SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSct Vac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSct Vac could be a candidate COVID-19 vaccine.

Characterization of alfalfa mosaic virus capsid protein using Cryo-EM.

VLPs are virus-like particles that comprise viral capsid proteins that can self-assemble and mimic the shape and size of real viral particles; however, because they do not contain genetic material they cannot infect host cells. VLPs have great potential as safe drug/vehicle candidates; therefore, they are gaining popularity in the field of preventive medicine and therapeutics. Indeed, extensive studies are underway to examine their role as carriers for immunization and as vehicles for delivery of therapeutic agents. Here, we examined the possibility of developing VLP-utilizing technology based on an efficient VLP production process and high-resolution structural analysis. Nicotiana benthamiana was used as an expression platform to produce the coat protein of the alfalfa mosaic virus (AMV-CP). About 250 mg/kg of rAMV-CP was produced from Nicotiana benthamiana leaves. Structural analysis revealed that the oligomeric status of rAMV-CP changed according to the composition and pH of the buffer. Size exclusion chromatography and electron microscopy analysis confirmed the optimal conditions for rAMV-CP VLP formation, and a 2.4 Å resolution structure was confirmed by cryo-EM analysis. Based on the efficient protein production, VLP manufacturing technology, and high-resolution structure presented herein, we suggest that rAMV-CP VLP is a useful platform for development of various new drugs.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

A classical swine fever virus E2 fusion protein produced in plants elicits a neutralizing humoral immune response in mice and pigs.

Classical swine fever (CSF) is one of the most important viral diseases of swine worldwide. Although live or attenuated virus vaccines have been used to control CSFV, it is difficult to distinguish vaccinated pigs from infected pigs; this leads to restrictions on import and export. Subunit vaccines based on the CSFV E2 glycoprotein have been developed using baculovirus or insect cell systems, but some weaknesses remain. Here, we describe production of an E2 recombinant protein using a Nicotiana benthamiana plant expression system. To do this, we took advantage of the ability of the swine Fc domain to increase solubility and stability of the fusion protein and to strengthen immune responses in target animals. N. benthamiana expressed high amounts of pFc2-fused E2 proteins, which were isolated and purified by affinity chromatography to yield a high pure recombinant protein in a cost-effective manner. Native-polyacrylamide gel electrophoresis and size exclusion chromatography confirmed that the pmE2:pFc2 fusion exists as a multimer rather than as a dimer. Injection of recombinant pmE2 protein into mice or piglets generated anti-pmE2 antibodies with efficient neutralizing activity against CSFV. These results suggest that a purified recombinant E2 protein produced in N. benthamiana generates high titers of neutralizing antibodies in vivo; as such, the protein could be developed as a subunit vaccine against CSFV.

Production of bacteriophage-encoded endolysin, LysP11, in Nicotiana benthamiana and its activity as a potent antimicrobial agent against Erysipelothrix rhusiopathiae.

KEY MESSAGE: We produced a biologically active phage-encoded endolysin, LysP11, in N. benthamiana. Plant-produced LysP11 exhibited robust antimicrobial activity against E. rhusiopathiae, and C-terminal domain of LysP11 bound specifically to E. rhusiopathiae. Bacterial resistance to antibiotics, a serious issue in terms of global public health, is one of the leading causes of death today. Thus, new antimicrobial agents are needed to combat pathogens. Recent research suggests that bacteriophages and endolysins derived from bacteriophages are potential alternatives to traditional antibiotics. Here, we examined the antimicrobial activity of LysP11, which is encoded by Propionibacterium phage P1.1 and comprises an N-terminal amidase-2 domain and a C-terminal domain with no homology to other bacteriophage endolysins. LysP11 was produced in Nicotiana benthamiana (N. benthamiana) using an Agrobacterium-mediated transient expression strategy. LysP11 was purified on microcrystalline cellulose-binding resin after attachment of the Clostridium thermocellum-derived family 3 cellulose-binding domain as an affinity tag. The affinity tag was removed using the small ubiquitin-related modifier (SUMO) domain and SUMO-specific protease. Plant-produced LysP11 showed strong antimicrobial activity toward Erysipelothrix rhusiopathiae (E. rhusiopathiae), mediated via lysis of the cell wall. Lytic activity was optimal at pH 8.0-9.0 (37 °C) and increased at higher concentrations of NaCl up to 400 mM. Furthermore, the C-terminal domain of LysP11 bound specifically to the E. rhusiopathiae cell wall. Based on these results, we propose that LysP11 is a potential candidate antimicrobial agent against E. rhusiopathiae.

