For that reason, recently we proposed a new vaccine candidate against CSFV, which is based on viral E2 gly- coprotein fused to the extracellular domain of the por- cine CD154 protein (Toledo et al. 2007; Pujol et al. 2015). This last protein has been used as a molecular adjuvant since several studies have claimed that it potentiates the immune system response (Xiang et al. 2001; Ramos et al. 2011). The signals triggered by the binding of this mol- ecule to its receptor (CD154-CD40) are crucial for the proliferation and differentiation of the antigen-specific B cells, as well as for the change of isotype and maturation of the antibody’s affinity. All this is essential for the effi- cient generation of both memory B cells and long-lived plasma cells (Henn et al. 2001). The pig immunization using a unique dose of the chimeric protein E2-CD154 produced by HEK-293 cells growing in a serum-free sus- pension culture and the challenge experiment performed only 7 days after by infection with a high virulent CSF virus strain without clinical manifestations of the disease demonstrated the early protective capacity of this antigen in pigs (Suárez et al. 2017).
forming enzymes . Thus, disulfide bond formation of recombinant Pfs48/45 most likely occurs spontaneously after secretion and is not only dependent on the primary structure but also on environmental factors that deter- mine cysteine oxidation. By carefully optimizing the ratio of different redox couples in the fermentation broth, we have identified conditions, which render the extracel- lular milieu more oxidizing allowing formation of struc- tural disulfide bonds in the Pfs48/45-6C domain of the fusion protein. These conditions seem to favor the forma- tion of intramolecular disulfide bonds as secreted R0.6C was mainly present in the monomer state. However, the finding that crude R0.6C shows reduced reactivity with mAb45.1 compared to the reference material consisting of mAb45.1 immune-purified R0.6C  suggests that the monomer fraction contains a mixture of conformers with native and non-native cysteine connectivity. A work- flow has therefore been developed using batch fermenta- tion in lab-scale stirred bioreactor to produce discreet batches of recombinant R0.6C purified by a simple 2-step purification process. The first step captures the recombi- nant protein from the culture supernatant and the second
Production of heterologous proteins holds great promise. In the realm of human recombinant therapeutic proteins, the first protein to be approved was insulin in 1982. Since then over 120 other proteins have been prepared and are used to treat human illness. A major concern with these drugs was an undesired immune response in patient that could lead to neutralization and reduced efficacy of the protein to anaphylactic reactions. Such effects were not observed and recombinant proteins play an important role in human health (Dingermann, 2008). Another source of concern in the production of heterologous proteins comes from the production of transgenic plants. An example of such genetic manipulation is the creation of ‘Golden Rice’- a new strain of rice engineered to have much higher β- carotene levels. Vitamin A deficiency is an important health concern, often leading to blindness. It is prevalent in many parts of the world especially Southeast Asia and Africa. Consumption of Golden Rice can reduce levels of vitamin A deficiency and save many from going blind. However, due to public concern regarding transgenic crops, Golden Rice is not marketed and not available to the populations that can benefit directly from the crop (Yonekura-Sakakibara and Saito, 2006). Public acceptance is an important consideration in the planning and production of genetically modified crops.
