Investigation of Kluyveromyces marxianus as a novel host for large-scale production of porcine parvovirus virus-like particles.

BACKGROUND: Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines. RESULTS: After optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice. CONCLUSIONS: This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.

Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of porcine parvovirus capsid proteins.

Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. Importance: Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.

Production and immunogenicity of VP2 protein of porcine parvovirus expressed in Pichia pastoris.

Viral protein 2 (VP2) of porcine parvovirus (PPV) is the major viral structural protein and is responsible for eliciting neutralizing antibodies in immunized animals. In this study, we constructed and characterized a recombinant yeast vector encoding the VP2 protein, designated as pGAPZαA-VP2. The construct was confirmed by restriction enzyme digestion, PCR, and sequencing and then introduced into P. pastoris strain SMD1168 by electroporation. The expressed VP2 protein was analyzed by SDS-PAGE and western blot. Immunization of mice with the VP2 protein elicited a PPV-specific humoral immune response. Notably, a preparation of VP2 protein containing adjuvant induced a much better antibody response than VP2 alone. Clearly, the adjuvant strongly enhanced the immunogenicity of VP2. This study provides a foundation for the application of the VP2 protein in the clinical diagnosis of PPV and in vaccination against PPV in the future.

TLR2-mediated NF-κB signaling pathway is involved in PPV1-induced apoptosis in PK-15 cells.

Porcine parvovirus 1 (PPV1) mainly induces severe reproductive failure in pregnant swine, and causes huge economic losses to the swine industry. Cell apoptosis induced by PPV1 infection has been identified the major cause of reproductive failure. However, the molecular mechanism was not fully elucidated. In this study, the potential mechanism of PPV1 induced apoptosis in PK-15 cells was investigated. Our results showed that PPV1 induced apoptosis in PK-15 cells. Further studies revealed toll-like receptor 2 (TLR2) was involved in the PPV1-mediated apoptosis. TLR2 siRNA significantly decreased the apoptosis. Finally, our study showed NF-κB was activated by TLR2 during PPV1-induced apoptosis. The activation of NF-κB signaling was demonstrated by the phosphorylation of p65, p65 nuclear translocation and degradation of inhibitor of kappa B α (IκBα). Together, these results provided evidence that the recognition between PPV1 and PK-15 cells was mainly through TLR2, and then induction of the NF-κB signaling pathway activation, which further induces apoptosis. Our study could provide information to understand the molecular mechanisms of PPV1 infection.

Porcine parvovirus replication is suppressed by activation of the PERK signaling pathway and endoplasmic reticulum stress-mediated apoptosis.

Endoplasmic reticulum (ER) stress is associated with numerous mammalian diseases, especially viral diseases. Porcine parvovirus (PPV) is the causative agent of reproductive failure in swine. Here, we observed that the PPV infection of porcine kidney 15 and porcine testis cells resulted in the activation of ER stress sensors mediated by protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 and activating transcription factor 6 (ATF6). ER stress activation obviously blocked PPV replication. Depletion of proteins, such as PERK, eukaryotic initiation factor 2, and ATF4, by small interfering RNA significantly enhanced PPV replication. Moreover, the pro-apoptotic factor C/EBP homologous protein was identified a key factor in the inhibition of PPV replication. These data demonstrate that PPV infection activates ER stress through the PERK signaling pathway and that ER stress inhibits further PPV replication by promoting apoptosis.

Ultraviolet Light (UV) Inactivation of Porcine Parvovirus in Liquid Plasma and Effect of UV Irradiated Spray Dried Porcine Plasma on Performance of Weaned Pigs.

