Integrated transcriptomic and metabolomic analysis provides insight into the pollen development of CMS-D1 rice.

BACKGROUND: Cytoplasmic male sterility (CMS) has greatly improved the utilization of heterosis in crops due to the absence of functional male gametophyte. The newly developed sporophytic D1 type CMS (CMS-D1) rice exhibits unique characteristics compared to the well-known sporophytic CMS-WA line, making it a valuable resource for rice breeding. RESULTS: In this research, a novel CMS-D1 line named Xingye A (XYA) was established, characterized by small, transparent, and shriveled anthers. Histological and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays conducted on anthers from XYA and its maintainer line XYB revealed that male sterility in XYA is a result of delayed degradation of tapetal cells and abnormal programmed cell death (PCD) of microspores. Transcriptome analysis of young panicles revealed that differentially expressed genes (DEGs) in XYA, compared to XYB, were significantly enriched in processes related to chromatin structure and nucleosomes during the microspore mother cell (MMC) stage. Conversely, processes associated with sporopollenin biosynthesis, pollen exine formation, chitinase activity, and pollen wall assembly were enriched during the meiosis stage. Metabolome analysis identified 176 specific differentially accumulated metabolites (DAMs) during the meiosis stage, enriched in pathways such as α-linoleic acid metabolism, flavone and flavonol biosynthesis, and linolenic acid metabolism. Integration of transcriptomic and metabolomic data underscored the jasmonic acid (JA) biosynthesis pathway was significant enriched in XYA during the meiosis stage compared to XYB. Furthermore, levels of JA, MeJA, OPC4, OPDA, and JA-Ile were all higher in XYA than in XYB at the meiosis stage. CONCLUSIONS: These findings emphasize the involvement of the JA biosynthetic pathway in pollen development in the CMS-D1 line, providing a foundation for further exploration of the molecular mechanisms involved in CMS-D1 sterility.

The vesicle trafficking gene, OsRab7, is critical for pollen development and male fertility in cytoplasmic male-sterility rice.

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.

OsCBL1 modulates rice nitrogen use efficiency via negative regulation of OsNRT2.2 by OsCCA1.

BACKGROUND: For cereal crop breeding, it is meaningful to improve utilization efficiency (NUE) under low nitrogen (LN) levels while maintaining crop yield. OsCBL1-knockdown (OsCBL1-KD) plants exhibited increased nitrogen accumulation and NUE in the field of low N level. RESULTS: OsCBL1-knockdown (OsCBL1-KD) in rice increased the expression of a nitrate transporter gene OsNRT2.2. In addition, the expression of OsNRT2.2, was suppressed by OsCCA1, a negative regulator, which could directly bind to the MYB-binding elements (EE) in the region of OsNRT2.2 promoter. The OsCCA1 expression was found to be down-regulated in OsCBL1-KD plants. At the low Nitrogen (N) level field, the OsCBL1-KD plants exhibited a substantial accumulation of content and higher NUE, and their actual biomass remained approximately as the same as that of the wild type. CONCLUSION: These results indicated that down-regulation of OsCBL1 expression could upregulate the expression of OsNRT2.2 by suppressing the expression of OsCCA1and then increasing the NUE of OsCBL1-KD plants under low nitrogen availability.

CBL-Interacting Protein Kinase OsCIPK18 Regulates the Response of Ammonium Toxicity in Rice Roots.

