Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes

<p>Abstract</p> <p>Background</p> <p>The yellow fever virus, a member of the genus <it>Flavivirus</it>, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.</p> <p>Results</p> <p>YF 17D EGFP recombinant virus was grew in Vero cells and reached a peak titer of approximately 6.45 ± 0.4 log10 PFU/mL at 96 hours post-infection. Immunoprecipitation and confocal laser scanning microscopy demonstrated the expression of the EGFP, which was retained in the endoplasmic reticulum and not secreted from infected cells. The association with the ER compartment did not interfere with YF assembly, since the recombinant virus was fully competent to replicate and exit the cell. This virus was genetically stable up to the tenth serial passage in Vero cells. The recombinant virus was capable to elicit a neutralizing antibody response to YF and antibodies to EGFP as evidenced by an ELISA test. The applicability of this cloning strategy to clone gene foreign sequences in other flavivirus genomes was demonstrated by the construction of a chimeric recombinant YF 17D/DEN4 virus.</p> <p>Conclusion</p> <p>This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow <it>in vivo </it>studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.</p

DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2

Limited replication of yellow fever 17DD and 17D-Dengue recombinant viruses in rhesus monkeys

For the development of safe live attenuated flavivirus vaccines one of the main properties to be established is viral replication. We have used real-time reverse transcriptase-polymerase chain reaction and virus titration by plaque assay to determine the replication of yellow fever 17DD virus (YFV 17DD) and recombinant yellow fever 17D viruses expressing envelope proteins of dengue virus serotypes 2 and 4 (17D-DENV-2 and 17D-DENV-4). Serum samples from rhesus monkeys inoculated with YFV 17DD and 17D-DENV chimeras by intracerebral or subcutaneous route were used to determine and compare the viremia induced by these viruses. Viral load quantification in samples from monkeys inoculated by either route with YFV 17DD virus suggested a restricted capability of the virus to replicate reaching not more than 2.0 log10 PFU mL-1 or 3.29 log10 copies mL-1. Recombinant 17D-dengue viruses were shown by plaquing and real-time PCR to be as attenuated as YF 17DD virus with the highest mean peak titer of 1.97 log10 PFU mL-1 or 3.53 log10 copies mL-1. These data serve as a comparative basis for the characterization of other 17D-based live attenuated candidate vaccines against other diseases

Percentage of survival of Balb/c mice immunized with pE1D2 and pE2D2 and challenged with DENV2.

<p>Mice were i.m. immunized with two DNA doses and challenged 4 weeks after the first plasmid inoculation. Non-immunized and pcTPA-injected mice followed the same virus infection procedure. Mice were daily monitored and deaths were recorded. Differences between pE1D2- and pE2D2-vaccinated animals were statistical significant (p = 0.0027), as well as between these groups and control animals (p<0.0001). Data represent compilation of two independent experiments, with groups of 10 animals in each test (n = 20).</p

Analysis of the expression <i>in vitro</i> of recombinant proteins.

<p>BHK cells were transfected with plasmids pE1D2 (A, D), pE2D2 (B, E) and pcTPA (C, F). Cells were permeabilized, fixed and treated with DENV2 hiperimmune mouse ascitic fluid (A–C) or the monoclonal DENV2 3H5 antibody (D–F), followed by incubation with anti-mouse fluorescein-conjugated goat IgG. Magnification 1000x (A, B, D, E) and 400x (C, F).</p

Plaque reduction neutralization test.

<p>The neutralizing antibody titrations (PRNT₅₀) against DENV2 was evaluated in serum samples collected from pE1D2- and pE2D2-vaccinated mice (n = 20), before and after virus challenge. Individual samples were serially diluted from 1∶5 to 1∶640 and PRNT₅₀ were performed in 96-well plates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020528#s2" target="_blank">material and methods</a>. Asterisks indicate differences that are statistically significant between pE1D2- and pE2D2-immunized animals (*, p = 0.0029) or between vaccinated mice before and after virus challenge (***, p<0.0001).</p

The morbidity in vaccinated mice after DENV2 challenge.

<p>The percentage (A) and degree (B) of morbidity of Balb/c mice immunized with pE1D2, pE2D2 and controls groups (non-immunized and pcTPA-inoculated animals) were analyzed after i.c. challenge with DENV2. Clinical signs of infection, mainly hind leg paralysis, alterations in spinal column and deaths, were monitored during 21 days post challenge (A). Differences in morbidity rates between pE1D2- and pE2D2-vaccinated mice were statistical significant (p<0.0001), as well as between pE1D2 and control animals (p<0.0001). The semi-quantitative analysis of morbidity degrees after virus challenge (B) were performed using a subjective scale ranging from 0 to 3 (0 = none, 1 =  mild paralyses in one hind leg or alteration of the spinal column with a small hump, 2 =  one severe hind leg paralyses and alteration of the spinal column with a small hump or two severe hind leg paralyses, 3 =  two severe hind leg paralyses and deformed spinal column or death). Asterisks indicate statistically significant differences between vaccinated animals and mice inoculated with the control vector pcTPA (* p = 0.0051; ** p = 0.0002; *** p<0.0001). Data represent compilation of two independent experiments, with groups of 10 animals in each test (n = 20).</p

Schematic representation of the DENV2 envelope protein sequence.

<p>The protein is composed by the three domains (I, II and III) and the steam-anchor region (adapted from Modis <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020528#pone.0020528-Modis1" target="_blank">[12]</a>). The sequence coding 80% of the N-terminal E protein, which contains domains I, II and III, was used for engineering the pE1D2 plasmid, while the pE2D2 plasmid encoded only the domain III sequence. Numbers indicated in the figure highlight amino acids corresponding the beginning and end positions at the primary protein sequence that compose each domain.</p

Molecular cloning of EGFP protein expression cassete in the chimeric YF17D/DEN4 virus genome

<p><b>Copyright information:</b></p><p>Taken from "Construction and characterization of recombinant flaviviruses bearing insertions between E and NS1 genes"</p><p>http://www.virologyj.com/content/4/1/115</p><p>Virology Journal 2007;4():115-115.</p><p>Published online 30 Oct 2007</p><p>PMCID:PMC2173888.</p><p></p> (A) Schematic representation of YF 17D/DEN4/Esa/EGFP/6 recombinant virus genome and the genetic elements fused to EGFP gene. (B) Growth of recombinant YF17D/DEN4 viruses in Vero cells. Three independent experiments were performed to measure viral spread in Vero cells after infection with an multiplicity of infection (MOI) of 0.02. Cell culture supernatant aliquots were taken at 24, 48, 72, 96, 120 and 140 hour post-infection (p.i.) and titrated by plaque formation on Vero cell monolayers. (C) Analysis of recombinant YF 17D/DEN4/Esa/6 virus genetic stability after serial passaging on Vero cell monolayers. Electrophoretic analysis of RT-PCR amplicons from viral RNA extracted from samples of the supernatant of cultures according to the passage numbering indicated on top of each lane. The first lane corresponds to cDNA-derived YF17D/DEN4 virus RNA; the remaining lanes are RT-PCR profiles from YF17D/DEN4/Esa/6 virus RNA at different passage levels with lanes 2 and 3 corresponding to amplicons from RNAs of viral stocks (1P, transfection supernatant) or passage two (2P, first passage of transfection supernatant), respectively. Lanes 4 to 11 represent RT-PCR products, which were obtained from viral RNA in the fifth, tenth, 15and 20passages of the two independent passage lineages (5P1 and 5P2; 10P1 and 10P2, 15P1 and 15P2, 20P1 and 20P2, respectively)