Signal 1 Facilitators and Signal 2 Facilitators, Their Immunomodulatory Compounds, Receptors They Act on and Principal Immune Responses They Elicit
9.6 EXPRESSION METHODS FOR VACCINE DEVELOPMENT .1 B acterIal e xpressIon s ystem
136 Fish Vaccines for fish diseases including V. anguillarum and Photobacterium damselae subsp. piscicida.128,136 Using the primary capsid protein VP2 and RNA-dependent RNA polymerase (RdRp) genes as well as immunodominant T-cell epitopes and immunoinformatics, Islam et al.121 developed a vaccine against Lates calcarifer birnavirus (MABV). The vaccine was nonallergenic, had a high level of immunogenicity, and had good solubility. Based on conserved proteins in Streptococcus agalactiae, Kawasaki137 developed candidate serotype-independent preventive vaccines using a pan-genome reverse vaccinology technique (Group B Streptococcus; GBS). Vibrio parahaemolyticus multivalent vaccination was created by Wang et al.138 using a bioinformatics technique and the OMP protein for signaling peptides, transmembrane (TM)-helix, and subcellular location.
9.6 EXPRESSION METHODS FOR VACCINE DEVELOPMENT
Biotechnological Approaches to Vaccines 137
and posttranslational modifications such as glycosylations, are just a few advantages that yeast expression has over bacterial expression system.139 In addition, due to their nonpathogenic nature, comprehensive genome sequence, well-established genetics, and intrinsic natural adjuvant, yeast species are the best model system for creating vaccines. For expressing the recombinant proteins, yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe are excellent.150 One of the well-characterized types of signal transduction at the biochemical, genetic, and molecular biology levels is the galactose induction system in yeasts, which serves as a model transcriptional induction system in eukaryotes.151 P. pastoris has a more effective expression system than Saccharomyces because it has a strong oxidase promoter called AOX1, which assisted to post- translate proteins.139 With the help of Pichia, greater concentrations of vaccine antigens have been made from a range of bacterial and viral proteins.152 In S. cerevisiae, heterologous glycoprotein synthesis typically leads in a hyper-mannosylated glycan structure, which may result in decreased activity and increased immunogenicity. For a higher yield or better recombinant product quality, this yeast species’ various selective promoter elements and mutations were examined.153 It is com- mon practice to overexpress heterologous proteins using well-characterized inducible or constitutive promoters with strong transcriptional activity seen in diverse yeast strains.154 The well-known and potent promoters GAL1, GAL10, JUB1, SNR52, MET17, TDH3, TPI1, ENO1, and PDC1 have been employed for high-level expression of foreign genes in S. cerevisiae.155,156 In addition, promising for enhancing the expression of recombinant proteins in S. cerevisiae are dual promoter systems.157 Yeast expression system has several advantages compared to bacterial and baculovirus mode of expression for veterinary and fish vaccines. Some of the advantages are: there is no need of purifica- tion, largely scalable, easily deliverable for oral vaccine, low cost, etc.158 In addition, yeast expres- sion system is naturally robust to severe conditions and have rigid cell walls, which indicates that recombinant antigens produced in yeast may have a higher probability of coming into touch with the immune cells of the gastrointestinal (GI) tract in intact form.159 The extra benefits of yeast cell walls acting as immune stimulators make them stronger candidates for vaccines. Because yeast cells con- tain polysaccharides such as mannan, beta-1, and 3-D-glucan (BGs), it was thought that yeast cells were immunogenic. Zhu et al.160 expressed hemolysin of V. harveyi SF-1 through