in mice vaccinated with HA alone (Fig. 5A). In contrast, TLR4 ⫹/⫹ mice vaccinated and boosted with HA⫹F or HA-F had signifi- cantly (P ⬍ 0.001 and P ⬍ 0.01, respectively) higher antibody titers than mice vaccinated and boosted with HA alone, suggesting an F protein adjuvant effect through TLR4 (Fig. 5B). TLR4 ⫺/⫺ mice vaccinated with PBS, HA, HA⫹F, or HA-F and challenged with X31 had reduced X31 lung titers at day 3 postchallenge com- pared to the PBS control mice (Fig. 6A). Despite the increased HA-specific antibody response in HA ⫹ F-immunized mice, there was no detectable increase in virus clearance attributable to F pro- tein, as was observed in wild-type (TLR4 ⫹/⫹ ) mice (Fig. 3A). Viral titers were also evaluated at 6 and 8 days p.i. to determine if mice vaccinated with HA ⫹ F and HA-F exhibited increased viral clear- ance. At day 6 p.i., wild-type mice vaccinated with HA⫹F and HA-F had enhanced lung virus clearance compared to replication of HA-alone X31 virus at day 6 in mice vaccinated with HA alone (Fig. 3B), and all mice cleared virus by 8 days p.i. (data not shown). In contrast, at day 6 p.i. in the absence of TLR4 (day 6 knockout [KO]), HA, HA ⫹ F, and HA-F lung titers were equivalent, indi- cating that RSV F protein mediates an HA adjuvant effect in the FIG 4 Histopathology in vaccinated and challenged mice. (A) The lungs from vaccinated and challenged mice were scored for histopathology using a scoring system from 1 to 4, where 1 indicates minimal pathology and 4 indicates maximum pathology. (B) Mice vaccinated with PBS, HA, HA ⫹ F, HA-F, X31, F, or PR8 were challenged with X31, or (C) mice vaccinated with PBS, RSV A2, RSV B1, HA, F, HA ⫹ F, or HA-F were challenged with RSV A2. Three or 5 days postchallenge, respectively, the lungs were sectioned, stained, and scored. Representative data from two independent experiments with 3 mice per group are shown. Student’s t test and one-way ANOVA were performed in GraphPad Prism version 5.
particle and will then covalently to attach to a protein modified with the spytag (ST) sequence at pH 5 – 8 (Figure 4.11). Spycatcher protein is designed to be produced then treated to create a free thiol for conjugation. SC was conjugated to PRINT particles using the same protocol used for RecE-tCys (Figure 4.9). Spycatcher was effectively conjugated to the particles after reduction with a TCEP solution (the optimal TCEP molarity is between 0 and 1 mM) when reduced for 30 minutes (Figure 4.12). An initial test of the ability of spycatcher conjugated particles (NP-SC) to capture spytag RecE (RecE-ST) was successful as NP-SC treated with RecE-ST (NP-SC + RecE-ST) in PBST then washed multiple times was able to bind DENV specific antibodies with no background from RecE-ST adsorption in a blank particle control group (Figure 4.13). More importantly, the NP-SC/T-RecE group was able to bind 2D22 antibody, specific to a highly neutralizing epitope found on the quaternary
Recombinant production of eukaryotic membrane proteins in heterologous expression systems can be a difficult task (Grisshammer, 2006). Membrane protein synthesis most commonly requires the polypeptide to be targeted to a protein-conducting channel (translocon) in the ER, to then be released into the ER membrane where it folds into its correct conformation. Only proteins that are correctly folded pass into the Golgi, otherwise proteins are degraded (reviewed by Grisshammer, 2006). The detection of incorrectly folded proteins triggers the unfolded protein response (UPR), which either targets these proteins for degradation, or causes cell apoptosis, in both mammalian and plant cells (Iwata and Koizumi, 2005). In addition, the use of strong promoters resulting in a large number of mRNAs which are translated on ribosomes and inserted into the ER may overwhelm the ER protein folding machinery, leading to increased misfolding and UPR. This may result in reduced accumulation of membrane proteins. To confound matters further, not all membrane proteins elicit UPR (Grisshammer, 2006). Why some membrane proteins insert and fold better than others is not understood. The ER
PP1 controls numerous cellular processes by the dephos- phorylation of key regulatory proteins. One substrate is the eukaryotic translation initiation factor (eIF-2 ␣ ). The ability of the HSV ICP34.5 and GADD34 proteins to target the catalytic subunit of PP1 to dephosphorylate eIF-2 ␣ has been previously described (4, 18). This requires the carboxyl termini of the ICP34.5 and GADD34 proteins, which share sequence simi- larity with DP71L (6, 20). The increase in PP1 activity we observed during ASFV infection might result in increased de- phosphorylation of PP1 substrates. We demonstrated that eIF-2 ␣ phosphorylation levels were reduced by more than two- fold in ASFV-infected cells compared with the background levels observed in controls, and this reduction correlated with the increase of PP1 activity. The DP71L protein may possibly act to increase dephosphorylation of eIF-2 ␣ indirectly by caus- ing a general increase in PP1 activity or might specifically target PP1 to dephosphorylate eIF-2 ␣ . Further studies are in progress to define the role of DP71L-PP1 interaction in viral infection and associated pathogenesis.
