Design of cocktail peptide vaccine against Cytomegalovirus infection

Document Type : Short Communication


1 Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran

2 Department of Medical Biochemistry, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

3 Antimicrobial Resistance Research Center, Avicenna Research Institute, Mashhad University of Medical Science, Mashhad, Iran


Objective(s):Human Cytomegalovirus (HCMV) remains a major morbidity and mortality cause in immuno suppressed patients. Therefore, significant effort has been made towards the development of a vaccine. In this study, the expression of the pp65 and gB fusion peptides and Fc domain of mouse IgG2a as a novel delivery system for selective uptake of antigens by antigen-presenting cells (APCs) in Pichia pastoris yeast system were studied.
Materials and Method: In this study, four immune dominant sequences in pp65 protein and 3 immuno dominant sequences in gB protein were selected according to literature review. Peptide linker -GGGGS- was used for construction of fusion peptide. This fusion peptide was cloned in the pPICZαA expression vector and transfected into P. pastoris host cells.
Results: Dot blot and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques showed that a high level of pp65-gB-Fc fusion peptide was expressed.
Conclusion: This CMV pp65-gB-Fc fusion peptide could be a promising candidate for the development of a novel peptide vaccine.


1.McVoy MA. Cytomegalovirus Vaccines. Clin Infect Dis 2013; 57:196–199.
2. Kotton CN, Kumar D, Caliendo AM, Asberg A, Chou S, Danziger-Isakov L, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation 2013; 96: 333-360.
3.Tomtishen JP 3rd. Human cytomegalovirus tegument proteins (pp65, pp71, pp150, pp28). Virol J 2012; 9:22.
4.Li W, Joshi MD, Singhania S, Ramsey KH, Murthy AK.Peptide Vaccine: Progress and Challenges. Vaccine 2014; 2:515-536.
5.Polz-Dacewicz M, Fołtyn S, Macieląg P, Polz D. Cytomegalovirus (CMV) – a new prospect for prevention. Pre-Clinical and Clinical Research 2013; 7:118-123.
6.Rieder F, Steininger C. Cytomegalovirus vaccine: phase II clinical trial results. Clin Microbiol Infect 2013; 10.1111:1469-0691.
7.Lu L, Palaniyandi S, Zeng R, Bai Y, Liu X, Wang Y. A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection. Virology 2012; 85:10542–10553.
8.Fickers P. Pichia pastoris: a workhorse for recombinant protein production. Curr Res Microbiol Biotechnol 2014; 2:354-363.
9.Dasari V, Smith C, Zhong J, Scott G, Rawlinson W, Khanna R. Recombinant glycoprotein B vaccine formulation with Toll-like receptor 9 agonist and immunestimulating complex induces specific immunity against multiple strains of cytomegalovirus. Gen Virol 2011; 92:1021–1031.
10.Kern F, Bunde T, Faulhaber N, Kiecker F, Khatamzas E, Rudawski I , et al. Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T Cell repertoire in CMV-exposed individuals. Infect Dis 2002; 185:1709–1716.
11.Provenzano M , Sais G, Bracci L, Egli A, Anselmi M, Viehl C, Schaub S, et al. A HCMV pp65 polypeptide promotes the expansion of CD4+and CD8+ T cells across a wide range of HLA specificities. Cell Mol Med 2009; 13:2131-2147.
12.Slezak S, Bettinotti M, Selleri S, Adams S, Marincola F, Stroncek D. CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects. J Translat Med 2007; 5:17.
13.Mariz F, Coimbra E, Jesus A, Nascimento L, Torres F, Freitas C. Development of an IP-Free biotechnology platform for constitutive production of HPV16 L1 capsid protein using the pichia pastoris PGK1 promoter. Hindawi 2015; 594120.
14.Li X, Guo L, Kong M, Su X, Yang D, Zou M, Liu Y, Lu L. Design and Evaluation of a Multi-Epitope Peptide of Human Metapneumovirus. Intervirology. 2015; 58: 403-412
15.Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 2014; 98:5301–5317.
16.Cai M, Zhu F, Wu H, Shen P. Expression, purification and glycosylation analysis of chicken 
infectious bursal disease virus VP2 in yeast. Iran J Vet Res 2013; 3:211-219.
17.Brein F, Heintel T, Schumacher A, Meyerhans A, Schmitt M. Specific activation of CMV-primed human T lymphocytes by cytomegalovirus pp65 expressed in fussion yeast. FEMS Immunol Med Microbiol 2003; 38:231-239.
18.Do BC, Dang TT, Berrin JG, Haltrich D, To KA, Sigoillot JC, et al. Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from AspergillusnigerBK01. Microb Cell Fact 2009; 8:59.
19.Krainer FW, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A. .Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Fact 2012; 11:22.
20.Fazlalipour M, Fazlalipour H, Monavari H, Mollaie H. Recombinant Coree1e2 protein expressed in pichia pastoris yeast a candidate vaccine for hepatitis C virus. Antiretrovir 2014; 6:3.
21.Battista M, Bergamini G, Campanini F, Landini M, Ripalti A. Intracellular production of a major cytomegalovirus antigenic protein in the methylotrophic yeast Pichia pastoris. Gene 1996; 176:197-201.
22.Reap EA, Dryga SA, Morris J, Rivers B, Norberg PK, Olmsted RA, et al.  Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins. Clin Vaccine Immunol 2007; 14:748–755.
23.Pass RF. Development and evidence for efficacy of CMV glycoprotein B vaccine with MF59 adjuvant. J Clin Virol 2009; 46:73–76.
24.La Rosa C1, Longmate J, Lacey SF, Kaltcheva T, Sharan R, Marsano D, et al. Clinical evaluation of safety and immunogenicity of PADRE-cytomegalovirus (CMV) and tetanus-CMV fusion peptide vaccines with or without PF03512676 adjuvant. J Infect Dis 2012; 205:1294–12304.
25.Yamada A, Yamada T, Noguchi M, Itoh K. Next-generation peptide vaccines for advanced cancer. Cancer Sci 2013; 104:15-21.
26.Baker K, Qiao SW, Kuo T, Kobayashi K, Yoshida M, Blumberg RS.. Immune and non-immune functions of the (not so) neonatal Fc receptor, FcRn. Semin Immunopathol 2009; 31:223–236.
27.Lu L, Palaniyandi S, Zeng R, Bai Y, Liu X, Wang Y, et al. A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection. J Virol 2011; 85:10542-10553.