Evaluation of Antibody and Cytokines Responses in Intranasally and Intramuscularly Administrated BALB/C Mice With Influenza Virus-Like Particle

  • Haideh Nadmdari Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
  • Mohsen Keshavarz Department of Medical Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
  • Talat Mokhtari-Azad Department of Virology, School of Public Health, National Influenza Center, Tehran University of Medical Sciences, Tehran, Iran.
  • Farhad Rezaei Mail Department of Virology, School of Public Health, National Influenza Center, Tehran University of Medical Sciences, Tehran, Iran.
Influenza virus-like particle, Vaccine, Immune responses, Mucosal antibody, Cytokines


We previously developed an influenza virus like particle with HA, M, and NA proteins using Bac-to-Bac expression system and SF9 cell line. To evaluate the immunogenicity of our construct, we assessed the humoral, cytokine induced by H1N1-VLP in BALB/c mice immunized intranasally and intramuscularly. Enzyme-linked immunosorbent assay and Relative quantitative real-time PCR were used to evaluate the antibody (IgG and IgA) and mRNA levels of IL-6, IL-4, IL-10 and IFN-g in PBMCs. Our results showed that VLP was capable of intranasal (I.N.) and intramuscular (I.M.) induction of serum IgG and IgA responses. Interestingly, I.N. route induced higher IgG and IgA titer than I.M. route, which was statistically significant. Moreover, mRNA levels of IL-6 (4.2-4.5 folds), IFN-g (5.5-5.7 folds), and the anti-inflammatory cytokine IL-10 (2.5-3 folds) and IL-4 (2.4-2.8 folds) were significantly elevated in mice immunized I.N. and I.M. with H1N1-VLP compared to the control group. Our findings indicated that a non-infectious genome-less VLP approach mimics parenteral virus with multiple viral antigens and epitopes that stimulate a diverse set of immune responses such as innate immunity, specific serum IgG antibody, cell-mediated immunity, and local antibodies.


Quan FS, Kim YC, Song JM, Hwang HS, Compans RW, Prausnitz MR, et al. Long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch. Clin Vaccine Immunol 2013;20:1433-9.

Wen Z, Ye L, Gao Y, Pan L, Dong K, Bu Z, et al. Immunization by influenza virus-like particles protects aged mice against lethal influenza virus challenge. Antiviral Res 2009;84:215-24.

Pushko P, Kort T, Nathan M, Pearce MB, Smith G, Tumpey TM. Recombinant H1N1 virus-like particle vaccine elicits protective immunity in ferrets against the 2009 pandemic H1N1 influenza virus. Vaccine 2010;28:4771-6.

Rezaei F, Mirshafiey A, Shahmahmoodi S, Shoja Z, Ghavami N, Mokhtari-Azad T. Influenza Virus-like Particle Containing Two Different Subtypes of Hemagglutinin Confers Protection in Mice Against Lethal Challenge With A/PR8 (H1N1) and A/HK (H3N2) Viruses. Iran Red Crescent Med J 2013;15:75-82.

Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, et al. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009;360:2605-15 .

Peiris JS, Tu WW, Yen HL. A novel H1N1 virus causes the first pandemic of the 21st century. Eur J Immunol 2009;39:2946-54.

Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet 2004;363:617-9.

Qin G, Liu Y, Zheng J, Ng IH, Xiang Z, Lam KT, et al. Type 1 responses of human Vgamma9Vdelta2 T cells to influenza A viruses. J Virol 2011;85:10109-16.

Inn KS, Lee GJ, Quan FS. A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice. Immunol Invest 2014;43:236-54 .

Hossain MJ, Bourgeois M, Quan FS, Lipatov AS, Song JM, Chen LM, et al. Virus-like particle vaccine containing

hemagglutinin confers protection against 2009 H1N1 pandemic influenza. Clin Vaccine Immunol 2011;18:2010- 7.

Prabakaran M, Velumani S, He F, Karuppannan AK, Geng GY, Yin LK, et al. Protective immunity against influenza H5N1 virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HA and recombinant CTB as an adjuvant. Virology 2008;380:412- 20

Kang SM, Song JM, Compans RW. Novel vaccines against influenza viruses. Virus Res 2011;162:31-8.

Kang SM, Song JM, Quan FS, Compans RW. Influenza vaccines based on virus-like particles. Virus Res 2009;143:140-6.

