








key: cord-302679-wytog9cv
authors: Panda, Somnath; Banik, Urmila; Adhikary, Arun K.
title: Bioinformatics analysis reveals four major hexon variants of human adenovirus type-3 (HAdV-3) as the potential strains for development of vaccine and siRNA-based therapeutics against HAdV-3 respiratory infections
date: 2020-06-23
journal: Infect Genet Evol
DOI: 10.1016/j.meegid.2020.104439
sha: 
doc_id: 302679
cord_uid: wytog9cv

Human adenovirus type 3 (HAdV-3) encompasses 15–87% of all adenoviral respiratory infections. The significant morbidity and mortality, especially among the neonates and immunosuppressed patients, demand the need for a vaccine or a targeted antiviral against this type. However, due to the existence of multiple hexon variants (3Hv-1 to 3Hv-25), the selection of vaccine strains of HAdV-3 is challenging. This study was designed to evaluate HAdV-3 hexon variants for the selection of potential vaccine candidates and the use of hexon gene as a target for designing siRNA that can be used as a therapy. Based on the data of worldwide distribution, duration of circulation, co-circulation and their percentage among all the variants, 3Hv-1 to 3Hv-4 were categorized as the major hexon variants. Phylogenetic analysis and the percentage of homology in the hypervariable regions followed by multi-sequence alignment, zPicture analysis and restriction enzyme analysis were carried out. In the phylogram, the variants were arranged in different clusters. The HVR encoding regions of hexon of 3Hv-1 to 3Hv-4 showed 16 point mutations resulting in 12 amino acids substitutions. The homology in HVRs was 81.81–100%. Therefore, the major hexon variants are substantially different from each other which justifies their inclusion as the potential vaccine candidates. Interestingly, despite the significant differences in the DNA sequence, there were many conserved areas in the HVRs, and we have designed functional siRNAs form those locations. We have also designed immunogenic vaccine peptide epitopes from the hexon protein using bioinformatics prediction tool. We hope that our developed siRNAs and immunogenic vaccine peptide epitopes could be used in the future development of siRNA-based therapy and designing a vaccine against HAdV-3.

In this study, we have analysed all the hexon variants of HAdV-3 based on various criteria, followed by the molecular analysis of their HVRs to assess their appropriateness as potential vaccine candidates. Then, we have identified the conserved locations in HVR encoding regions of the hexon gene and fr om those locations we have designed functional siRNAs. Next, we have also designed immunogenic vaccine peptide epitopes from the hexon protein that can be used to design a vaccine. We anticipate that our developed siRNAs and peptide epitopes could be used in the development of siRNA-based therapy and designing a future vaccine against HAdV-3.

The amino acid (AA) sequences that include the seven HVRs of 25 hexon variants and prototype strain (GB-GenBank accession no. AB330084) were collected from NCBI (http://www.ncbi.nlm.nih.gov/). Hexon variants were selected based on their duration of circulation, co-circulation and worldwide distribution. The selection process is depicted in Fig. 1 .

To explore the variations, the 319 AA long sequence (extending from 132 to 450) that included the seven HVRs of the GB strain and 3Hv-1 to 3Hv-4 were aligned by Genetyx software (www.genetyx.co.jp). After alignment the number of AA variations in all the HVRs was observed. Then the differences in AA sequences in all the HVRs were tabulated and the percentage of homologies were calculated manually as compared to the GB strain.

A phylogenetic tree was constructed with the help of the Phylogeny.fr website (http://www.phylogeny.fr/documentation.cgi) [20] using the "One Click mode". It has been designed to provide a high-performance platform that transparently chains programs relevant to phylogenetic analysis in a comprehensive and flexible pipeline. By default, the pipeline is already set up to run and connect programs recognized for their accuracy and speed (MUSCLE for multiple alignment and PhyML for phylogeny) to reconstruct a robust phylogenetic tree. The 319AA long (extending from 132 to 450) sequence of 25 hexon variants (3Hv-1 to 3Hv-25) and GB strain were uploaded to the website in FASTA format to build the phylogram.

The variations among the HVR encoding regions of the hexon gene of 3Hv-1 to 3Hv-4 were shown by multi-sequence alignment (MSA), in silico RE analysis Numerous tools are available to design functional siRNAs such as MysiRNA-designer [22] , siDirect [23] and siMAX-siRNA Designer (https://eurofinsgenomics.eu/en/dnarna-oligonucleotides/custom-dna-rna-oligos/simax-sirna/) etc. In this study, siDirect 2.0 (http://sidirect2.rnai.jp) has been used to design functional siRNAs from the conserved regions found in all hexon variants (3Hv-1 to 3Hv-25). siDirect 2.0 algorithm eliminates off-target effects by reflecting the recent finding that the capability of siRNA to induce off-target effect is highly correlated to the thermodynamic stability, or the melting temperature (Tm), of the seed-target duplex. Hence, the selection of siRNAs with lower seed-target duplex stabilities (benchmark Tm < 21.5°C) minimizes the offtarget effects. It generates and filters siRNAs in three selection steps: step 1 involves the selection of highly functional siRNAs, step 2 involves the reduction of seed-dependent off-target effects and step 3 involves the elimination of near -perfect matched genes [24] .

We used NetMHC 4.0 (http://www.cbs.dtu.dk/services/NetMHC/) to predict MHC class I binding epitopes. For this, the 319 AA long sequences (extending from 132 to 450) that included the seven HVRs of 3Hv-1 to 3Hv-4 were uploaded in FASTA format. We chose 9mer peptides as most HLA molecules have a strong preference for binding to 9mer peptides. The peptides were identified as a strong binder if the % rank is below the specified threshold for the strong binders, by default 0.5%. On the other hand, the peptide was identified as a weak binder if the % rank is above the threshold of the strong binders but below the specified threshold for the weak binders, by default 2%.