Ammonium ( NH 4 + ) is one of the major nitrogen sources for plants. However, excessive ammonium can cause serious harm to the growth and development of plants, i.e., ammonium toxicity. The primary regulatory mechanisms behind ammonium toxicity are still poorly characterized. In this study, we showed that OsCIPK18, a CBL-interacting protein kinase, plays an important role in response to ammonium toxicity by comparative analysis of the physiological and whole transcriptome of the T-DNA insertion mutant (cipk18) and the wild-type (WT). Root biomass and length of cipk18 are less inhibited by excess NH 4 + compared with WT, indicating increased resistance to ammonium toxicity. Transcriptome analysis reveals that OsCIPK18 affects the NH 4 + uptake by regulating the expression of OsAMT1;2 and other NH 4 + transporters, but does not affect ammonium assimilation. Differentially expressed genes induced by excess NH 4 + in WT and cipk18 were associated with functions, such as ion transport, metabolism, cell wall formation, and phytohormones signaling, suggesting a fundamental role for OsCIPK18 in ammonium toxicity. We further identified a transcriptional regulatory network downstream of OsCIPK18 under NH 4 + stress that is centered on several core transcription factors. Moreover, OsCIPK18 might function as a transmitter in the auxin and abscisic acid (ABA) signaling pathways affected by excess ammonium. These data allowed us to define an OsCIPK18-regulated/dependent transcriptomic network for the response of ammonium toxicity and provide new insights into the mechanisms underlying ammonium toxicity.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

OsCBL1 affects rice seedling growth by modulating nitrate and phosphate responses.

To sustain high crop yield, a comprehensive understanding of the processes by which plants sense and acquire nutrients is of great importance. For the efficiency of crop fertilizer, it is essential to exploring the the signaling networks that coordinate the usage of nitrogen and phosphorus, the most demanding two mineral nutrients in plants. Here, we found that a protein OsCBL1 (Calcineurin B-like protein 1) is involved in the regulation of nitrogen and phosphorus signaling in rice. The nitrogen element, existing as ammonium or nitrate in the environment, affects nitrate signaling in vivo and root growth. Compared with the wild type, knockdown of OsCBL1 inhibit the growth of rice to the same extent, when nitrogen is deficient or nitrogen is present in the form of ammonium-nitrate mixture. The growth inhibition by OsCBL1-knockdown is more pronounced when nitrogen is present as ammonium. The phosphorus starvation-responsive genes is also regulated by the compound of nitrogen present in vitro and OsCBL1, while the phosphorus content is not affected. These results suggest that OsCBL1 may be involved in the response of rice to nitrogen and phosphorus nutrition in the environment, as well as the regulation of rice growth by environmental nutrition.

Grass carp (Ctenopharyngodon idellus) PIAS1 inhibits innate immune response via interacting with STAT1.

Protein inhibitor of activated signal transducer and activator of transcription (PIAS) family protein involved in gene transcriptional regulation acts as negative regulator in Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway. But until now, the roles of PIAS in fish are not clear. In this study, we identified the two mammalian PIAS1 orthologs from Ctenopharyngodon idellus, namely CiPIAS1a and CiPIAS1b, respectively. They can respond to the stimulation from Polyribocytidylic acid (Poly I:C), Grass Carp Reovirus (GCRV) and Lipopolysaccharides (LPS) respectively, so we suggested that they could participate in interferon (IFN)-mediated antiviral and antibacterial immune response. The subcellular localization and nuclear cytoplasm extraction showed that CiPIAS1a and CiPIAS1b were mainly distributed in the nucleus. In addition, Co-IP showed that they separately inhibited the phosphorylation of STAT1 via interacting with it, which leads to the reduction of IFN1 expression.

Comparison of wild rice (Oryza longistaminata) tissues identifies rhizome-specific bacterial and archaeal endophytic microbiomes communities and network structures.