Adaptor protein complex 2-mediated endocytosis is crucial for male reproductive organ development in Arabidopsis.

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2-dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.

The immediate upstream region of the 5'-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana.

The nucleotide sequence around the translational initiation site is an important cis-acting element for post-transcriptional regulation. However, it has not been fully understood how the sequence context at the 5'-untranslated region (5'-UTR) affects the translational efficiency of individual mRNAs. In this study, we provide evidence that the 5'-UTRs of Arabidopsis genes showing a great difference in the nucleotide sequence vary greatly in translational efficiency with more than a 200-fold difference. Of the four types of nucleotides, the A residue was the most favourable nucleotide from positions -1 to -21 of the 5'-UTRs in Arabidopsis genes. In particular, the A residue in the 5'-UTR from positions -1 to -5 was required for a high-level translational efficiency. In contrast, the T residue in the 5'-UTR from positions -1 to -5 was the least favourable nucleotide in translational efficiency. Furthermore, the effect of the sequence context in the -1 to -21 region of the 5'-UTR was conserved in different plant species. Based on these observations, we propose that the sequence context immediately upstream of the AUG initiation codon plays a crucial role in determining the translational efficiency of plant genes.

Oral immunization of haemaggulutinin H5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza A virus infection.

Pandemics in poultry caused by the highly pathogenic avian influenza (HPAI) A virus occur too frequently globally, and there is growing concern about the HPAI A virus due to the possibility of a pandemic among humans. Thus, it is important to develop a vaccine against HPAI suitable for both humans and animals. Various approaches are underway to develop such vaccines. In particular, an edible vaccine would be a convenient way to vaccinate poultry because of the behaviour of the animals. However, an edible vaccine is still not available. In this study, we developed a strategy of effective vaccination of mice by the oral administration of transgenic Arabidopsis plants (HA-TG) expressing haemagglutinin (HA) in the endoplasmic reticulum (ER). Expression of HA in the ER resulted in its high-level accumulation, N-glycosylation, protection from proteolytic degradation and long-term stability. Oral administration of HA-TG with saponin elicited high levels of HA-specific systemic IgG and mucosal IgA responses in mice, which resulted in protection against a lethal influenza virus infection with attenuated inflammatory symptoms. Based on these results, we propose that oral administration of freeze-dried leaf powders from transgenic plants expressing HA in the ER together with saponin is an attractive strategy for vaccination against influenza A virus.

Trafficking of vacuolar proteins: the crucial role of Arabidopsis vacuolar protein sorting 29 in recycling vacuolar sorting receptor.

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.

Functional identification of sorting receptors involved in trafficking of soluble lytic vacuolar proteins in vegetative cells of Arabidopsis.