Swinedysentery caused by Brachyspira hyodysenteriae, results in substantial economic losses in swine producing countries worldwide. Although a number of different vaccine approaches have been explored with regard to this dis- ease, they show limitations and none of them have reached the market. We here determine the vaccine potential of a weakly haemolytic B. hyodysenteriae strain. The virulence of this strain was assessed in experimental infection trials and its protection against swinedysentery was quantified in a vaccination-challenge experiment using a seeder infection model. Systemic IgG production and local IgA production were monitored in serum and faeces respectively. Across all trials, pigs that were colonized by virulent, strongly haemolytic B. hyodysenteriae strains consistently developed swinedysentery, in contrast to none of the pigs colonized by the weakly haemolytic B. hyodysenteriae vaccine strain. In the seeder vaccination trial nearly all immunised animals developed swinedysentery on subsequent challenge with a virulent strain, but the speed of spread of swinedysentery and faecal score were significantly reduced in animals immunised with the weakly haemolytic strain compared to sham-immunised animals. The IgA response of immu- nised animals upon challenge with a virulent B. hyodysenteriae strain significantly correlated to a later onset of disease. The correlation between local IgA production and protection induced by a weakly haemolytic B. hyodysenteriae strain provides leads for future vaccine development against swinedysentery.
in cereal grain can be achieved by various forms of feed processing that cause starch gelatinisation. Starch in the grain can be altered by physical processes including grind- ing, milling, moist-heat treatment, flaking and extrusion (Bengala Freire et al. 1991; Carter & Leibholz, 1991; Gill et al. 1996; Pluske et al. 1996). Studies intended to reduce LI fermentation and hence SD after experimental infection have used cereal grains that were steam-flaked (Siba et al. 1996), or hammer-milled, extruded or steam- flaked (Pluske et al. 1996). Extrusion was found to be the most effective method to achieve this with wheat- and sorghum-based diets. These studies also showed that diets that were low in RS still resulted in a high incidence of SD in experimentally-infected pigs, an effect which was then thought to be due to residual sNSP. The anti-nutritive effect of sNSP can be reduced by the dietary inclusion of specific sNSP-degrading enzymes which partially depoly- merise sNSP, resulting in lower digesta viscosity (Bedford & Classen, 1992; Choct et al. 1995, 1996). Improved growth, weight gain, protein and starch digestibility, and better feed conversion have been achieved in pigs by supplementing wheat- or barley-based diets with sNSP- degrading enzymes, as a result of a better nutrient avail- ability in the small intestine (Graham et al. 1989; Inborr et al. 1991a,b). Various treatments have been applied to sorghum-based diets to improve their nutritive value for animals. Heat treatments such as extrusion and steam-flak- ing increased starch digestibility (Buchanan-Smith et al. 1968; Mills et al. 1994; Kemm & Brand, 1996), while reduction of the sorghum particle size by fine grinding resulted in an increase of the apparent ileal digestibility of DM, starch, gross energy and protein (Owsley et al. 1981). The addition of enzymes to sorghum-based diets to enhance performance in finishing pigs has been limited to dietary supplementation of cellulases (Kim et al. 1994). The overall aim of the present study was to identify a cost-effective, commercially applicable pig diet which would provide protection against SD. Three experiments were undertaken using diets based on wheat or sorghum, investigating the effects of grain extrusion and addition of an RS-degrading enzyme (both to reduce RS), and/or addition of an sNSP-degrading enzyme (to reduce the vis- cous effect of sNSP). The influences of these diets on pig performance, fermentation in the large intestine, and on the expression of SD after experimental infection were recorded. Results of the influences of these diets on the microflora have been published elsewhere (Durmic et al. 2000).