yeast cells and immunized with turbot and the immunized fishes had significant protection. Jha et al.161 expressed WSSV protein in eukaryotic expression systems such as yeast Pichia pastoris and delivered to Procambarus clarkia. Viral capsid protein (VP2) of infectious pancreatic necrosis (IPN) expressed through S. cerevisiae and immunized with rainbow trout. The immunized fishes had improved immunity and reduction of viral load after IPN challenge.162 The IPNV capsid protein VP2, pro- duced in yeast, self-assembles into SVPs, and the injection of SVPs into rainbow trout causes an immunological response, as shown by Dhar et al.114 Mao et al. 163 successfully immunized sea bass Lateolabrax japonicas with V. harveyi OmpK, which expressed in P. pastoris GS115. Recombinant main capsid protein (r-MCPMCP) of RBIV was generated in yeast after the virus was challenged in rock bream, Oplegnathus fasciatus, and this helped to reduce viral load and develop gut mucosal protection.164 Ananphongmanee et al.165 successfully displayed WSSV pVP28 through the yeast expression in S. cerevisiae and P. pastoris. Nervous necrosis virus (NNV) VLPs expressed through S. cerevisiae and delivered to E. septemfasciatus had higher survival rate and improved immu- nity against NNV challenge.166 Cyprinid herpesvirus 2 (CyHV-2) recombinant truncated proteins, TCID50 and tORF25, expressed through P. pastoris and vaccinated with C. gibelio. The vaccinated fishes had improved survival and upregulated the immune genes.167
9.6.3 BaculoVIrus expressIon
Recombinant baculoviruses with circular double-stranded DNA genomes have been widely used to produce recombinant proteins in insect cells and larvae. The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most often used baculovirus species in biotechnological
138 Fish Vaccines applications.168 A baculovirus genome’s enormous genetic storage capacity allows for the inclusion of many genes.169 Inserting foreign genes downstream of the polyhedron promoter will result in high-level expression upon infection of permissive cells. The polyhedron protein is replaced by the foreign gene of interest and integrated into the viral genome by homologous recombination.139 The Spodoptera frugiperda (Sf-9 and Sf-21) cell lines are capable of producing the recombinant virus easily, and after infection, the replication cycle is accompanied by the production of the foreign protein driven by either the polyhedron or p10 promoters.170 This protein is expressed at very high levels so that it can package assembled viral particles into occlusion bodies that protect them from proteolytic degradation by the decomposing host.139 Both insect cells and larvae can effectively synthesize heterologous proteins.171 Compared to other expression systems, such as the bacterial expression system, they have a number of advantages. Utilizing the function of the polyhedron protein in the viral life cycle, high-level production of a foreign protein, often in a soluble form, in baculoviruses is possible.139 Glycosylation, fatty acid acylation, phosphorylation, and proteolytic processing are among the posttranslational alterations that insect cells infected with baculovirus are capable of doing. Since baculovirus vectors are simple to make, there is no risk of endotoxin contamination, unlike with bacterial expression. Two WSSV structural genes, VP19 and VP466 (VP28), were cloned and expressed in Sf21 insect cells using a baculovirus expression system. The efficacy of the vaccine was further investigated using both intramuscular and oral delivery meth- ods, and the vaccinated shrimp had a survival rate of greater than 50%.101 Brevibacillus brevis was modified to express the WSSV VP28, which was then given to P. japonicus and challenged with the virus by Caipang et al.100 Around 72.5% of the shrimp fed pure protein at a dose of 50 mg survived.