Compared with root-associated habitats, little is known about the role of microbiota inside other rice organs, especially the rhizome of perennial wild rice, and this information may be of importance for agriculture. Oryza longistaminata is perennial wild rice with various agronomically valuable traits, including large biomass on poor soils, high nitrogen use efficiency, and resistance to insect pests and disease. Here, we compared the endophytic bacterial and archaeal communities and network structures of the rhizome to other compartments of O. longistaminata using 16S rRNA gene sequencing. Diverse microbiota and significant variation in community structure were identified among different compartments of O. longistaminata. The rhizome microbial community showed low taxonomic and phylogenetic diversity as well as the lowest network complexity among four compartments. Rhizomes exhibited less phylogenetic clustering than roots and leaves, but similar phylogenetic clustering with stems. Streptococcus, Bacillus, and Methylobacteriaceae were the major genera in the rhizome. ASVs belonging to the Enhydrobacter, YS2, and Roseburia are specifically present in the rhizome. The relative abundance of Methylobacteriaceae in the rhizome and stem was significantly higher than that in leaf and root. Noteworthy type II methanotrophs were observed across all compartments, including the dominant Methylobacteriaceae, which potentially benefits the host by facilitating CH4-dependent N2 fixation under nitrogen nutrient-poor conditions. Our data offers a robust knowledge of host and microbiome interactions across various compartments and lends guidelines to the investigation of adaptation mechanisms of O. longistaminata in nutrient-poor environments for biofertilizer development in agriculture.

Haemagglutinin displayed on the surface of Lactococcus lactis confers broad cross-clade protection against different H5N1 viruses in chickens.

BACKGROUND: The highly pathogenic avian influenza (HPAI) H5N1 virus poses a potential threat to the poultry industry. The currently available avian influenza H5N1 vaccines for poultry are clade-specific. Therefore, an effective vaccine for preventing and controlling H5N1 viruses belonging to different clades needs to be developed. RESULTS: Recombinant L. lactis/pNZ8148-Spax-HA was generated, and the influenza virus haemagglutinin (HA) protein of A/Vietnam/1203/2004 (H5N1) was displayed on the surface of Lactococcus lactis (L. lactis). Spax was used as an anchor protein. Chickens vaccinated orally with unadjuvanted L. lactis/pNZ8148-Spax-HA could produce significant humoral and mucosal responses and neutralizing activities against H5N1 viruses belonging to different clades. Importantly, unadjuvanted L. lactis/pNZ8148-Spax-HA conferred cross-clade protection against lethal challenge with different H5N1 viruses in the chicken model. CONCLUSION: This study provides insights into the cross-clade protection conferred by unadjuvanted L. lactis/pNZ8148-Spax-HA, and the results might help the establishment of a promising platform for the development of a safe and effective H5N1 cross-clade vaccine for poultry.

GmFAD3A, A ω-3 Fatty Acid Desaturase Gene, Enhances Cold Tolerance and Seed Germination Rate under Low Temperature in Rice.

Low temperature is an environmental stress factor that is always been applied in research on improving crop growth, productivity, and quality of crops. Polyunsaturated fatty acids (PUFAs) play an important role in cold tolerance, so its genetic manipulation of the PUFA contents in crops has led to the modification of cold sensitivity. In this study, we over-expressed an ω-3 fatty acid desaturase from Glycine max (GmFAD3A) drove by a maize ubiquitin promoter in rice. Compared to the wild type (ZH11), ectopic expression of GmFAD3A increased the contents of lipids and total PUFAs. Seed germination rates in GmFAD3A transgenic rice were enhanced under low temperature (15 °C). Moreover, cold tolerance and survival ratio were significantly improved in GmFAD3A transgenic seedlings. Malondialdehyde (MDA) content in GmFAD3A transgenic rice was lower than that in WT under cold stress, while proline content obviously increased. Meanwhile, the activities of superoxide dismutase (SOD), hydroperoxidase (CAT), and peroxidase (POD) increased substantially in GmFAD3A transgenic rice after 4 h of cold treatment. Taken together, our results suggest that GmFAD3A can enhances cold tolerance and the seed germination rate at a low temperature in rice through the accumulation of proline content, the synergistic increase of the antioxidant enzymes activity, which finally ameliorated the oxidative damage.

OsDCL3b affects grain yield and quality in rice.