infection, prevent translational shutoff, and prolong host life. For example, vaccinia virus E3L and K3L inhibit PKR-medi- ated phosphorylation of eIF2 ␣ (23), herpes simplex virus (HSV) protein Us11 inhibits PKR activation, and HSV ICP34.5 protein recruits PP1 ␣ to dephosphorylate eIF2 ␣ (18). African swine fever virus (ASFV) is the sole member of the Asfaviridae family. It is a large cytoplasmic DNA virus with similarity to poxviruses in genome organization and replication strategy. ASFV infects domestic pigs and causes an acute dis- ease with a high mortality, for which there is no vaccine. It has a genome of between 170 and 190 kb, depending on the strain, and encodes more than 150 genes, of which many are not essential for virus replication but are involved in modulating virus-host interaction, immune evasion, and pathogenesis (36). The DP71L gene is encoded as either a 184-amino-acid long form, DP71L(l), or a 70- to 72-amino-acid short form, DP71L(s). The ASFV DP71L(l) form has been shown to be expressed late during the replication cycle (11). Deletion of either the DP71L(l) or the DP71L(s) gene did not reduce ASFV replication in either primary pig macrophages or tissue culture cell lines (32, 44). Deleting the short form of this gene from the virulent E70 isolate resulted in a dramatic reduction of virulence following infection of pigs (44). However, deletion of the DP71L gene from other isolates, Malawi Lil20/1 and Pr4, did not significantly reduce virulence (1). The C-terminal 56-amino-acid domain of DP71L is highly conserved among different isolates and shares about 40% amino acid identity with the C terminus of the herpes simplex virus ICP34.5 pro- tein and the cellular protein GADD34. Notably, the PP1 dock- ing site and the regulatory motif Vx(7,8)Rx3Wx5DRxRFxRR x11L are conserved between these proteins, suggesting that DP71L may have a similar function and also acts to direct PP1c to dephosphorylate substrates, including eIF2 ␣ . By using the yeast two-hybrid system and by direct binding studies, we have previously shown that DP71L interacts with PP1c (32). ASFV infection of Vero cells was shown to decrease the phosphory- lation level of eIF2 ␣ at late times postinfection (5, 32), but so far there is no direct evidence to show that DP71L is respon- sible for this dephosphorylation of eIF2 ␣ . We reported previ- ously that extracts from ASFV-infected cells have increased PP1 activity compared to that of mock-infected cells, as as- sayed by dephosphorylation of the substrate phosphorylase a, and that this is dependent on the presence of the DP71L gene (32). However, the relevance of this increase in PP1 activity to ASFV virulence and pathogenesis is unclear.
play a role, since loss of glycan residues in the ectodomain of GP5 enhanced both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of a nearby neutralization epitope (76). It is also possible that reduced neutralization and the prolonged duration of viremia might be associated with the phenomenon of antibody-dependent enhancement (ADE) (77). In ADE, low titers of NAs can increase the virus infectivity by facilitating uptake of the virus into permissive macrophages through binding of virus- antibody complex to the Fc receptor (78). Another explanation for the lack of NA to PRRSV concerns the presence of decoy epitopes. Ostrowski et al identified a non-neutralizing epitope (aa 27-30, epitope A) which is located near the neutralizing epitope (aa 37-45, epitope B) in the ectodomain of the GP5 protein (74). In fact, PRRSV-infected pigs first develop antibodies against epitope A and neutralizing antibodies against epitope B appear later suggesting that epitope A can impede and diminish humoral immunity against PRRSV (74, 79). Liurong et al tested whether enhanced NA develops when the decoy effect is minimized or eliminated using the modified GP5 of PRRSV by inserting a pan-DR helper T cell epitope (PADRE) between the neutralizing and decoy epitopes in order to allow efficient display of the neutralizing epitope (80). They demonstrated that the DNA vaccine encoding modified GP5 induced a significantly higher level of NA compared to naïve GP5 in vaccinated mice. Lastly, the role of PRRSV-specific antibodies in eliminating PRRSV- infected cells may be limited because viral proteins are not efficiently incorporated into the plasma membrane of PRRSV-infected macrophages, which make infected cells invisible to PRRSV-specific antibodies (81).