In eukaryotic cells, protein trafficking plays an essential role in biogenesis of proteins that belong to the endomembrane compartments. In this process, an important step is the sorting of organellar proteins depending on their final destinations. For vacuolar proteins, vacuolar sorting receptors (VSRs) and receptor homology-transmembrane-RING H2 domain proteins (RMRs) are thought to be responsible. Arabidopsis (Arabidopsis thaliana) contains seven VSRs. Among them, VSR1, VSR3, and VSR4 are involved in sorting storage proteins targeted to the protein storage vacuole (PSV) in seeds. However, the identity of VSRs for soluble proteins of the lytic vacuole in vegetative cells remains controversial. Here, we provide evidence that VSR1, VSR3, and VSR4 are involved in sorting soluble lytic vacuolar and PSV proteins in vegetative cells. In protoplasts from leaf tissues of vsr1vsr3 and vsr1vsr4 but not vsr5vsr6, and rmr1rmr2 and rmr3rmr4 double mutants, soluble lytic vacuolar (Arabidopsis aleurain-like protein:green fluorescent protein [GFP] and carboxypeptidase Y:GFP and PSV (phaseolin) proteins, but not the vacuolar membrane protein Arabidopsis ßFructosidase4:GFP, exhibited defects in their trafficking; they accumulated to the endoplasmic reticulum with an increased secretion into medium. The trafficking defects in vsr1vsr4 protoplasts were rescued by VSR1 or VSR4 but not VSR5 or AtRMR1. Furthermore, of the luminal domain swapping mutants between VSR1 and VSR5, the mutant with the luminal domain of VSR1, but not that of VSR5, rescued the trafficking defects of Arabidopsis aleurain-like protein:GFP and phaseolin in vsr1vsr4 protoplasts. Based on these results, we propose that VSR1, VSR3, and VSR4, but not other VSRs, are involved in sorting soluble lytic vacuolar and PSV proteins for their trafficking to the vacuoles in vegetative cells.

Exposure of iron nanoparticles to Arabidopsis thaliana enhances root elongation by triggering cell wall loosening.

In this study, we investigated the effect of nZVI on plant root elongation in Arabidopsis thaliana and showed, for the first time, that nZVI enhanced root elongation by inducing OH radical-induced cell wall loosening. Exposure of plants to 0.5 g/L nZVI enhanced root elongation by 150-200% over that in the control, and further mechanistic studies showed that this occurred via nZVI-mediated OH radical-induced cell wall loosening. The oxidation capacity of nZVI, leading to release of H2O2, allowed it to cause OH radical-induced cell wall loosening in roots. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis clearly revealed that pectin-polysaccharides in roots were degraded; they are one of the main matrix-polysaccharide-connecting and load-bearing polymers in cell walls. Rapid root elongation led to structural changes in root cell walls: reduction of cell wall thickness and a bias on the orientation of cellulose microfibrils. Additionally, the asymmetrical distribution of tensional strength resulted from the OH radical-induced cell wall loosening enhanced endocytosis. These findings emphasize that OH radical-induced cell wall loosening is important for mechanical regulation of the cell wall and provide new insights into the cellular responses of plants exposed to reactive metal nanoparticles.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Methods: Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. Results: E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. Conclusion: E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Development and evaluation of two rapid lateral flow assays for on-site detection of African swine fever virus.

Introduction: African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. In the absence of a widely available and definitively proven vaccine, rapid and early detection is critical for ASF control. The quick and user-friendly lateral flow assay (LFA) can easily be performed by following simple instructions and is ideal for on-site use. This study describes the development and validation of two LFAs for the rapid detection of ASF virus (ASFV) in pig serum. Methods: The highly immunogenic antigens (p30 and p72) of ASFV Georgia 2007/1 (genotype II) were expressed in plants (Nicotiana benthamiana) and were used to immunize BALB/c mice to generate specific monoclonal antibodies (mAbs) against the p30 and p72 proteins. mAbs with the strongest binding ability to each protein were used to develop p30_LFA and p72_LFA for detecting the respective ASFV antigens. The assays were first evaluated using a spike-in test by adding the purified p30 or p72 protein to a serum sample from a healthy donor pig. Further validation of the tests was carried out using serum samples derived from experimentally infected domestic pigs, field domestic pigs, and feral pigs, and the results were compared with those of ASFV real-time PCR. Results: p30_LFA and p72_LFA showed no cross-reaction with common swine viruses and delivered visual results in 15 min. When testing with serially diluted proteins in swine serum samples, analytical sensitivity reached 10 ng/test for p30_LFA and 20 ng/test for p72_LFA. Using real-time PCR as a reference, both assays demonstrated high sensitivity (84.21% for p30_LFA and 100% for p72_LFA) with experimentally ASFV-infected pig sera. Specificity was 100% for both LFAs using a panel of PBS-inoculated domestic pig sera. Excellent specificity was also shown for field domestic pig sera (100% for p30_LFA and 93% for p72_LFA) and feral pig sera (100% for both LFAs). Conclusion: The results obtained in this study suggest that p30_LFA and p72_LFA hold promise as rapid, sensitive, user-friendly, and field-deployable tools for ASF control, particularly in settings with limited laboratory resources.