KEY MESSAGE: We reported that knockdown of OsDCL3b decreased grain yield but increased grain quality in rice, which is helpful for molecular breeding in crops. Multiple DICER-LIKE (DCL) genes usually exist and show diverse biochemical and phenotypic functions in land plants. In rice, the biochemical function of OsDCL3b is known to process 24-nucleotide panicle phased small RNAs, however, its phenotypic functions are unclear. Here we reported that knockdown of OsDCL3b led to reduced pollen fertility, seed setting rate, and decreased grain yield but increased grain quality in rice. To reveal the molecular mechanism of the above phenomena, extracted RNAs from rice panicles of the wild type (WT) and OsDCL3b-RNAi line S6-1 were analyzed by deep sequencing. It showed that knockdown of OsDCL3b affected the biogenesis of both 21- and 24-nucleotide small RNAs including miRNAs and phased small RNAs. Using RNA-seq, 644 up- and 530 down-regulated mRNA genes were identified in panicles of line S6-1, and 550 and 273 differentially spliced genes with various alternative splicing (AS) events were observed in panicles of line S6-1 and WT, respectively, suggesting that OsDCL3b involved in influencing the transcript levels of mRNA genes and the AS events in rice panicles. Thus, our results show that knockdown of OsDCL3b will affect the biogenesis of small RNAs, which is involved in regulating the transcription of mRNA genes, and consequently influence the grain yield and quality in rice.

Comparative proteomic analysis of Methanothermobacter thermautotrophicus reveals methane formation from H2 and CO2 under different temperature conditions.

The growth of all methanogens is limited to a specific temperature range. However, Methanothermobacter thermautotrophicus can be found in a variety of natural and artificial environments, the temperatures of which sometimes even exceed the temperature growth ranges of thermophiles. As a result, the extent to which methane production and survival are affected by temperature remains unclear. To investigate the mechanisms of methanogenesis that Archaea have evolved to cope with drastic temperature shifts, the responses of Methanothermobacter thermautotrophicus to temperature were investigated under a high temperature growth (71°C) and cold shock (4°C) using Isobaric tags for relative and absolute quantitation (iTRAQ). The results showed that methane formation is decreased and that protein folding and degradation are increased in both high- and low-temperature treatments. In addition, proteins predicted to be involved in processing environmental information processing and in cell membrane/wall/envelope biogenesis may play key roles in affecting methane formation and enhancing the response of M. thermautotrophicus to temperature stress. Analysis of the genomic locations of the genes corresponding to these temperature-dependent proteins predicted that 77 of the genes likely to form 32 gene clusters. Here, we assess the response of M. thermautotrophicus to different temperatures and provide a new level of understanding of methane formation and cellular putative adaptive responses.

Over-expression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice.

High temperature (HT) stress is a major environmental stress that limits agricultural production worldwide. Discovery and application of genes promoting high temperature tolerance is essential to enhance crop tolerance to heat stress. Proteins associated with chaperone and protein folding plays an important role in the high temperature stress response of plants. MTH1745 (MtPDI), a disulfide isomerase-like protein (PDI) with a chaperone function and disulfide isomerase activity from Methanothermobacter thermautotrophicus delta H, was selected for studying the heat stress tolerance using an ectopic expression method in rice. Through molecular identification via quantitative real-time PCR and western blot, we demonstrated that the MtPDI gene was expressed stably in transgenic rice. Heat stress tolerance and survival ratio were significantly improved in seedling transgenic rice. At the same time, proline content, superoxide dismutase (SOD) and peroxidase (POD) activities were increased in MtPDI transgenic rice with a reduced malondialdehyde (MDA) content. Moreover, increased content of thiols group was discovered in transgenic plants. These results indicate that heterologous expression of MtPDI from extremophiles could confer heat stress tolerance of transgenic rice through the accumulation of proline content, the synergistic increase of the antioxidant enzymes activity and elevated production of more thiols group, which finally ameliorated the oxidative damage.

The chimeric mitochondrial gene orf182 causes non-pollen-type abortion in Dongxiang cytoplasmic male-sterile rice.

