Engineering PRINT® Nanoparticle Subunit Vaccine to Induce Antitumor Immune Response

Educating our immune system via vaccination is an attractive approach to combat cancer. Tumor-specific cytotoxic T cells (CTLs) such as CD8+ effector T cells play a critical role in tumor control. However, vaccination aimed at eliciting a potent CD8+ T cell response with tumor-associated peptide antigens, are typically ineffective due to poor immunogenicity. Nanoparticle delivery of antigens and adjuvants can enhance their uptake by antigen presenting cells (APCs) and facilitate their intracellular delivery to induce antigen specific CD8+ cells. PRINT® (Particle Replication in Non-Wetting Template) is a unique platform to fabricate nano and microparticles with exquisite control over size, shape, and surface chemistry. The goal of this study is to design a PRINT® nanoparticle based subunit vaccine for the intracellular delivery of antigenic peptides and adjuvants to induce potent CTLs response. The aims of project include, i) formulation design of nanoparticle subunit vaccine, and induction of ii) in vitro and in vivo immune response. Under the first aim of this study we have engineered a reduction sensitive PRINT® hydrogel based subunit vaccine for intracellular delivery of antigenic peptide SIINFEKL (ovalbumin-derived CTL epitope [OVA257-264 –SIINFEKL]) and an immunostimulatory adjuvant, CpG ODNs (TLR9 agonist). SIINFEKL and CpG ODN were conjugated to PRINT® hydrogel via disulfide linkages. These NPs were successfully internalized and processed by BMDCs, resulting in BMDC maturation, subsequent cross-presentation of antigenic peptide, and induction of potent antigen-specific T cells in mice. Under the second aim of this study we further demonstrated induction of SIINFEKL specific IFN-γ producing CD8+ T cells as well as antitumor protective immune response in EG7 tumor mouse model by delivering sustained release formulations of CSIINFEKL and CpG ODN via PRINT® hydrogel NPs. Taken together, this study provides a highly effective approach, i) to induce potent CTLs response by reduction sensitive NP subunit vaccine and, ii) to induce antitumor immunity by tuning the release of antigenic peptide by changing the conjugation chemistry.Doctor of Philosoph

Pseudomonas aeruginosa inhibits quorum-sensing mechanisms of soft rot pathogen Lelliottia amnigena RCE to regulate its virulence factors and biofilm formation

The quorum-sensing (QS) cascade is responsible for the colonization and phenotypic behavior of the pathogenic organism and the regulation of diverse signal molecules. The disruption of the quorum-sensing system is an effective strategy to overcome the possibility of antibiotic resistance development in the pathogen. The quorum quenching does not kill the microbes. Instead, it hinders the expression of pathogenic traits. In the present experiment, Pseudomonas aeruginosa RKC1 was used to extract the metabolites responsible for quorum-sensing inhibition in soft rot pathogen Lelliottia amnigena RCE. During the initial screening, P. aeruginosa RKC1 was found to be most promising and inhibits violacein of Chromobacterium violaceum MTCC2656 pyocyanin, swarming-swimming motility of P. aeruginosa MTCC2297. The characterization of metabolites produced by the microbes which are responsible for quorum-sensing inhibition through GC-MS is very scarce in scientific literature. The ethyl acetate extract of P. aeruginosa RKC1 inhibits biofilm formation of L. amnigena RCE while inhibiting growth at higher concentrations. The GC-MS analysis suggested that Cyclic dipeptides (CDPs) such as Cyclo (L-prolyl-L-valine), Cyclo (Pro-Leu), and Cyclo(D-phenylalanyl-L-prolyl) were predominantly found in the ethyl acetate extract of the P. aeruginosa RKC1 (93.72%). This diketopiperazine (DKPs) exhibited quorum-sensing inhibition against the pathogen in liquid media during the active growth phase and regulated diverse metabolites of the pathogen. Moreover, the metabolites data from the clear zone around wells showed a higher concentration of DKSs (9.66%) compared to other metabolites. So far, very few reports indicate the role of DKPs or CDPs in inhibiting the quorum-sensing system in plant pathogenic bacteria. This is one such report that exploits metabolites of P. aeruginosa RKC1. The present investigation provided evidence to use quorum-sensing inhibitor metabolites, to suppress microbes' pathogenesis and thus develop an innovative strategy to overcome antibiotic resistance.Peer reviewe

Evaluation of Plant Growth-Promoting and Salinity Ameliorating Potential of Halophilic Bacteria Isolated From Saline Soil

Among the biotic and abiotic stress affecting the physical, chemical, and biological properties of soil, salinity is a major threat that leads to the desertification of cultivable land throughout the world. The existence of diverse and versatile microbial populations inhabiting the nutrient-rich soil and varied soil conditions affects the soil dynamism. A normal soil constitutes 600 million bacteria belonging to about 20,000 species, which is reduced to 1 million with 5,000-8,000 species in stress conditions. Plant growth-promoting rhizobacteria (PGPR) are in symbiotic association with the plant system, which helps in combating the abiotic stress and increases the overall productivity and yield. These microorganisms are actively associated with varied cellular communication processes through quorum sensing and secondary metabolites such as the production of Indole-3-acetic acid (IAA), exopolysaccharide (EPS) siderophore, ammonia, ACC deaminase, and solubilization of phosphate. The present study focused on the isolation, identification, and characterization of the microorganisms isolated from the seacoast of Dandi, Navsari. Twelve isolates exhibited PGP traits at a high salt concentration of 15-20%. AD9 isolate identified as Bacillus halotolerans showed a higher ammonia production (88 +/- 1.73 mu g/mL) and phosphate solubilization (86 +/- 3.06 mu g/mL) at 15% salt concentration, while AD32* (Bacillus sp. clone ADCNO) gave 42.67 +/- 1.20 mu g/mL IAA production at 20% salt concentration. AD2 (Streptomyces sp. clone ADCNB) and AD26 (Achromobacter sp. clone ADCNI) showed ACC deaminase activity of 0.61 +/- 0.12 and 0.60 +/- 0.04 nM alpha-ketobutyrate/mg protein/h, respectively. AD32 (Bacillus sp. clone ADCNL) gave a high siderophore activity of 65.40 +/- 1.65%. These isolates produced salinity ameliorating traits, total antioxidant activities, and antioxidant enzymes viz. superoxide dismutase (SOD), Glutathione oxidase (GSH), and catalase (CAT). Inoculation of the multipotent isolate that produced PGP traits and salinity ameliorating metabolites promoted the plant growth and development in rice under salinity stress conditions. These results in 50% more root length, 25.00% more plant dry weight, and 41% more tillers compared to its control.Peer reviewe

Nanoparticle delivery of chemosensitizers improve chemotherapy efficacy without incurring additional toxicity

We demonstrate proof of principle that nanoparticle delivery of chemosensitizers can improve efficacy of chemotherapy without increasing toxicity

Halotolerant microbial consortia for sustainable mitigation of salinity stress, growth promotion, and mineral uptake in tomato plants and soil nutrient enrichment

Salinity significantly impacts the growth, development, and reproductive biology of various crops such as vegetables. The cultivable area is reduced due to the accumulation of salts and chemicals currently in use and is not amenable to a large extent to avoid such abiotic stress factors. The addition of microbes enriches the soil without any adverse effects. The effects of microbial consortia comprising Bacillus sp., Delftia sp., Enterobacter sp., Achromobacter sp., was evaluated on the growth and mineral uptake in tomatoes (Solanum Lycopersicum L.) under salt stress and normal soil conditions. Salinity treatments comprising Ec 0, 2, 5, and 8 dS/m were established by mixing soil with seawater until the desired Ec was achieved. The seedlings were transplanted in the pots of the respective pH and were inoculated with microbial consortia. After sufficient growth, these seedlings were transplanted in soil seedling trays. The measurement of soil minerals such as Na, K, Ca, Mg, Cu, Mn, and pH and the Ec were evaluated and compared with the control 0 days, 15 days, and 35 days after inoculation. The results were found to be non-significant for the soil parameters. In the uninoculated seedlings’ (control) seedling trays, salt treatment significantly affected leaf, shoot, root dry weight, shoot height, number of secondary roots, chlorophyll, and mineral contents. While bacterized seedlings sown under saline soil significantly increased leaf (105.17%), shoot (105.62%), root (109.06%) dry weight, leaf number (75.68%), shoot length (92.95%), root length (146.14%), secondary roots (91.23%), and chlorophyll content (−61.49%) as compared to the control (without consortia). The Na and K intake were higher even in the presence of the microbes, but the beneficial effect of the microbe helps plants sustain in the saline environment. The inoculation of microbial consortia produced more secondary roots, which accumulate more minerals and transport substances to the different parts of the plant; thus, it produced higher biomass and growth. Results of the present study revealed that the treatment with microbial consortia could alleviate the deleterious effects of salinity stress and improve the growth of tomato plants under salinity stress. Microbial consortia appear to be the best alternative and cost-effective and sustainable approach for managing soil salinity and improving plant growth under salt stress conditions

Role of Linker Length and Antigen Density in Nanoparticle Peptide Vaccine

Multiple studies have been published emphasizing the significant role of nanoparticle (NP) carriers in antigenic peptide-based subunit vaccines for the induction of potent humoral and cellular responses. Various design parameters of nanoparticle subunit vaccines such as linker chemistry, the proximity of antigenic peptide to NPs, and the density of antigenic peptides on the surface of NPs play an important role in antigen presentation to dendritic cells (DCs) and in subsequent induction of CD8+ T cell response. In this current study, we evaluated the role of peptide antigen proximity and density on DC uptake, antigen cross-presentation, in vitro T cell proliferation, and in vivo induction of CD8+ T cells. To evaluate the role of antigen proximity, CSIINFEKL peptides were systematically conjugated to poly­(ethylene glycol) (PEG) hydrogels through N-hydroxysuccinimide–PEG–maleimide linkers of varying molecular weights: 2k, 5k, and 10k. We observed that the peptides conjugated to NPs via the 2k and 5k PEG linkers resulted in higher uptake in bone marrow-derived DCs (BMDCs) and increased p-MHC-I formation on the surface of bone marrow-derived DCs (BMDCs) as compared to the 10k PEG linker formulation. However, no significant differences in vitro T cell proliferation and induction of in vivo CD8+ T cells were found among linker lengths. To study the effect of antigen density, CSIINFEKL peptides were conjugated to PEG hydrogels via 5k PEG linkers at various densities. We found that high antigen density NPs presented the highest p-MHC-I on the surface of BMDCs and induced higher proliferation of T cells, whereas NPs with low peptide density resulted in higher DC cell uptake and elevated frequency of IFN-γ producing CD8+ T cells in mice as compared to the medium- and high-density formulations. Altogether, findings for these experiments highlighted the importance of linker length and peptide antigen density on DC cell uptake, antigen presentation, and induction of in vivo CD8+ T cell response

Reduction Sensitive PEG Hydrogels for Codelivery of Antigen and Adjuvant To Induce Potent CTLs

Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8⁺ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8⁺ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA_257–264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8⁺ effector T cells in mice (∼6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases

Nanoparticle delivery of chemosensitizers improve chemotherapy efficacy without incurring additional toxicity

Chemosensitizers can improve the therapeutic index of chemotherapy and overcome treatment resistance. Successful translation of chemosensitizers depends on the development of strategies that can preferentially deliver chemosensitizers to tumors while avoiding normal tissue. We hypothesized that nanoparticle (NP) formulation of chemosensitizers can improve their delivery to tumors which can in turn improve their therapeutic index. To demonstrate the proof of principle of this approach, we engineered NP formulations of two chemosensitizers, the PI3-kindase inhibitor wortmanin (Wtmn) and the PARP inhibitor olaparib. NP Wtmn and NP olaparib were evaluated as chemosensitizers using lung cancer cells and breast cancer cells respectively. We found Wtmn to be an efficient chemosensitizer in all tested lung-cancer cell lines reducing tumor cell growth between 20 and 60% compared to drug alone. NP formulation did not decrease its efficacy in vitro. Olaparib showed less consistent chemosensitization as a free drug or in NP formulation. NP Wtmn was further evaluated as a chemosensitizer using mouse models of lung cancer. We found that NP Wtmn is an effective chemosensitizer and more effective than free Wtmn showing a 32% reduction in tumor growth compared to free Wtmn when given with etoposide. Importantly, NP Wtmn was able to sensitize the multi-drug resistant H69AR cells to etoposide. Additionally, the combination of NP Wtmn and etoposide chemotherapy did not significantly increase toxicity. The present study demonstrates the proof of principle of using NP formulation of chemosensitizing drugs to improve the therapeutic index of chemotherapy