In shrimp treated by Syed and Kwang172 using oral and immersion vaccinations, which expressed VP28 on the surface of a baculovirus, protection against WSSV challenge was seen 15 days after the final booster. White spot syndrome virus (WSSV) protein VP28 was delivered in a recombinant form inside the spores of the gram-positive bacteria B. subtilis using a unique technique by Fu et al.173 The baculovirus-expressed M. rosenbergii nodavirus (MrNV) capsid protein produces protec- tive immunity and increases survival against MrNV challenge in M. rosenbergii.106 Recombinant baculovirus BmNPV-VP35-VP4 was created using a Bac-to-Bac baculovirus expression method to express the VP35 and VP4 proteins from grass carp reovirus (GCRV) type II, and the expressed protein was then inoculated with Ctenopharyngodon idella. The immune-stimulated fish exhibited a greater percentage of survival, better immunity, and an upregulation of immune genes.174
9.6.4 mammalIan cell lIne expressIon
The various vector systems, including plasmid-based expression vectors, adenovirus vectors, vac- cinia vectors, retroviral vectors, and baculovirus as vectors, that transfer the gene into mamma- lian cells were investigated.175 Viral-mediated transduction is an effective method for achieving protein expression in mammalian cells.176 Recombinant baculoviruses are used in this technology to easily transduce mammalian cells and produce milligram amounts of protein.177 The benefits include a wide range of posttranslationally modified proteins with a higher degree of flexibility than other expression systems, correct protein folding, and genuine glycosylation.178 Mammalian cells carry out posttranslational alterations such as glycosylation, carbohydrate trimming, and pro- teolytic processing of the propeptide.179 Other alterations may involve carboxylation of glutamic acid residues,180,181 hydroxylation of aspartic acid and asparagine residues,182 sulfation of tyrosine residues,183 phosphorylation of proteins via cell receptor–protein interaction,184 fatty acid acyla- tion,185 and correct assembly of multimeric proteins. Chen et al.186 expressed the structural proteins (glycoprotein) of spring viremia of carp virus (SVCV) through mammalian cell lines. Red sea bream iridovirus’s (RSIV) transmembrane domain (pCMV-569) and capsid protein (pCMV-MCP) were expressed using the pCI-neo mammalian expression vector by Marlowe et al.187 The proteins immunized Pagrus major had improved cellular immune response and upregulated the MHC class I transcripts. Wolf et al.91 used pCI mammalian expression vector to express hemagglutinin-esterase
Biotechnological Approaches to Vaccines 139
of infectious salmon anemia virus (ISAV). The Atlantic salmon (Salmo salar) used to immunize the protein provided good protection from an upcoming ISAV test. Kayansamruaj et al.188 devel- oped α-enolase-based DNA vaccination against Streptococcus iniae using the mammalian expres- sion vector pCI-neo. The immune-relevant genes (IL-1, TNF-, COX-2, IL-12, and IL-13R-1) were upregulated in Oreochromis niloticus at 7 days after immunization, and it had the best survival rate.
9.6.5 mIcro-algal expressIon
A novel, affordable, and highly productive vaccine approach for many infectious diseases, including fish disease, is the algae-based vaccine. Algae have been found to collect quickly, fold a variety of vaccine antigens correctly, and produce recombinant algal fusion proteins that can increase the anti- genicity of vaccinations that are administered orally. It has been suggested that a chloroplast expres- sion system is a promising method of producing oral vaccinations.189 For the purpose of preventing and controlling infectious diseases, Chlamydomonas reinhardtii, Dunaliella salina, cyanobacteria, and other microalgae have been treated to express antigen genes in chloroplasts.190 Algal expres- sion involves physically introducing foreign DNA into the nuclear or chloroplast genome through transformation using electroporation, glass beads, or silicon carbide whiskers, microparticle bom- bardment, or agrobacterium-mediated DNA transfer.191 Then, the foreign gene is integrated into the chloroplast genome by homologous recombination.192 Algal expression has a number of benefits, including a reduction in the requirement for costlier purification, expensive fermentation, cold stor- age, and shipping, as well as simplicity, safety, and sterile administration.193 In addition, they have health-promoting advantages due to their high protein, fat, and nutritional contents, as well as the potential immunogenicity of their pigments for the hosts.194 Through the use of C. reinhardtii’s transgenic technology, Siripornadulsil et al.195 developed the p57 antigen of Renibacterium salmo- ninarum, the culprit behind BKD. Different forms of oral WSSV VP28 delivery systems have been created employing the cyanobacterium Anabaena sp., which had a 68% survival rate in crayfish against the WSSV challenge, and Dunaliella salina, which had a 59% survival rate.196,197 A hairpin DNA cassette encoding dsRNA targeted at the yellow head virus (YHV) was successfully inte- grated in the C. reinhardtii nucleus by Somchai et al.198 The transgenic algae fed L. vannamei had partial protection against YHV challenge. Transgenic Anabaena sp. PCC 7120, which expressed WSSV VP28 fed L. vannamei PL, had higher resistance against WSSV than the control.199