D1-cytoplasmic male sterility (CMS) rice is a sporophytic cytoplasmic male-sterile rice developed from Dongxiang wild rice that exhibits a no-pollen-grain phenotype. A mitochondrial chimeric gene (orf182) was detected by mitochondrial genome sequencing and a comparative analysis. Orf182 is composed of three recombinant fragments, the largest of which is homologous to Sorghum bicolor mitochondrial sequences. In addition, orf182 was found only in wild rice species collected from China. Northern blot analysis showed that orf182 transcripts were affected by Rf genes in the isocytoplasmic restorer line DR7. Western blot analysis showed that the ORF182 product was localized in the mitochondria of the CMS line. An expression cassette containing orf182 fused to a mitochondrial transit peptide induced the maintainer line of male sterility, which lacked pollen grains in the anthers. Furthermore, the in vivo expression of orf182 also inhibited the growth of Escherichia coli, with lower respiration rate, excess accumulation of reactive oxygen species and decreased ATP levels. We conclude that the mitochondrial chimeric gene orf182 possesses a unique structure and origin differing from other identified mitochondrial CMS genes, and this gene is connected to non-pollen type of sporophytic male sterility in D1-CMS rice.

BnSIP1-1, a Trihelix Family Gene, Mediates Abiotic Stress Tolerance and ABA Signaling in Brassica napus.

The trihelix family genes have important functions in light-relevant and other developmental processes, but their roles in response to adverse environment are largely unclear. In this study, we identified a new gene, BnSIP1-1, which fell in the SIP1 (6b INTERACTING PROTEIN1) clade of the trihelix family with two trihelix DNA binding domains and a fourth amphipathic α-helix. BnSIP1-1 protein specifically targeted to the nucleus, and its expression can be induced by abscisic acid (ABA) and different stresses. Overexpression of BnSIP1-1 improved seed germination under osmotic pressure, salt, and ABA treatments. Moreover, BnSIP1-1 decreased the susceptibility of transgenic seedlings to osmotic pressure and ABA treatments, whereas there was no difference under salt stress between the transgenic and wild-type seedlings. ABA level in the transgenic seedlings leaves was higher than those in the control plants under normal condition. Under exogenous ABA treatment and mannitol stress, the accumulation of ABA in the transgenic plants was higher than that in the control plants; while under salt stress, the difference of ABA content before treatment was gradually smaller with the prolongation of salt treatment time, then after 24 h of treatment the ABA level was similar in transgenic and wild-type plants. The transcription levels of several general stress marker genes (BnRD29A, BnERD15, and BnLEA1) were higher in the transgenic plants than the wild-type plants, whereas salt-responsive genes (BnSOS1, BnNHX1, and BnHKT) were not significantly different or even reduced compared with the wild-type plants, which indicated that BnSIP1-1 specifically exerted different regulatory mechanisms on the osmotic- and salt-response pathways in seedling period. Overall, these findings suggested that BnSIP1-1 played roles in ABA synthesis and signaling, salt and osmotic stress response. To date, information about the involvement of the Brassica napus trihelix gene in abiotic response is scarce. Here, we firstly reported abiotic stress response and possible function mechanisms of a new trihelix gene in B. napus.

Expression of a mitochondrial gene orfH79 from CMS-Honglian rice inhibits Escherichia coli growth via deficient oxygen consumption.

Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open frames (ORF), orfH79 is a mitochondrial chimeric gene responsible for the CMS trait in Honglian (HL) rice. In this study, the weakly produced ORFH79 protein significantly inhibited the growth of E. coli in an oxygen culture, however, the growth of the transformants producing ORFH79 was indistinguishable from the control under anaerobic incubation conditions. In addition, a lower respiration rate, wrinkled bacterial surfaces, and decreased pyruvate kinase and α-ketoglutarate dehydrogenase activities were observed in the ORFH79 produced E. coli. These results indicate that ORFH79 impairs the oxygen respiration of E. coli, which may inhibit E. coli growth.