1 Lee DH, Bae SW, Park JK, Kwon JH, Yuk SS, Song JM, et al. Virus-like particle vaccine protects against H3N2 canine influenza virus in dog. Vaccine 2013;31:3268-73.

Lee DH, Park JK, Lee YN, Song JM, Kang SM, Lee JB, et al. H9N2 avian influenza virus-like particle vaccine provides protective immunity and a strategy for the differentiation of infected from vaccinated animals. Vaccine 2011;29:4003-7 .

Mahmood K, Bright RA, Mytle N, Carter DM, Crevar CJ, Achenbach JE, et al. H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses. Vaccine 2008;26:5393-9.

Marashi SM, Jalilvand S, Mollaei-Kandelous Y, Shahmahmoodi S, Rezaei F, Salimi V, et al. Intra-peritoneal and intra-rectal immunogenicity induced by rotavirus virus like particles 2/6/7 in mice. Microb Pathog 2014;67-68:48-54 .

Park JK, Lee DH, Youn HN, Kim MS, Lee YN, Yuk SS, et al. Protective efficacy of crude virus-like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy. Influenza.Other Respir.Viruses 2013;7:340-8.

Quan FS, Huang C, Compans RW, Kang SM. Virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus. J Virol 2007;81:3514-24 .

Maroof A, Yorgensen YM, Li Y, Evans JT. Intranasal vaccination promotes detrimental Th17-mediated immunity against influenza infection. PLoS Pathog 2014;10:e1003875 .

Quan FS, Steinhauer D, Huang C, Ross TM, Compans RW, Kang SM. A bivalent influenza VLP vaccine confers complete inhibition of virus replication in lungs. Vaccine 2008;26:3352-61.

Tumpey TM, Renshaw M, Clements JD, Katz JM. Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection. J Virol 2001;75:5141-50.

Bright RA, Carter DM, Daniluk S, Toapanta FR, Ahmad A, Gavrilov V, et al. Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin. Vaccine 2007;25:3871-8.

Galarza JM, Latham T, Cupo A. Virus-like particle vaccine conferred complete protection against a lethal influenza virus challenge. Viral Immunol 2005;18:365-72 .

Latham T, Galarza JM. Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J Virol 2001;75:6154-65.

Pushko P, Tumpey TM, Bu F, Knell J, Robinson R, Smith G. Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice. Vaccine 2005;23:5751-9.

Pushko P, Tumpey TM, Van HN, Belser JA, Robinson R, Nathan M, et al. Evaluation of influenza virus-like particles and Novasome adjuvant as candidate vaccine for avian influenza. Vaccine 2007;25:4283-90.

Wang BZ, Xu R, Quan FS, Kang SM, Wang L, Compans RW. Intranasal immunization with influenza VLPs incorporating membrane-anchored flagellin induces strong heterosubtypic protection. PLoS One 2010;5:e13972.

Lv J, Wang D, Hua YH, Pei SJ, Wang J, Hu WW, et al. Pulmonary immune responses to 2009 pandemic influenza A (H1N1) virus in mice. BMC Infect Dis 2014;14:197.

Mazanec MB, Coudret CL, Fletcher DR. Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies. J Virol 1995;69:1339-43.

Mazanec MB, Kaetzel CS, Lamm ME, Fletcher D, Nedrud JG. Intracellular neutralization of virus by immunoglobulin A antibodies. Proc Natl Acad Sci U S A 1992;89:6901-5 .

Mazanec MB, Kaetzel CS, Lamm ME, Fletcher D, Peterra J, Nedrud JG. Intracellular neutralization of Sendai and influenza viruses by IgA monoclonal antibodies. Adv Exp Med Biol 1995;371A:651-4.

Perrone LA, Ahmad A, Veguilla V, Lu X, Smith G, Katz JM, et al. Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge. J Virol 2009;83:5726-34.

See H, Wark P. Innate immune response to viral infection of the lungs. Paediatr Respir Rev 2008;9:243-50.

Eriksson JC, Cox RJ, Szyszko E, Davidsson A, Brokstad KA. Local and systemic cytokine and chemokine responses after parenteral influenza vaccination. Influenza Other Respir Viruses 2007;1:139-46.

How to Cite
Nadmdari H, Keshavarz M, Mokhtari-Azad T, Rezaei F. Evaluation of Antibody and Cytokines Responses in Intranasally and Intramuscularly Administrated BALB/C Mice With Influenza Virus-Like Particle. Acta Med Iran. 55(10):604-611.
Original Article(s)