High efficient production of Pr55(gag) virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A.
The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.
Buonaguro
L
L
Division of Viral Oncology and AIDS Reference Center, 1st. Naz. Tumori Fond. G. Pascale, Naples, Italy.
Buonaguro
F M
FM
Tornesello
M L
ML
Mantas
D
D
Beth-Giraldo
E
E
Wagner
R
R
Michelson
S
S
Prevost
M C
MC
Wolf
H
H
Giraldo
G
G
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D000939:Epitopes / Q000235:genetics; D015683:Gene Products, gag / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015754:Genes, nef / Q000235:genetics; D015751:Genes, pol / Q000235:genetics; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D014454:Uganda; D014614:Vaccines, Synthetic / Q000235:genetics; D014771:Virion / Q000648:ultrastructure
D000939:Epitopes; D015683:Gene Products, gag; D015699:HIV Envelope Protein gp120; D011498:Protein Precursors; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C059767:p55 gag precursor protein, Human immunodeficiency virus 1