A novel ultraviolet light irradiation (UV-C, 254 nm) process was designed as an additional safety feature for manufacturing of spray dried porcine plasma (SDPP). In Exp. 1, three 10-L batches of bovine plasma were inoculated with 10(5.2 ± 0.12) tissue culture infectious dose 50 (TCID50) of porcine parvovirus (PPV) per mL of plasma and subjected to UV-C ranging from 0 to 9180 J/L. No viable PPV was detected in bovine plasma by micro-titer assay in SK6 cell culture after UV-C at 2295 J/L. In Exp. 2, porcine plasma was subjected to UV-C (3672 J/L), then spray dried and mixed in complete mash diets. Diets were a control without SDPP (Control), UV-C SDPP either at 3% (UVSDPP3) or 6% (UVSDPP6) and non-UV-C SDPP at 3% (SDPP3) or 6% (SDPP6). Diets were fed ad libitum to 320 weaned pigs (26 d of age; 16 pens/diet; 4 pigs/pen) for 14 d after weaning and a common diet was fed d 15 to 28. During d 0 to 14, pigs fed UVSDPP3, UVSDPP6, or SDPP6 had higher (P < 0.05) weight gain and feed intake than control. During d 0 to 28, pigs fed UVSDPP3 and UVSDPP6 had higher (P < 0.05) weight gain and feed intake than control and SDPP3, and SDPP6 had higher (P < 0.05) feed intake than control. Also, pigs fed UVSDPP had higher (P < 0.05) weight gain than pigs fed SDPP. In conclusion, UV-C inactivated PPV in liquid plasma and UVSDPP used in pig feed had no detrimental effects on pig performance.

Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity.

The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.

Porcine parvovirus flocculation and removal in the presence of osmolytes.

Viruses can be modified into viral vaccines or gene therapy vectors in order to treat acquired or genetic diseases. To satisfy the current market demand, an improvement in current vaccine manufacturing is needed. Chromatography and nanofiltration are not suitable for all types of viruses. In this study, we propose to use virus flocculation with osmolytes, followed by microfiltration, as a potential virus purification process. We hypothesize that osmolytes strongly bind to water, thus leading to the formation of a hydration layer around the virus particles and stimulation of aggregation. We have discovered that osmolytes, including sugars, sugar alcohols and amino acids, preferentially flocculate porcine parvovirus (PPV), and demonstrate a >80% removal with a 0.2 µm filter while leaving model proteins in solution. This large pore size filter increases the flux and decreases the transmembrane pressure of typical virus filters. The best flocculants were tested for their ability to aggregate PPV at different concentrations, shear stress, pH and ionic strength. We were able to remove 96% of PPV in 3.0M glycine at a pH of 5. Glycine is also an excipient, and therefore may not require removal later in the process. Virus flocculation using osmolytes, followed by microfiltration could be used as an integrated process for virus purification.

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma.

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.

[The surface display of porcine parvovirus VP2 protein in Lactobacillus casei].

Lactobacillus casei 393 was selected as a bacterial carrier for the expression of Porcine Parvovirus (PPV) protective antigen VP2 protein. The gene encoding PPV VP2 protein was cloned into the Lactobacillus casei surface expression vector pPG, and then the constructed recombinant vector pPG-VP2 was electrotransformed into Lactobacillus casei 393 generating the recombinant system pPG-VP2/L. casei393 expressing PPV VP2 protein. The recombinant strain was induced by 2% Lactose in MRS and about 74kD protein was detected with SDS-PAGE. The result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescent test showed that the expressed protein was secreted on the cell surface Lactobacillus casei.

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system.

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained. Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.

In vivo, dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway.

Recombinant parvovirus-like particles (PPV-VLPs) are particulate exogenous Ags that induce strong CTL response in the absence of adjuvant. In the present report to decipher the mechanisms responsible for CTL activation by such exogenous Ag, we analyzed ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by dendritic cells (DCs). In vivo, PPV-VLPs are very efficiently captured by CD8alpha- and CD8alpha+ DCs and then localize in late endosomes of DCs. Macropinocytosis and lipid rafts participate in PPV-VLPs capture. Processing of PPV-VLPs does not depend upon recycling of MHC class I molecules, but requires vacuolar acidification as well as proteasome activity, TAP translocation, and neosynthesis of MHC class I molecules. This study therefore shows that in vivo DCs can cross-present PPV-VLPs using an endosome-to-cytosol processing pathway.