Sequence analysis, databases and De Novo chimeric construction Design: HCMV genome

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Methods:

study design:

The present study consists of two parts. In the first part, analysis and database search were performed to find a conserved and specific regions of HCMV genome leading to selection of immunogenic and proper region expressed in several species of HCMV. The second part consists of various analyses of the selective gene such as epitope mapping, secondary and tertiary structure, antigenicity and B-cell epitope prediction, etc.

Sequence analysis, databases and De Novo chimeric construction Design:

Immunological studies that determine HCMV antigens importance in immunity, have demonstrated that the tegument phosphoprotein pp65 is the most dominant target for cellular immune responses against HCMV(Gyulai et al., 2000). Another usually recognized antigen of HCMV is pp150, that can induce specific CTLs responses (Elkington et al., 2003). Also similarity researches have shown that glycoprotein B (gB) is a principal target for neutralizing antibodies against HCMV (Reap et al., 2007).

We selected a pp65-derived epitope (RQYDPVAALFFFDIDL), defined by specific features such as: the capability to induce immune responses in both CD8+ and CD4+ T cells even in pharmacologically immunosuppressive individuals, high immunogenicity, wide range of HLA class I and II, ability to stimulate of producing TNF-α , INF-γ and proliferating CD4 T cells (M. Provenzano et al., 2009). Other information on T-cell Epitope sequences was obtained from researches by (Elkington et al., 2003) and (Jelinek et al., 2011) involving the recognition of epitopes and efficient sites.

Conformationally and linear binding sites on the glycoprotein B molecule have been mapped by Bioinformatic analysis. Linear binding sites have two conservative linear epitopes on SU segment and also two linear antibody sites on TM segment (Britt, Jarvis, Drummond, & Mach, 2005).

Accordingly, we chose two regions that efficiently stimulate the production of antibodies against HCMV.

All sequences were obtained from Uniprot Database at http:// www.uniprot.org.

T-cell Epitope sequences

HLA

HCMV antigens

ORF Names

Reference

RQYDPVAALFFFDIDL

pp65340-355

UL83

(M. Provenzano et al., 2009)

(Elkington et al., 2003)

(Maurizio Provenzano et al., 2003)

ARNLVPMVATVQGQNLKYQEFFW

A2

pp65493-515

UL83

(M. Provenzano et al., 2009)

(Diamond, York, Sun, Wright, & Forman, 1997)

(Jelinek et al., 2011)

WPRERAWAL

B7/B8

pp150

UL32

(Elkington et al., 2003)

Antibody-binding site

HCMV antigens

ORF Names

Reference

AD2 27-86

gB

UL55

(Beninga, Kropff, & Mach, 1995)

(Wagner et al., 1992)

(Meyer, Masuho, & Mach, 1990)

AD1 540-645

gB

UL55

(Gicklhorn, Eickmann, Meyer, Ohlin, & Radsak, 2003)

(Ohlin, Sundqvist, Mach, Wahren, & Borrebaeck, 1993)

(Britt et al., 2005)

(Zhao et al., 2009)

The five selected segments were fused together for designing a synthetic chimeric construction. In order to increase translation and exact expression of mRNA in the eukaryotic host, the Kozak sequence was inserted at the 5' end. For efficient accumulation of the recombinant protein in Endoplasmic Reticulum (ER), the sequence KDEL was added at the 3' end of the synthetic chimeric construct (Amani, Mousavi, Rafati, & Salmanian, 2009). Arrangements of fragment junctions are shown.

De Novo Peptide Design and Bioinformatics Analysis of the Chimeric Protein

Subsequently, the multi parameter gene characterizations and basic physico-chemical properties of the synthetic chimera protein were determined using standalone software, such as Gene Designer v.1.1 (http://www. dnatwopointo.com) and CLC Protein Workbench v.5.0.1 (http://www.clcbio.com) and an online database, such as Prot-Param (http://expasy.org/tools/protparam.html).