Elucidation of Cross-Talk and Specificity of Early Response Mechanisms to Salt and PEG-Simulated Drought Stresses in Brassica napus Using Comparative Proteomic Analysis.

To understand the cross-talk and specificity of the early responses of plants to salt and drought, we performed physiological and proteome analyses of Brassica napus seedlings pretreated with 245 mM NaCl or 25% polyethylene glycol (PEG) 6000 under identical osmotic pressure (-1.0 MPa). Significant decreases in water content and photosynthetic rate and excessive accumulation of compatible osmolytes and oxidative damage were observed in response to both stresses. Unexpectedly, the drought response was more severe than the salt response. We further identified 45 common differentially expressed proteins (DEPs), 143 salt-specific DEPs and 160 drought-specific DEPs by isobaric tags for relative and absolute quantitation (iTRAQ) analysis. The proteome quantitative data were then confirmed by multiple reaction monitoring (MRM). The differences in the proteomic profiles between drought-treated and salt-treated seedlings exceeded the similarities in the early stress responses. Signal perception and transduction, transport and membrane trafficking, and photosynthesis-related proteins were enriched as part of the molecular cross-talk and specificity mechanism in the early responses to the two abiotic stresses. The Ca2+ signaling, G protein-related signaling, 14-3-3 signaling pathway and phosphorylation cascades were the common signal transduction pathways shared by both salt and drought stress responses; however, the proteins with executive functions varied. These results indicate functional specialization of family proteins in response to different stresses, i.e., CDPK21, TPR, and CTR1 specific to phosphorylation cascades under early salt stress, whereas STN7 and BSL were specific to phosphorylation cascades under early drought stress. Only the calcium-binding EF-hand family protein and ZKT were clearly identified as signaling proteins that acted as cross-talk nodes for salt and drought signaling pathways. Our study provides new clues and insights for developing strategies to improve the tolerance of crops to complex, multiple environmental stresses.

Cross-protection of Lactococcus lactis-displayed HA2 subunit against homologous and heterologous influenza A viruses in mice.

Current influenza vaccines provide strain-specific protection against homologous subtypes and need to be updated annually. Therefore, it is essential to develop a universal vaccine that would induce broadly cross-protective immunity against homologous and heterologous influenza A viruses. The highly conserved HA2 subunit is a promising candidate for developing a universal influenza vaccine. Here, we hypothesized that the HA2 subunit could be displayed on the surface of Lactococcus lactis (L. lactis), using Spax as an anchor protein (L. lactis/pNZ8008-Spax-HA2) and that L. lactis/pNZ8008-Spax-HA2 would have immunogenicity by oral administration without the use of adjuvant in the mouse model. To address this hypothesis, we show that oral vaccination of mice with L. lactis/pNZ8008-Spax-HA2 elicited significant humoral and mucosal immune responses. Importantly, L. lactis/pNZ8008-Spax-HA2 provided 100% protection against homologous H5N1 or heterologous H1N1 virus challenge. These results suggest that an HA2 subunit presented on the surface of L. lactis is an effective universal vaccine candidate against influenza A viruses in the poultry industry and in humans.

Broadly protective immunity against divergent influenza viruses by oral co-administration of Lactococcus lactis expressing nucleoprotein adjuvanted with cholera toxin B subunit in mice.

