RickA Expression Is Not Sufficient to Promote Actin-Based Motility of Rickettsia raoultii

Background: Rickettsia raoultii is a novel Rickettsia species recently isolated from Dermacentor ticks and classified within the spotted fever group (SFG). The inability of R. raoultii to spread within L929 cells suggests that this bacterium is unable to polymerize host cell actin, a property exhibited by all SFG rickettsiae except R. peacocki. This result led us to investigate if RickA, the protein thought to generate actin nucleation, was expressed within this rickettsia species. Methodology/Principal Findings: Amplification and sequencing of R. raoultii rickA showed that this gene encoded a putative 565 amino acid protein highly homologous to those found in other rickettsiae. Using immunofluorescence assays, we determined that the motility pattern (i.e. microcolonies or cell-to-cell spreading) of R. raoultii was different depending on the host cell line in which the bacteria replicated. In contrast, under the same experimental conditions, R. conorii shares the same phenotype both in L929 and in Vero cells. Transmission electron microscopy analysis of infected cells showed that non-motile bacteria were free in the cytosol instead of enclosed in a vacuole. Moreover, western-blot analysis demonstrated that the defect of R. raoultii actin-based motility within L929 cells was not related to lower expression of RickA. Conclusion/Significance: These results, together with previously published data about R. typhi, strongly suggest that another factor, apart from RickA, may be involved with be responsible for actin-based motility in bacteria from the Rickettsi

Measurement of VP1 specific IgG antibody response in the serum of immunized mice by indirect ELISA.

<p>(A) <b>Orally immunized mice:</b> Groups of mice (n = 10) were orally vaccinated three times on days 0, 7, and 21 with 200 µl containing live or inactivated recombinant baculovirus associated or non-associated with bilosomes. Bac-wt and PBS were given as negative controls. Each point represents the arithmetic mean value (n = 6) ±SD. *, P<0.05; **, P<0.01; ***, P<0.001 between live Bac-VP1 associated with bilosomes and live Bac-VP1 alone (a) or ***, P<0.001 between live Bac-VP1 associated with bilosomes and inactive Bac-VP1 associated with bilosomes (b) or ***, P<0.001 between live Bac-VP1 and inactive Bac-VP1 (c). (B) Subcutaneously immunized mice: Groups of mice (n = 10) were subcutaneously vaccinated two times on days 0 and 21 with 200 µl of live Bac-VP1 or inactivated EV71 or Bac-wt and PBS as negative controls. VP1-specific IgG antibody levels were determined by indirect ELISA. Each point represents the arithmetic mean value (n = 6) ± SD. *, P<0.05 between inactive EV71 and live Bac-VP1.</p

Confirmation of VP1 expression in purified baculoviruses by Coomassie staining and Western blotting.

<p>Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified baculoviruses, showing the presence of VP1. Lane 1, prestained protein marker; lane 2, Coomassie staining; lane 3, Western blot using anti-VP1 guinea pig polyclonal antibody and respective secondary antibody (a) Bac-VP1, (b) Bac-wt.</p

Neutralization antibody titers of vaccinated sera against homologous EV71 strain.

<p>Sera were collected from mice on days 0, 14, 28, 42, and 56. 25 µl of serial two-fold dilutions of sera were then mixed with 25 µl of 100 TCID50 of virus (Fuyang EV71, C4 virus) and incubated at 37°C for 2 h to neutralize infectious virus. The mixtures were then transferred to 96-well plates with more than 90% confluent monolayers of RD cells grown in DMEM containing 5% FBS. After incubation for 5 days at 37°C, the neutralizing antibody titers were read as the highest dilution of sera that completely inhibited virus growth. (A) Neutralization titers of orally vaccinated mice candidate’s sera. Each point represents the arithmetic mean value (n = 6) ±SD. **, P<0.01; ***, P<0.001 between live Bac-VP1 associated with bilosomes and live Bac-VP1 alone (a) or ***, P<0.001 between bilosomes associated live Bac-VP1 and bilosomes associated inactive Bac-VP1 (b) or ***, P<0.001 between live Bac-VP1 and inactive Bac-VP1 (c). (B) Neutralization titers of subcutaneously vaccinated mice candidate’s sera. Each point represents the arithmetic mean value (n = 6) ± SD. **, P<0.01; ***P<0.001 between inactive EV71 and live Bac-VP1.</p

Measurement of VP1 specific mucosal IgA antibody response in orally immunized mice by indirect ELISA.

