Differential Immune Responses in Mice Immunized with Recombinant Neutralizing Epitope Protein of Hepatitis E Virus Formulated with Liposome and Alum Adjuvants.
In the developing countries, Hepatitis E virus (HEV) is a predominant cause of sporadic acute hepatitis in adults and waterborne epidemics leading to high mortality in pregnant women. Vaccine development mainly focuses on the structural capsid protein open-reading-frame-2 (ORF-2) of the virus. We successfully evaluated liposome-adjuvanted recombinant neutralizing epitope protein (rNEp), a part of ORF-2, 458-607aa, in mice and rhesus macaques. We compared immune response to adjuvants alone, rNEp alone, or adjuvanted with liposome (lipo-rNEp)/alum (al-rNEp) in mice following intramuscular administration of two doses of 5 μg each. IgG anti-HEV titers (enzyme-linked immunosorbent assay), immunophenotyping (flow cytometry, CD3(+)CD4(+), CD3(+)CD8(+), CD11c(+), CD11b(+), CD19(+) cells; costimulatory markers CD80, CD86, MHC-I, MHC-II, and early activation marker CD69), and levels of Th1/Th2 cytokines (IL-2/IFN-γ/IL-4/IL-5 and additionally IL-1β/IL-6/IL-10/TNF for early time points) were determined at early (4/12/24-h postdose-1) and later time points (2 weeks post-both doses). IgG anti-HEV titers were higher in the lipo-rNEp group than al-rNEp post-both doses (p < 0.05). At early time points, cell type proportions were comparable at the site of injection; IL-Iβ levels increased in lipo-rNEp, 24 h, while IL-6 levels rose in lipo-rNEp/al-rNEp/alum-alone groups, 4 h, compared to controls. In the draining lymph nodes (DLNs), CD11c(+)CD86(+) cells increased at 24 h in liposome-alone/lipo-rNEp groups. A rise in the CD11c(+)CD69(+) cells was noted in the lipo-rNEp group compared to other groups (p < 0.05). Cytokine levels in the spleen/sera remained unchanged in all the groups (p > 0.05). At 2 weeks postdose-2, CD11c(+)MHC-II(+)/CD11b(+)MHC-II(+) cells increased in the spleen in the lipo-rNEp and al-rNEp groups, respectively. In the DLNs, CD19(+)MHC-II(+) cells increased in rNEp/al-rNEp/lipo-rNEp groups post-both doses and CD11c(+)CD86(+) cells in the lipo-rNEp group. A balanced Th1/Th2 response was evident in the lipo-rNEp, while a Th2 bias was noted in al-rNEp. Different immune response gene clustering patterns were noted in uncultured spleens from immunized mice and cultured-stimulated splenocytes. In conclusion, lipo-rNEp is a better immunogen, works through dendritic cells, and elicits a balanced Th1/Th2 response, while alum functions through macrophages and induces a Th2 response.
Hepatitis Division, National Institute of Virology , Pune, India .
Shivali Shirish
SS
Joshi
Hepatitis Division, National Institute of Virology , Pune, India .
Vidya Avinash
VA
Arankalle
D000276:Adjuvants, Immunologic / Q000008:administration & dosage*; D000534:Alum Compounds / Q000008:administration & dosage*; D000818:Animals; D016207:Cytokines / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D006508:Hepatitis Antibodies / Q000097:blood; D016751:Hepatitis E / Q000276:immunology / Q000517:prevention & control*; D007115:Immunization Schedule; D007074:Immunoglobulin G / Q000097:blood; D016130:Immunophenotyping; D007273:Injections, Intramuscular; D008081:Liposomes / Q000008:administration & dosage*; D008253:Macaca mulatta; D008807:Mice, Inbred BALB C; D016176:T-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology; D013997:Time Factors; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology*; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*
D000276:Adjuvants, Immunologic; D000534:Alum Compounds; D016207:Cytokines; D000939:Epitopes; D006508:Hepatitis Antibodies; D007074:Immunoglobulin G; D008081:Liposomes; C400971:ORF2 protein, Hepatitis E virus; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D014765:Viral Vaccines; C041524:aluminum sulfate