195 expected, the total DNA copy number was significantly higher when CL was added (~2.5 × 196 10 6 ) in comparison to the group without CL pretreatment (1 × 10 5 ).

Collectively, these data indicate that HAdV-20-42-42 has a good safety profile with 198 only spleen tropism found in the studies, while the adenoviral DNA copies in other organs 199 tested were poorly detectable. Based on these initial data we set out to generate a vector system based on HAdV-20- 

Luciferase activity detected ranged from low (shown in blue) to high (shown in red) levels.

After the imaging was completed, animals were sacrificed and their organs (liver, heart, spleen, 417 kidney, intestine, pancreas and lungs) were collected for the quantification of the vector 418 genomes as described before (13). 

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The polypeptides of adenovirus: I. Evidence for 587 multiple protein components in the virion and a comparison of types 2, 7A, and 12

Figure 3. HAdV-20-42-42 vector generation and characterization

Overlapping regions allowed homologous recombination events between the HAdV-622 20-42-42 sequences in HEK293 production cell line. E and L represent the "early" and "late" 623 genes of the adenoviral genome

CPE development in HEK293 cells upon transfection of three HAdV-20-42-42 plasmids. C)

Verification of transgene (LacZ) expression. A549 cells were infected with HAdV-20-42-42-LacZ at various MOIs (indicated as vp per well)

Lysates of a previous HAdV-20-42-42-LacZ infection were used as controls 629 (+ ctrl A and B), while lysates of HAdV-20-42-42-GFP infected cells (-ctrl A) or uninfected 630 cells (-ctrl B) were used as negative controls

light gray bars) CAR, sialic 638 acid-containing glycans or DSG2 receptors were infected with HAdV-20-42-42-Luc or HAdV-639 5-Luc as a control vector. B) CHO cells lacking CD46 (K1) or expressing various CD46 640 isoforms (BC1, BC2, C1, or C2) were infected with HAdV-20-42-42-Luc or HAdV-5-Luc as 641 a control vector. A, B) One day post-infection, cells were lysed to determine intracellular 642 luciferase activity. Luciferase activity is presented as relative light units (RLU) per milligram 643 (mg) protein. All results represent averaged data from several times performed experiments, 644 with four replicates for each condition. Error bars are presented as standard error of the mean 645 (SEM). Two-sample, two-tailed Student's t-tests

Figure 5. Transduction efficiency of HAdV-20-42-42 in vascular cells

HAdV-20-42-42-Luc and -LacZ vectors were tested for transduction capacity in HSVEC 655 (human saphenous vein endothelial cells). A) HSVEC were infected with

Luc or the control vectors HAdV-35-Luc and HAdV-5-Luc at various doses (1000, 5000, or 657 10000 vp/cell) for 3 hours. Where indicated (dark gray bars), the vectors were incubated for 30

After 2 days, cells were lysed to measure intracellular luciferase activity, 660 which is presented as relative light units (RLU) per milligram (mg) protein. Bars represent the 661 means plus standard errors of the means (SEM) (error bars) for quadruplicate values

Student's t-tests

All results represent averaged data from several experiments, with four replicates 664 for each condition. B) HSVEC were infected with various doses of

After 2 days, cells were stained for LacZ expression

Mice were pre-treated with clodronate (CL+) or untreated (CL-) and injected intravenously 670 with HAdV-5-Luc or HAdV-20-42-42-Luc vectors. A) At 48 hours post virus delivery, 671 luciferin was injected to the mice and luciferase activity was imaged with the method IVIS 672

which ranged from low activity (shown in blue) to high activity (shown in red) 673 levels. B) After imaging, animals were sacrificed and organs were collected to determine 674 adenoviral genome copy numbers with qPCR. Data represent viral copy number per 100 ng of 675 total DNA. Bars represent the means plus

relative number of IFN-γ-secreting cells. shown as Spot Forming Units (SFU) per million cells

Each dot represents a mouse, the bar indicates the geometric mean and the dotted line is the 691 95 th percentile based on the medium control samples. B) Cross-neutralization between HAdV-692 20-42-42 and HAdV-26. Mice antisera against hAd20V-42-42 and HAdV-26 were cross-tested 693 against both vectors in an adenovirus neutralization assay. Starting from a 1:16 dilution, the 694 sera were 2-fold serially diluted

luciferase, and subsequently incubated overnight with A549 cells at MOI 500 virus particles

Luciferase activity levels in infected cell lysates measured 24 hours post-infection represented 697 vector infection efficiencies. The neutralization titers were arbitrarily divided into the 698 categories shown