Among the 25 hexon variants, 3Hv-1 to 3Hv-4 comprised 80% of all the variants. We found that they are most prevalent in 7 countries (Japan, Korea, Taiwan, Germany, China, USA and India) as depicted in Fig. 2 . Hence, considering the percentage among the variants, global distribution and duration of circulation, we considered 3Hv -1 to 3Hv-4 as the major hexon variants.

The alignment data showed remarkable AA sequence variations among the HVRs of 3Hv-1 to 3Hv-4 as compared to the GB strain. The highest number of 7 variations was found in HVR7 followed by 2 variations in HVR6 (Fig. 3) . Table 2) .

The different hexon variants (3Hv-1 to 3Hv-25) were segregated into multiple clusters in the phylogenetic tree as shown in Fig. 4 . Two of the four major hexon variants (3Hv-1 and 3Hv-4) were incorporated in the same cluster. However, 3Hv-2 and 3Hv-3 were included in a different cluster. Multiple major clusters are formed due to their heterogeneity of the AA sequence in the HVRs.

The NT alignment data of the GB strain and the four major hexon variants of HAdV-3 (3Hv-1 to 3Hv-4) showed a total number of 16 point mutation on the seven HVRs (Fig.   5 ). There are substantial numbers of conserved regions within the variation in the gene which are depicted in Table 3 . The rest of the variants (3Hv-5 to 3Hv-25) also showed similar conserved regions.

In silico DNA restriction patterns with restriction endonuclease BccI, BcoDI, Bsp1286I

and BstNI clearly differentiated GB from 3Hv-1 to 3Hv-4 (Fig. 6A) . Similarly, the zPicture analysis also clearly depicted the variations (Fig. 6B) .

We found 10 conserved regions in the HVR encoding portion among all HAdV -3 hexon variants as presented in Table 3 . For example, the functional siRNAs designed from the first sequence is shown in Fig. 7 . The functional siRNAs designed from the other conserved regions are also shown in the supplementary data 1. However, we could not obtain any functional siRNAs from the conserved region numbers 4 and 6 when the seed-duplex stability (Tm) was < 21.5°C. We did not try to design functional siRNAs by relaxing the parameters as Tm < 21.5°C is the minimum requirement for getting offtarget reduced functional siRNAs.

MHC class I binding epitopes of 9mer peptides were predicted from NetMHC 4.0. All HLA-A alleles were selected for this. A total number of 311 epitopes were predicted for HLA-A0101. Among them there were 2 strong binders and 5 weak binders based on their %rank as shown in Table 4 . The complete dataset of MHC class I binding epitopes prediction from 3Hv-1 has been shown in the supplementary data 2.

HAdV-3 respiratory infections have become a global concern, especially among Asian countries [25] . The only adenovirus live vaccine was developed to prevent HAdV -4 and In case of HAdV-3, the selection of vaccine strains is complicated due to the existence of a large number of hexon variants [11] . In the present study, we have selected 3Hv-1 to 3Hv-4, as they 1) comprise 80% of all hexon variants, 2) have been circulating over longer periods when the circulation of others was shorter and 3) were co-circulating among different countries. Therefore, we have designated them as the major hexon Designing siRNAs from the conserved regions to inhibit the viral replication has been used against several pathogenic viruses such as HIV, HCV, HBV, SARS coronaviruses [17, 18, 27] . After a successful clinical trial, the siRNA-based drug (Onpatrro) is now available for the treatment of systemic disease like amyloidosis [31] . Due to the presence of multiple hexon variants of HAdV-3, we have selected their conserved regions for designing siRNAs, as they will work best against all the variants. siRNA fro m the conserved region has been successfully used against influenza and HCV [15, 16, [32] [33] [34] . Hexon protein constitutes 95% of the viral capsomere [35] . If the hexon gene can be knocked down by siRNAs [36] , the formation of complete viral particles will be prevented and without a complete capsid, HAdV will be unable to infect new host cells

Journal Pre-proof [29] . We have also predicted MHC Class I epitopes from the AA sequence of the HVRs (132-450 AA) of each major hexon variant which will save time and cost of future biological work of vaccine development.

In this study, we have found that the major hexon variants (3Hv-1 to 3Hv-4) are the most appropriate vaccine candidates against HAdV-3 and the several conserved regions located in the HVR encoding portion of the hexon gene are the suitable sites for designing siRNAs against all the hexon variants. We have designed functional siRNAs from those conserved regions and immunogenic vaccine peptide epitopes from the hexon protein. We expect that our findings could pave the way for the development of vaccine and siRNA-based therapeutics against HAdV-3 respiratory infections. The GenBank accession nos. are mentioned in the parentheses of each selected hexon variant.

J o u r n a l P r e -p r o o f

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Analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotypespecific residues

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Worldwide increased prevalence of human adenovirus type 3 (HAdV-3) respiratory infections is well correlated with heterogeneous hypervariable regions (HVRs) of hexon

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zPicture: dynamic alignment and visualization tool for analyzing conservation profiles

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Fields virology -NLM Catalog -NCBI

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 Table 4 : MHC class I binding epitopes as predicted from the AA sequence of the HVR of 3Hv-1 for HLA-A0101. The peptides with a % rank below 0.5 are strong binders and the peptides with a % rank between 0.5-2 are weak binders. 