preventing ZIKV infection. Several vaccines developed using different platforms and targets have also advanced into phase I clinical trials [3, 11, 31]. However, none of these candidates have been tested for their ability to pro- tect the fetus or infants born to infected mothers, espe- cially from devastating sequelae such as microcephaly. Han et al. showed that co-administration of ZIKV and sera from mice immunized with a ZIKV envelope protein sub- unit vaccine (E90) reduced the lethality of the infection, protecting neonates from death . However, we sought to directly explore the potential of this vaccine to confer protection from pregnant dams to offspring in utero and reduce ZIKV-associated complications.
186 Woo J.H., Liu Y.Y., Mathias A., Stavrou S., Wang Z., Thompson J., Neville D.M. (2002). Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris. Protein Expression and Purification 25, 270–282.
IL-6, mainly produced by cells such as Th2 cells, mono- cytes, B cells and muscle tissue , is often regarded as a useful biomarker of bacterial infections in pigs, and has been reported to be present for several days after an Actin- obacillus pleuropneumoniae infection . This is partly due to its slow and stable plasma kinetics (for review see ). Elevated serum levels of IL-6 have been observed in pigs after injection with LPS and endotoxin extracts from B. hyodysenteriae . In the present study it seemed that serum IL-6 was not a reliable marker of swinedysentery, as only three of the sick animals showed detectable levels of this cytokine in the blood during dysentery. Still, it can- not be excluded that the other pigs also produced IL-6 on an occasion other than those covered by the sampling fre- quency. Both the presence of IL-6 and TNF-α after inocu- lation in the two animals that remained clinically healthy could indicate a subclinical infection with B. hyodysente- riae.
4 Yeast transformation and expression were carried out and led to the isolation of positive clones expressing the synthetic genes. Protein was isolated from both the cell pellet and the medium, indicating that the product was being secreted from the cells. Yields varied and reached nearly 14.5 mg/L for BoNT/C and more than 3 mg/L in the case of BoNT/D.
4. Diagnosis Diagnosis by clinical manifestation shows difficulties in separating swine flu from classical influenza and other common respiratory tract infections. This is a serious problem for clinical diagnosis. The basic immunological diagnostic tool can confirm only H1N1 infection, but not typical or atypical. Molecular-based detection is required. A real-time PCr for diagnosis is available. However, the problems of over-register or under-diagnosis in setting due to lack of a gold standard can be expected.
cine did not show protection (8). New oral vaccine strain CVD 111, which is a live-attenuated El Tor biotype, Ogawa serotype strain, provided 80% pro- tection in adult volunteers (9). This vaccine is being evaluated collectively with CVD 103-HgR to estab- lish if the mixture can give more protection against both biotypes in a single dose(10). Two problems of live vaccine strains, in spite of their advantages, exist:1) the live-attenuated strains cause side effects such as abdominal pain, moderate diarrhea, and low fever, 2) reversion to virulence, because these live strains are attenuated by deleting the ctx genes that are carried on a bacteriophage, vaccine strains can obtain ctx-carrying phage in endemic area (11). An alternative approach to cholera vaccine design is the development of a subunitvaccine. Much research has focused on Vibrio cholerae colonization factors such as Toxin co-regulated pili (TCP), for develop- ment subunit vaccines. TCP is a thin filamentous pi- lus forming distinctive clusters on the surface of V. cholerae when cultured under high toxin-expressing conditions. TcpA a critical role in colonization in the infant mouse cholera model and following in human volunteers (1).This antigen, TcpA, could be a can- didate for research and development of subunit vac- cine.