Recombinant dengue 2 virus NS3 protein conserves structural antigenic and immunological properties relevant for dengue vaccine design.
The NS3 protein is a multifunctional non-structural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2. The full-length NS3 gene was cloned and expressed as a His-tagged fusion protein in Escherichia coli. The pNS3 protein was purified by two chromatography steps. The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-α in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques. The successfully purified recombinant protein was able to preserv the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.
PAHO/WHO Collaborating Centre for the Study of Dengue and its Vector, Institute of Tropical Medicine "Pedro Kourí", IPK, Autopista Novia del Mediodía Km 6 1/2, 17100, Havana, Cuba.
Rosa
R
Ramírez
Rosabel
R
Falcón
Alienys
A
Izquierdo
Angélica
A
García
Mayling
M
Alvarez
Ana Beatriz
AB
Pérez
Yudira
Y
Soto
Mayra
M
Muné
Emiliana Mandarano
EM
da Silva
Oney
O
Ortega
Ronaldo
R
Mohana-Borges
María G
MG
Guzmán
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D015153:Blotting, Western; D003001:Cloning, Molecular; D053059:Dengue Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003716:Dengue Virus / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008807:Mice, Inbred BALB C; D020365:RNA Helicases / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000914:Antibodies, Viral; D053059:Dengue Vaccines; D007371:Interferon-gamma; D016753:Interleukin-10; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014409:Tumor Necrosis Factor-alpha; D014614:Vaccines, Synthetic; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases