In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade.

Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.

Production of cytoplasmic type citrus leprosis virus-like particles by plant molecular farming.

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60-70 nm × 110-120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure-function studies to compare with the 30-nm icosahedrons of other VLPs.

A Fast-Track Phenotypic Characterization of Plasmodium falciparum Vaccine Antigens through Lyse-Reseal Erythrocytes Mediated Delivery (LyRED) of RNA Interference for Targeted Translational Repression.

The minimal success of the malaria vaccine with available antigens indicates the need for intensive and accelerated research to identify and characterize new antigens that confer protection against infection, clinical manifestation, and even malaria transmission. Further, the genetic manipulation tools to characterize such antigens are very time-consuming and laborious due to the very low efficiency of transfection in the malaria parasite. Here, we report a human miRNA-mediated translational repression of antigens in Plasmodium falciparum as a fast-track method for understanding and validating their function. In this method, candidate miRNAs are designed based on favorable hybridization energy against a parasite gene, and miRNA mimics are delivered to the parasite by loading them as cargo in the erythrocytes by simple lyse-reseal method. Incubation of the miRNA loaded erythrocytes with purified mature trophozoites or schizonts results in the loaded erythrocytes' infection. The miRNA mimics are translocated to parasites, and the effect of miRNA-mediated translation repression can be monitored within 48-72 h post-invasion. Unlike other transfection based methods, this method is fast, reproducible, and robust. We call this method as lyse-reseal erythrocytes for delivery (LyRED) of miRNA, which is a rapid and straight-forward method providing an efficient alternative to the existing genetic tools for P. falciparum to characterize the function of antigens or genes. The identification of crucial antigens from the different stages of the Plasmodium falciparum life cycle by the miRNA targeting approach can fuel the development of efficacious subunit vaccines against malaria.

Plasmodium falciparum Antigen Expression in Leishmania Parasite: A Way Forward for Live Attenuated Vaccine Development.

Live attenuated vaccines (LAVs) are among the most critical interventions in modern medicine and have already proven their potential to save millions of lives. LAVs are always explored as potential vaccine candidates since they induce an immune response, which is as good as the wild type pathogen. For parasitic diseases, the efficacy of LAVs is still under investigation and needs extensive research to mark their presence in the field. In malaria, live attenuated sporozoites have been evaluated for a vaccine against the liver stage. This vaccine approach is limited due to the highly cumbersome technique of sporozoite isolation and related relapse issues. We have developed a novel vaccine against malaria by expressing Plasmodium falciparum antigens in Leishmania donovani promastigotes. These hybrid, recombinant L. donovani parasites mimicking P. falciparum parasite antigens were analyzed for their anti-malarial efficacy in preclinical studies. We demonstrate the potential of Leishmania spp. parasites in developing an important live vector vaccine against malaria for the induction of protective immune responses. Herein, we describe a method to express malaria parasite antigens in L. donovani promastigotes and analyze its potential for a vaccine against malaria. This methodology can be extended to live, attenuated Leishmania promastigotes parasites to develop LAV against malaria.

Recombinant FimH, a fimbrial tip adhesin of Vibrio parahaemolyticus, elicits mixed T helper cell response and confers protection against Vibrio parahaemolyticus challenge in murine model.

Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (rVpFimH) expressed in E. coli. Immunization of BALB/c mice with the rVpFimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-rVpFimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the rVpFimH-immunized mice with 2×LD50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus.

Immunogenic characterization and protective efficacy of recombinant CsgA, major subunit of curli fibers, against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is one of the major pathogens responsible for vibriosis and zoonotic infections in teleosts, marine invertebrates, and also humans through consumption of contaminated or unprocessed seafood. Emergence of resistance against current accessible antibiotics and spread to the food chain and environment necessitate the development of safe and effective subunit vaccine against this bacterium. Many bacteria including V. parahaemolyticus produce extracellular curli fibrils, heteropolymeric filaments of major and minor subunit, which have been implicated in adhesion, biofilm formation, and virulence. Adhesins are the primary contact points with the host which help in establishing infection and colonization. CsgA, an adhesin, is the major structural component of the curli fiber that forms homopolymers of several hundred units. Due to their exposure on the cell surface, the curli fibers are recognized by the host's immune system, would generate high immune response, and therefore can serve as effective vaccine candidate. In the present study, we describe characterization of the csgA gene, and preparation of recombinant soluble CsgA of V. parahaemolyticus (rVpCsgA), and evaluation of its vaccine potential. Immunization of BALB/c mice with the rVpCsgA mounted a strong immune response. Cellular immune assays such as antibody isotyping, in vitro splenocyte proliferation analysis, and cytokine profiling revealed mixed T-helper cell immune response. The anti-rVpCsgA antiserum was agglutination positive and specifically cross-reacted with the curli CsgA present on the outer membrane of V. parahaemolyticus cells, thus demonstrating its neutralization potential. One hundred percent survival of the immunized mice upon challenge with the lethal dosage of the bacterium established that the rVpCsgA could serve as an effective vaccine against the bacterium. KEY POINTS: • Recombinant histidine-tagged CsgA of V. parahaemolyticus, rVpCsgA, was purified. • The rVpCsgA immunization produced mixed immune response and agglutinating antibodies. • Immunization with the rVpCsgA protected mice against V. parahaemolyticus challenge.

Structural-functional characterization of recombinant Apolipoprotein A-I fromLabeo rohitademonstrates heat-resistant antimicrobial activity.

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose-dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 µg/ml or 0.81 µM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 µg/ml or 3.23 µM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Structural and Functional Characterization of Recombinant Interleukin-10 from Indian Major Carp Labeo rohita.

Interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. Though it is one of the major cytokines, IL-10 from the Indian major carp, Labeo rohita, has not yet been characterized. In the present study, we report large scale production and purification of biologically active recombinant IL-10 of L. rohita (rLrIL-10) using a heterologous expression system and its biophysical and functional characterization. High yield (~70 mg/L) of soluble rLrIL-10 was obtained at shake flask level. The rLrIL-10 was found to exist as a dimer. Far-UV CD spectroscopy showed presence of predominantly alpha helices. The tertiary structure of the purified rLrIL-10 was verified by fluorescence spectroscopy. Two-dimensional gel analysis revealed the presence of six isoforms of the rLrIL-10. The rLrIL-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1ß, TNFα, and IL-8, and augmented the NKEF transcript levels in spleen of L. rohita. Anti-inflammatory role of the rLrIL-10 was further established by inhibition of phagocytosis using NBT reduction assay in vitro. The data indicate that the dimeric alpha helical structure and function of IL-10 of L. rohita as a key regulator of anti-inflammatory response have remained conserved during evolution.

Apolipoprotein A-I in Labeo rohita: Cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection.

Apolipoprotein A-I (ApoA-I) is the most abundant and multifunctional high-density lipoprotein (HDL) having a major role in lipid transport and potent antimicrobial activity against a wide range of microbes. In this study, a complete CDS of 771 bp of Labeo rohita (rohu) ApoA-I (LrApoA-I) encoding a protein of 256 amino acids was amplified, cloned and sequenced. Tissue specific transcription analysis of LrApoA-I revealed its expression in a wide range of tissues, with a very high level of expression in liver and spleen. Ontogenic study of LrApoA-I expression showed presence of transcripts in milt and 3 h post-fertilization onwards in the larvae. The expression kinetics of LrApoA-I was studied upon infection with three different types of pathogens to elucidate its functional significance. Its expression was found to be up-regulated in the anterior kidney of L. rohita post-infection with Aeromonas hydrophila. Similarly following poly I:C (poly inosinic:cytidylic) stimulation, the transcript levels increased in both the anterior kidney and liver tissues. Significant up-regulation of LrApoA-I expression was observed in skin, mucous, liver and anterior kidney of the fish challenged with the ectoparasite Argulus siamensis. Immunomodulatory effect of recombinant LrApoA-I (rApoA-I) produced in Escherichia coli was demonstrated against A. hydrophila challenge in vivo. L. rohita administered with rApoA-I at a dose of 100 µg exhibited significantly higher protection (∼55%) upon challenge with A. hydrophila 12 h post-administration of the protein, in comparison to that observed in control group, along with higher level of expression of immune-related genes. The heightened expression of ApoA-I observed post-infection reflected its involvement in immune responses against a wide range of infections including bacterial, viral as well as parasitic pathogens. Our results also suggest the possibility of using rApoA-I as an immunostimulant, particularly rendering protection against A. hydrophila.

Genomic cloning and sequence analysis of Interleukin-10 from Labeo rohita.

UNLABELLED: Interleukin-10 (IL-10) is a pleiotropic cytokine and plays an important role in inflammation, immunoregulation and the pathogenesis of various diseases. Therefore, it is our interest to isolate, clone, sequence and characterize IL-10 gene from the fish Labeo rohita (Lr). The gene was amplified using genomic DNA isolated from head kidney with primers designed on conserved sequence homologues of fishes belonging to Cyprinidae family. The gLrIL-10 is 1467 nucleotides long with five exons and four introns sharing the same organization as of mammalian IL-10 genes. An open reading frame of 537 bp was found to encode a putative 179 amino acid protein with a signal peptide of 22 amino acids with conserved signature sequence motif. Sequence analysis showed similarity with the IL-10 from most fresh water fishes of Cyprinidae family. LrIL-10 has 27.2 % identity and 54.95 % similarity with the human IL-10. Sequence analysis followed by phylogenetic studies showed highest identity with Catla catla (98%) followed by Cyprinus carpio (93%), Hypophthalmichthys molitrix (89%) and is distantly related to human, rhesus monkey and frog. These data from primary sequence characterization may be used to further understand transcriptional regulation and functional characterization of LrIL-10 in relation to species-specific molecular immunology. ABBREVIATIONS: IL-10 - Interleukin-10, Lr - Labeo rohita, nt - nucleotides.