<p>Groups of mice (n = 10) were orally vaccinated three times on days 0, 7, and 21 with 200 µl containing live or inactivated recombinant baculovirus associated or non-associated with bilosomes or Bac-wt. Each point represents the arithmetic mean value (n = 6) ± SD. **, P<0.01 between live Bac-VP1 associated with bilosomes and live Bac-VP1 alone or ***, P<0.001 between live Bac-VP1 and inactive Bac-VP1 or ***, P<0.001 between bilosomes associated live Bac-VP1 and bilosomes associated inactive Bac-VP1.</p

Measurement of viral copies in brain by real-time PCR.

<p>Mice were treated with orally or subcutaneously vaccinated sera on day 1 post challenge with 5 MLD₅₀ (mouse lethal dose) of mouse adapted EV71 B4 strain HFM 41 virus. The viral loads were measured in the brain of dead and survived animals. (a) Mice treated with orally vaccinated mice sera (b) Mice treated with subcutaneously vaccinated mice sera. Each column represents the mean of triplicate assays with standard deviation.</p

Passive protection of neonatal mice by vaccinated mice sera against mouse adapted lethal HEV71 infection.

<p>Six day old BALB/c mice were inoculated i.p. with mouse adapted EV71-B4 strain at a dose of 5 MLD50 per mouse. 24 h later, each mouse was passively immunized with sera obtained from the vaccinated adult mice. The control groups received sera from Bac-wt and PBS immunized mice. Mortality was monitored until 21 days post infection. (a) Passive protection study using sera from orally vaccinated mice (b) Passive protection study using sera from subcutaneously vaccinated mice.</p

Anchoring of VP1 on the surface of infected Sf9 cells.

<p>(<b>a</b>) Confirmation of the anchoring of VP1 on the plasma membrane of Sf9-II cells infected with Bac-VP1. (b) No anchoring of VP1 was observed on the plasma membrane of Sf9- II cells infected with Bac-wt. The cells were cultured on sterile cover slips and infected at an MOI of 0.1. Cells were fixed with 4% PFA and blocked with 2% bovine serum albumin for 30 min at 37°C. VP1 was detected by a polyclonal primary antibody against bacterially expressed VP1 raised in guinea pig (1∶300 dilution; in house production) followed by a secondary FITC-conjugated rabbit anti-guinea pig mAb (1∶100 dilution; Dako). Plasma membrane staining was done using the CellMask™ Orange plasma membrane stain (Invitrogen).</p

Baculovirus Surface Display of Immunogenic Proteins for Vaccine Development

Vaccination is an efficient way to prevent the occurrence of many infectious diseases in humans. To date, several viral vectors have been utilized for the generation of vaccines. Among them, baculovirus&mdash;categorized as a nonhuman viral vector&mdash;has been used in wider applications. Its versatile features, like large cloning capacity, nonreplicative nature in mammalian cells, and broad tissue tropism, hold it at an excellent position among vaccine vectors. In addition to ease and safety during swift production, recent key improvements to existing baculovirus vectors (such as inclusion of hybrid promoters, immunostimulatory elements, etc.) have led to significant improvements in immunogenicity and efficacy of surface-displayed antigens. Furthermore, some promising preclinical results have been reported that mirror the scope and practicality of baculovirus as a vaccine vector for human applications in the near future. Herein, this review provides an overview of the induced immune responses by baculovirus surface-displayed vaccines against influenza and other infectious diseases in animal models, and highlights the strategies applied to enhance the protective immune responses against the displayed antigens