Identification of sorting motifs of AtßFruct4 for trafficking from the ER to the vacuole through the Golgi and PVC.

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtßFructosidase 4 (AtßFruct4), to the central vacuole in protoplasts. AtßFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtßFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtßFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.

Auxin-mediated ribosomal biogenesis regulates vacuolar trafficking in Arabidopsis.

In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis.

Field evaluation of novel plant-derived porcine circovirus type 2 vaccine related to subclinical infection.

BACKGROUND: The objective of this field trial was to evaluate the efficacy of a new plant-based porcine circovirus type 2a (PCV2a) vaccine. This vaccine was a recombinant capsid subunit PCV2a vaccine based on the Nicotiana benthamiana expression system. METHODS: Three farms were selected for the study based on their history of subclinical PCV2 infection. A total of 40 18-day-old pigs were randomly allocated to either vaccinated or unvaccinated groups (20 pigs per group; 10 = male and 10 = female). Pigs received a 2.0-mL dose of the plant-based PCV2a vaccine intramuscularly at 21 days of age in accordance with the manufacturer's recommendations, whereas unvaccinated pigs were administered a single dose of phosphate buffered-saline at the same age. RESULTS: Vaccination had a positive effect on pig growth performance compared to that of unvaccinated pigs on all three of the farms. Vaccination of pigs with a plant-based PCV2a vaccine induced high levels of neutralizing antibodies titres against PCV2d and PCV2d-specific interferon-γ secreting cells which resulted in the reduction of PCV2d viral load and reduced lymphoid lesions severity. CONCLUSIONS: The results of this field trial demonstrated cross-protection of PCV2d by a plant-based PCV2a vaccine and a positive effect of pig growth performance with vaccination.

Efficacy of Plant-Made Human Recombinant ACE2 against COVID-19 in a Golden Syrian Hamster Model.

Coronavirus disease 2019 (COVID-19) is a novel infectious respiratory disease caused by SARS-CoV-2. We evaluated the efficacy of a plant-based human recombinant angiotensin-converting enzyme 2 (hrACE2) and hrACE2-foldon (hrACE2-Fd) protein against COVID-19. In addition, we analyzed the antiviral activity of hrACE2 and hrACE2-Fd against SARS-CoV-2 using real-time reverse-transcription PCR and plaque assays. The therapeutic efficacy was detected using the Golden Syrian hamster model infected with SARS-CoV-2. Both hrACE2 and hrACE2-Fd inhibited SARS-CoV-2 by 50% at concentrations below the maximum plasma concentration, with EC50 of 5.8 µg/mL and 6.2 µg/mL, respectively. The hrACE2 and hrACE2-Fd injection groups showed a tendency for decreased viral titers in nasal turbinate tissues on day 3 after virus inoculation; however, this decrease was not detectable in lung tissues. Histopathological examination on day 9 after virus inoculation showed continued inflammation in the SARS-CoV-2 infection group, whereas decreased inflammation was observed in both the hrACE2 and hrACE2-Fd injection groups. No significant changes were observed at other time points. In conclusion, the potential therapeutic efficacy of plant-based proteins, hrACE2 and hrACE2-Fd, against COVID-19 was confirmed in a SARS-CoV-2-inoculated Golden Syrian hamster model. Further preclinical studies on primates and humans are necessary to obtain additional evidence and determine the effectiveness of these therapies.

A Plant-Derived Maternal Vaccine against Porcine Epidemic Diarrhea Protects Piglets through Maternally Derived Immunity.