BACKGROUND: Current influenza vaccines need to be annually reformulated to well match the predicated circulating strains. Thus, it is critical for developing a novel universal influenza vaccine that would be able to confer cross-protection against constantly emerging divergent influenza virus strains. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Influenza virus nucleoprotein (NP) is a structural protein which encapsidates the negative strand viral RNA, and anti-NP antibodies play role in cross-protective immunity. Lactococcus lactis (L. lactis) is an ideal vaccine delivery vehicle via oral administration route. However, L. lactis vectored vaccine exhibits poor immunogenicity without the use of mucosal adjuvant. To enhance the immunogenicity of L. lactis vectored vaccine, cholera toxin B (CTB) subunit, one of mucosal adjuvants, is a safe adjuvant for oral route, when combined with L. lactis vectored vaccine. In this study, we hypothesized that pNZ8008, a L. lactis expression plasmid, encoding NP antigen, would be able to elicit cross-protection with the use of CTB via oral administration route. RESULTS: To construct L. lactis vectored vaccine, nucleoprotein (NP) gene of A/California/04/2009(H1N1) was sub-cloned into a L. lactis expression plasmid, pNZ8008. The expression of recombinant L. lactis/pNZ8008-NP was confirmed by Western blot, immunofluorescence assay and flow cytometric analysis. Further, immunogenicity of L. lactis/pNZ8008-NP alone or adjuvanted with cholera toxin B (CTB) subunit was evaluated in a mouse model via oral administration route. Antibodies responses were detected by ELISA. The result indicated that oral administration of L. lactis/pNZ8008-NP adjuvanted with CTB could elicit significant humoral and mucosal immune responses, as well as cellular immune response, compared with L. lactis/pNZ8008-NP alone. To further assess the cross-protective immunity of L. lactis/pNZ8008-NP adjuvanted with CTB, we used L. lactis/pNZ8110-pgsA-HA1 alone or adjuvanted with CTB as controls. Mice that received L. lactis/pNZ8008-NP adjuvanted with CTB were completely protected from homologous H1N1 virus and showed 80% protection against heterologous H3N2 or H5N1 virus, respectively. By contrast, L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB also conferred 100% protection against H5N1 virus infection, but indicated no cross-protection against H1N1 or H5N1 virus challenge. As controls, mice vaccinated orally with L. lactis/pNZ8008-NP alone or L. lactis/pNZ8110-pgsA-HA1 alone could not survive. CONCLUSION: This study is the first to report the construction of recombinant L. lactis/pNZ8008-NP and investigate its immunogenicity with the use of CTB. Compared with L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB, our data support 5 × 10(11) CFU of L. lactis/pNZ8008-NP adjuvanted with 1 µg of CTB is a better combination for universal influenza vaccines development that would provide cross-protective immunity against divergent influenza A viruses.

Protective immunity against influenza H5N1 virus challenge in chickens by oral administration of recombinant Lactococcus lactis expressing neuraminidase.

BACKGROUND: Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat in poultry. Current influenza vaccines predominantly focus on hemagglutinin (HA) which anti-HA antibodies are often neutralizing, and are used routinely to assess vaccine immunogenicity. However, Neuraminidase (NA), the other major glycoprotein on the surface of the influenza virus, has historically served as the target for antiviral drug therapy and is much less studied in the context of humoral immunity. The aim of this study was to evaluate the protective immunity of NA based on Lactococcus lactis (L.lactis) expression system against homologous H5N1 virus challenge in a chicken model. RESULTS: L.lactis/pNZ2103-NA which NA is derived from A/Vietnam/1203/2004 (H5N1) (VN/1203/04) was constructed based on L.lactis constitutive expression system in this study. Chickens vaccinated orally with 10(12) colony-forming unit (CFU) of L.lactis/pNZ2103-NA could elicit significant NA-specific serum IgG and mucosa IgA antibodies, as well as neuraminidase inhibition (NI) titer compared with chickens administered orally with saline or L.lactis/pNZ2103 control. Most importantly, the results revealed that chickens administered orally with L.lactis/pNZ2103-NA were completely protected from a lethal H5N1 virus challenge. CONCLUSIONS: The data obtained in the present study indicate that recombinant L.lactis/pNZ2103-NA in the absence of adjuvant can be considered an effective mucosal vaccine against H5N1 infection in chickens via oral administration. Further, these findings support that recombinant L.lactis/pNZ2103-NA can be used to perform mass vaccination in poultry during A/H5N1 pandemic.