Bacillus subtilis spores expressing the VP28 antigen: a potential oral treatment to protect Litopenaeus vannamei against white spot syndrome.

The envelope protein VP28 of white spot syndrome virus (WSSV) is considered a candidate antigen for use in a potential vaccine to this important shrimp pathogen (the cause of white spot syndrome, WSS). Here, we used spores of Bacillus subtilis to display VP28 on the spore surface. Trials were conducted to evaluate their ability to protect shrimps against WSSV infection. The gene cotB-vp28 was integrated into the chromosome of the laboratory strain B. subtilis PY79, and expression of CotB-VP28 was detected by Western blotting and immunofluorescence. Expression of CotB-VP28 was equivalent to 1000 molecules per spore. PY79 and CotB-VP28 spores were mixed with pellets for feeding of whiteleg shrimps (Litopenaeus vannamei), followed by WSSV challenge. Superoxidase dismutase (SOD), phenoloxidase activities and mortality rates of the two shrimp groups were evaluated. Groups fed with PY79 and CotB-VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores (PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS.

Nguyen AT ，Pham CK ，Pham HT ，Pham HL ，Nguyen AH ，Dang LT ，Huynh HA ，Cutting SM ，Phan TN ... -  《-》

Protection of Penaeus monodon against white spot syndrome by continuous oral administration of a low concentration of Bacillus subtilis spores expressing the VP28 antigen.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB-VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB-VP28 spores (at ≥1 × 109  CFU per g pellet) to shrimps induced immune-relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore-coated pellets at ≥1 × 109  CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB-VP28 spores in feed pellets was reduced down to ≥5 × 107  CFU per g and ≥1 × 106  CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 109  CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB-VP28 spores at low dose of ≥1 × 106  CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. This study reports the protective efficacy of Bacillus subtilis CotB-VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB-VP28 spores (≥1 × 109  CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB-VP28 spores in feed pellets to ≥1 × 106  CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB-VP28 spores at a low dose of ≥1 × 106  CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.

Pham KC ，Tran HT ，Van Doan C ，Le PH ，Van Nguyen AT ，Nguyen HA ，Hong HA ，Cutting SM ，Phan TN ... -  《-》

First Litopenaeus vannamei WSSV 100% oral vaccination protection using CotC::Vp26 fusion protein displayed on Bacillus subtilis spores surface.

Litopenaeus vannamei do not have an adaptive immune response system. White spot syndrome virus (WSSV) is the most severe pathogen in shrimps. Bacillus subtilis spores carrying heterologous antigens on its surface have been evaluated as a vaccine inducing specific systemic responses on vertebrates. Orally administrated Vp28 vaccines have been investigated in crustaceans. Vp26 is also an important constituent of WSSV structure but little is known about its oral vaccination capacity in L. vannamei. In this study, for first time, L. vannamei WSSV protection was carried out using B. subtilis recombinant spores (RS), displaying CotC::Vp26 fusion protein (FP) on its surface. RS-expressing FP were coated on shrimp food pellets and used to feed L. vannamei. Results have shown that orally administered B. subtilis RS protected 100% L. vannamei against WSSV infection. Bacillus subtilis spores orally administrated expressing CotC::Vp26 fusion protein on its surface demonstrated the great capacity of Vp26 to induce immune protection, equally or even greater than Vp28 in L. vannamei. The biotechnological process developed represents an easy to produce, practical to handle, environmentally stable, human-safe and economically feasible opportunity to apply a new Vp26 vaccine in a massively way in shrimp farms.

Valdez A ，Yepiz-Plascencia G ，Ricca E ，Olmos J ... -  《-》

Surface-displayed VP28 on Bacillus subtilis spores induce protection against white spot syndrome virus in crayfish by oral administration.

Surface-displayed heterologous antigens on Bacillus subtilis spores can induce the vertebrate animals tested to generate local and systematic immune response through oral immunization. Here, the protection potential of the recombinant spores displaying the VP28 protein of white spot syndrome virus (WSSV) was investigated in the invertebrate crayfish (Cambarus clarkii). The VP28 protein was successfully displayed on the surfaces of B. subtilis spores using CotB or CotC as a fusion partner. Crayfish were administrated orally by feeding the feed pellets coated with B. subtilis spores for 7 days and immediately followed by WSSV challenge. Oral administration of either spores expressing CotB-VP28 or CotC-VP28 resulted in significantly higher relative survival rates of 37.9 and 44.8% compared with the crayfish orally administrated with the spores nonexpressing VP28 (10.3% relative survival rate). When challenges were separately conducted at 7 and 21 days after oral administration, the relative survival rates increased to 46.4 and 50% at 7 days post-oral administration, but decreased to 30 and 33.3% at 21 days after oral administration. These evidences indicate that the surface-displayed VP28 on B. subtilis spore could induce protection of crayfish against WSSV via oral administration. This is the first report to use the spore surface display system to deliver orally a heterologous antigen in an aquatic invertebrate animal, crayfish. The results presented here suggest that the spore-displayed VP28 might be suitable for an oral booster vaccine on prevention of WSSV infection in shrimp farming.

Ning D ，Leng X ，Li Q ，Xu W ... -  《-》

Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans.

Solís-Lucero G ，Manoutcharian K ，Hernández-López J ，Ascencio F ... -  《-》