Amani, J., Mousavi, S. L., Rafati, S., & Salmanian, A. H. (2009). In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications. Theor Biol Med Model, 6, 28. doi: 10.1186/1742-4682-6-28

Beninga, J., Kropff, B., & Mach, M. (1995). Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response. J Gen Virol, 76 ( Pt 1)(1), 153-160.

Britt, W. J., Jarvis, M. A., Drummond, D. D., & Mach, M. (2005). Antigenic domain 1 is required for oligomerization of human cytomegalovirus glycoprotein B. J Virol, 79(7), 4066-4079.

Diamond, D. J., York, J., Sun, J.-Y., Wright, C. L., & Forman, S. J. (1997). Development of a candidate HLA A* 0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood, 90(5), 1751-1767.

Elkington, R., Walker, S., Crough, T., Menzies, M., Tellam, J., Bharadwaj, M., & Khanna, R. (2003). Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol, 77(9), 5226-5240.

Gicklhorn, D., Eickmann, M., Meyer, G., Ohlin, M., & Radsak, K. (2003). Differential effects of glycoprotein B epitope-specific antibodies on human cytomegalovirus-induced cell-cell fusion. J Gen Virol, 84(Pt 7), 1859-1862.

Gyulai, Z., Endresz, V., Burian, K., Pincus, S., Toldy, J., Cox, W. I., . . . Berencsi, K. (2000). Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs. J Infect Dis, 181(5), 1537-1546. doi: 10.1086/315445

Jelinek, J. í., Adkins, I., Mikulkova, Z., Jagosova, J., Pacasova, R., Michlickova, S., . . . Michálek, J. (2011). In vitro activation of CMV-specific human CD8+ T cells by adenylate cyclase toxoids delivering pp65 epitopes. Bone marrow transplantation, 47(2), 243-250.

Meyer, H., Masuho, Y., & Mach, M. (1990). The gp116 of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope. J Gen Virol, 71 ( Pt 10)(10), 2443-2450.

Ohlin, M., Sundqvist, V.-A., Mach, M., Wahren, B., & Borrebaeck, C. (1993). Fine specificity of the human immune response to the major neutralization epitopes expressed on cytomegalovirus gp58/116 (gB), as determined with human monoclonal antibodies. J Virol, 67(2), 703-710.

Provenzano, M., Lim, J. B., Mocellin, S., Monsurro, V., Bettinotti, M., Marincola, F. M., & Stroncek, D. F. (2003). The matrix protein pp65341-350: a peptide that induces ex vivo stimulation and in vitro expansion of CMV specific CD8+ T cells in subjects bearing either HLA A* 2402 or A* 0101 allele. Transfusion, 43(11), 1567-1574.

Provenzano, M., Sais, G., Bracci, L., Egli, A., Anselmi, M., Viehl, C. T., . . . Spagnoli, G. C. (2009). A HCMV pp65 polypeptide promotes the expansion of CD4+ and CD8+ T cells across a wide range of HLA specificities. J Cell Mol Med, 13(8B), 2131-2147. doi: 10.1111/j.1582-4934.2008.00531.x

Reap, E. A., Morris, J., Dryga, S. A., Maughan, M., Talarico, T., Esch, R. E., . . . Chulay, J. D. (2007). Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus. Vaccine, 25(42), 7441-7449. doi: 10.1016/j.vaccine.2007.08.016

Wagner, B., Kropff, B., Kalbacher, H., Britt, W., Sundqvist, V., Ostberg, L., & Mach, M. (1992). A continuous sequence of more than 70 amino acids is essential for antibody binding to the dominant antigenic site of glycoprotein gp58 of human cytomegalovirus. J Virol, 66(9), 5290-5297.

Zhao, P., Ma, D., Yan, S., Shao, N., Zhang, J., Bi, Z., . . . Ji, C. (2009). Towards a novel vaccine against human cytomegalovirus based on a chimeric Ad5F35 adenovirus vector expressing the immunodominant antigenic domain 1 epitope. Intervirology, 52(1), 35-42. doi: 10.1159/000212989

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