Newborn piglets are susceptible to a highly contagious enteritis caused by the porcine epidemic diarrhea virus (PEDV), associated with high levels of mortality worldwide. There is pressing need for a rapid, safe, and cost-effective vaccine to safeguard pigs from getting infected by PEDV. PEDV belongs to the coronavirus family and is characterized by high levels of mutability. The primary goal of a PEDV vaccine is to provide immunity to newborn piglets through vaccination of sows. Plant-based vaccines are becoming more popular because they have low manufacturing costs, are easily scalable, have high thermostability, and a long shelf life. This is in contrast to conventional vaccines which include inactivated, live, and/or recombinant types that can be expensive and have limited ability to respond to rapidly mutating viruses. The binding of the virus to host cell receptors is primarily facilitated by the N-terminal subunit of the viral spike protein (S1), which also contains several epitopes that are recognized by virus-neutralizing antibodies. As a result, we generated a recombinant S1 protein using a plant-based vaccine platform. We found that the recombinant protein was highly glycosylated, comparable to the native viral antigen. Vaccination of pregnant sows at four and two weeks before farrowing led to the development of humoral immunity specific to S1 in the suckling piglets. In addition, we noted significant viral neutralization titers in both vaccinated sows and piglets. When challenged with PEDV, piglets born from vaccinated sows displayed less severe clinical symptoms and significantly lower mortality rates compared to piglets born from non-vaccinated sows.

Plant-expressed Zika virus envelope protein elicited protective immunity against the Zika virus in immunocompetent mice.

Zika virus infection causes multiple clinical issues, including Guillain-Barré syndrome and neonatal malformation. Vaccination is considered as the only strategy for the prevention of ZIKV-induced clinical issues. This study developed a plant-based recombinant vaccine that transiently expressed the ZIKV envelope protein (ZikaEnv:aghFc) in Nicotiana benthamiana and evaluated the protective immunity afforded by it in immunocompetent mice. ZikaEnv:aghFc induced both humoral and cellular immunity at a low dose (1-5 µg). This immune-inducing potential was enhanced further when adjuvanted CIA09A. In addition, antigen-specific antibodies and neutralizing antibodies were vertically transferred from immunized females to their progeny and afforded both protective immunity to ZIKV and cross-protection to Dengue virus infection. These results suggest that our plant-based ZIKV vaccine provides a safe and efficient protective strategy with a competitive edge.

Small heat shock protein Hsp17.8 functions as an AKR2A cofactor in the targeting of chloroplast outer membrane proteins in Arabidopsis.

Plastid proteins that are encoded by the nuclear genome and synthesized in the cytosol undergo posttranslational targeting to plastids. Ankyrin repeat protein 2A (AKR2A) and AKR2B were recently shown to be involved in the targeting of proteins to the plastid outer envelope. However, it remains unknown whether other factors are involved in this process. In this study, we investigated a factor involved in AKR2A-mediated protein targeting to chloroplasts in Arabidopsis (Arabidopsis thaliana). Hsp17.8, a member of the class I (CI) cytosolic small heat shock proteins (sHsps), was identified in interactions with AKR2A. The interaction between Hsp17.8 and AKR2A was further confirmed by coimmunoprecipitation experiments. The carboxyl-terminal ankyrin repeat domain of AKR2A was responsible for AKR2A binding to Hsp17.8. Other CI cytosolic sHsps also interact with AKR2A to varying degrees. Additionally, Hsp17.8 binds to chloroplasts in vitro and enhances AKR2A binding to chloroplasts. HSP17.8 was expressed under normal growth conditions, and its expression increased after heat shock. Hsp17.8 exists as a dimer under normal physiological conditions, and it is converted to high oligomeric complexes, ranging from 240 kD to greater than 480 kD, after heat shock. High levels of Hsp17.8 together with AKR2A resulted in increased plastid targeting of Outer Envelope Protein7 (OEP7), a plastid outer envelope protein expressed as a green fluorescent protein fusion protein. In contrast, artificial microRNA suppression of HSP17.8 and closely related CI cytosolic sHSPs in protoplasts resulted in a reduction of OEP7:green fluorescent protein targeting to plastids. Based on these data, we propose that Hsp17.8 functions as an AKR2A cofactor in targeting membrane proteins to plastid outer membranes under normal physiological conditions.