First Characterization of Avian Memory T Lymphocyte Responses to Avian Influenza Virus Proteins

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein

Phytochemical investigation and determination of phytoconstituents in flower extract of Nelumbo nucifera

The aim of this study seeks to investigate the presence of qualitative and quantitative analysis of phytoconstituents of the flower of the plant Nelumbo nucifera. The present study provides evidence that successive solvent extract of Nelumbo nucifera contains medicinally important bioactive compounds and this justifies the use of plant species as traditional medicine for treatment of various diseases. Maximum phenolic and flavanoid content was observed in ethanolic extract of Nelumbo nucifera. The DPPH scavenging potential of the ethanolic extracts of the herbs ranged from 33%-55%. In the present study analysis of free radical scavenging activity and total phenolic and flavonoid content showed that mainly the ethanolic extract of flower extract can be the potent source of natural antioxidants. Keywords: Phytochemical, Ethanolic extract, Antioxidant activity, Nelumbo nucifer

Developments in Avian Influenza Virus Vaccines

The serologically diverse influenza A viruses, although transmissible among various susceptible species, mostly infect avian species. Avian influenza viruses (AIV) are also notorious for adapting to mammalian species, including humans. Although eradication of commercial birds infected with AIV is the preferred method of control, the ever presence of potential avian migratory, reservoir species makes worldwide spread inevitable and vaccine development a high priority for poultry. Live, attenuated vaccine strategies are of concern because of the potential of AIV to mutate, through point mutations and/or reassortment of their segmented genome. Both live and the safer, killed vaccines are of concern because of competition of natural and vaccine antigens in critical diagnostic assays. Subunit vaccines, which allow for protein distinction for diagnostic purposes, may consist of purified AIV protein or genes that encode individual viral proteins. Most vaccines have targeted the virus hemagglutinin protein, which is responsible for induction of the most effective neutralizing antibodies. Gene vaccines that include plasmid DNA and viral vector delivery of AIV genes allow for endogenous in vivo amplification of protein within cells. While fowlpox virus vectors have been licensed and proven to be efficacious even in field situations, other viral vectors that target the respiratory tract are in experimental development with promising practical application for poultry. Maximum exploitation of vectored vaccines may incorporate enhancing immune molecules

Response of potato crop to vrikshaayurveda based herbal kunapajala against black scurf and early blight disease

Potato is susceptible to diseases like early blight and black scurf caused by Alternaria solani (Ellis and Martin) and Rhizoctonia solani Kuhn, respectively which are known to reduce the quality, yield and price of the tubers. Since ancient times in India, cultivation of plants using fermented liquid organic fertilizers and amendments had been practiced which are well documented in various scriptures and books. One such formulation is the kunapajala mentioned in Vrikshayurveda which was prepared by fermenting animal remains. It was used not only to stimulate plant growth but also protect them from pests and diseases. The potato cultivar Kufri Bahar was used against early blight and black scurf disease under field conditions during the rabi season of 2020-21 at VRC, Pantnagar. The results revealed that 10 per cent solution of KJ2 (50% nettle grass + 50% seasonal local weed based KJ) at 2000 L/ha dose and KJ3 (seasonal weed based KJ) at 1000 L/ha were found effective against black scurf and early blight disease of potato, respectively showing 12.37 and 35.79% reduction disease severity over control, respectively. It was also found that kunapajala treated tubers were statistically at par in terms of germination per cent and tuber yield with the control treatment in which recommended dose of fertiliser was applied suggesting that kunapajala treatment as mentioned above effectively provided the nutrients required by the growing tubers.

Response of potato crop to vrikshaayurveda based herbal kunapajala against black scurf and early blight disease

239-244Potato is susceptible to diseases like early blight and black scurf caused by Alternaria solani (Ellis and Martin) and Rhizoctonia solani Kuhn, respectively which are known to reduce the quality, yield and price of the tubers. Since ancient times in India, cultivation of plants using fermented liquid organic fertilizers and amendments had been practiced which are well documented in various scriptures and books. One such formulation is the kunapajala mentioned in Vrikshayurveda which was prepared by fermenting animal remains. It was used not only to stimulate plant growth but also protect them from pests and diseases. The potato cultivar Kufri Bahar was used against early blight and black scurf disease under field conditions during the rabi season of 2020-21 at VRC, Pantnagar. The results revealed that 10 per cent solution of KJ2 (50% nettle grass + 50% seasonal local weed based KJ) at 2000 L/ha dose and KJ3 (seasonal weed based KJ) at 1000 L/ha were found effective against black scurf and early blight disease of potato, respectively showing 12.37 and 35.79% reduction disease severity over control, respectively. It was also found that kunapajala treated tubers were statistically at par in terms of germination per cent and tuber yield with the control treatment in which recommended dose of fertiliser was applied suggesting that kunapajala treatment as mentioned above effectively provided the nutrients required by the growing tubers

Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice

Infection by Mycobacterium tuberculosis (Mtb) remains a major global concern and the available Bacillus Calmette-Guerin (BCG) vaccine is poorly efficacious in adults. Therefore, alternative vaccines and delivery strategies focusing on Mtb antigens and appropriate immune stimulating adjuvants are needed to induce protective immunity targeted to the lungs, the primary sites of infections and pathology. We present here evidence in support of mucosal vaccination by the sublingual route in mice using the subunit Mtb antigens Ag85B and ESAT-6 adjuvanted with the glycolipid alpha-galactosylceramide (α-GalCer), a potent natural killer T (NKT) cell agonist. Vaccinated animals exhibited strong antigen-specific CD4 and CD8 T cells responses in the spleen, cervical lymph nodes and lungs. In general, inclusion of the α-GalCer adjuvant significantly enhanced these responses that persisted over 50 days. Furthermore, aerosolized Mtb infection of vaccinated mice resulted in a significant reduction of bacterial load of the lungs and spleens as compared to levels seen in naïve controls or those vaccinated with subunit proteins, adjuvant , or BCG alone. The protection induced by the Mtb antigens and-GalCer vaccine through sublingual route correlated with a TH1-type immunity mediated by antigen-specific IFN-γ and IL-2 producing T cells

Intranasal Vaccination Affords Localization and Persistence of Antigen-Specific CD8+ T Lymphocytes in the Female Reproductive Tract

Immunization strategies generating large numbers of antigen-specific T cells in the female reproductive tract (FRT) can provide barrier protection against sexually-transmitted pathogens, such as the human immunodeficiency virus (HIV) and human papillomaviruses (HPV). The kinetics and mechanisms of regulation of vaccine-induced adaptive T cell-mediated immune responses in FRT are less well defined. We present here evidence for intranasal delivery of the model antigen ovalbumin (OVA) along with alpha-galactosylceramide adjuvant as a protein vaccine to induce significantly higher levels of antigen-specific effector and memory CD8+ T cells in the FRT, relative to other systemic and mucosal tissues. Antibody blocking of the CXCR3 receptor significantly reduced antigen-specific CD8+ T cells subsequent to intranasal delivery of the protein vaccine suggesting an important role for the CXCR3 chemokine-receptor signaling for T cell trafficking. Further, intranasal vaccination with an adenoviral vector expressing OVA or HIV-1 envelope was as effective as intramuscular vaccination for generating OVA- or ENV-specific immunity in the FRT. These results support the application of the needle-free intranasal route as a practical approach to delivering protein as well as DNA/virus vector-based vaccines for efficient induction of effector and memory T cell immunity in the FRT

Enhancement of Mucosal Immunogenicity of Viral Vectored Vaccines by the NKT Cell Agonist Alpha-Galactosylceramide as Adjuvant

Gene-based vaccination strategies, specifically viral vectors encoding vaccine immunogens are effective at priming strong immune responses. Mucosal routes offer practical advantages for vaccination by ease of needle-free administration, and immunogen delivery at readily accessible oral/nasal sites to efficiently induce immunity at distant gut and genital tissues. However, since mucosal tissues are inherently tolerant for induction of immune responses, incorporation of adjuvants for optimal mucosal vaccination strategies is important. We report here the effectiveness of alpha-galactosylceramide (α-GalCer), a synthetic glycolipid agonist of natural killer T (NKT) cells, as an adjuvant for enhancing immunogenicity of vaccine antigens delivered using viral vectors by mucosal routes in murine and nonhuman primate models. Significant improvement in adaptive immune responses in systemic and mucosal tissues was observed by including α-GalCer adjuvant for intranasal immunization of mice with vesicular stomatitis virus vector encoding the model antigen ovalbumin and adenoviral vectors expressing HIV env and Gag antigens. Activation of NKT cells in systemic and mucosal tissues along with significant increases in adaptive immune responses were observed in rhesus macaques immunized by intranasal and sublingual routes with protein or adenovirus vectored antigens when combined with α-GalCer adjuvant. These results support the utility of α-GalCer adjuvant for enhancing immunogenicity of mucosal vaccines delivered using viral vectors

Sublingual Vaccination Induces Mucosal and Systemic Adaptive Immunity for Protection against Lung Tumor Challenge

<div><p>Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8⁺ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.</p></div

Nanofibrous MultiDomain Peptide Hydrogels Provide T Cells a 3D, Cytocompatible Environment for Cell Expansion and Antigen-Specific Killing

T cells have the ability to recognize and kill specific target cells, giving therapies based on their potential for treating infection, diabetes, cancer, and other diseases. However, the advancement of T cell-based treatments has been hindered by difficulties in their ex vivo activation and expansion, the number of cells required for sustained in vivo levels, and preferential localization following systemic delivery. Biomaterials may help to overcome many of these challenges by providing a combined means of proliferation, antigen presentation, and cell localization upon delivery. In this work, we studied self-assembling Multidomain Peptides (MDPs) as scaffolds for T cell culture, activation, and expansion. We evaluated the effect of different MDP chemistries on their biocompatibility with T cells and the maintenance of antigen specificity for T cells cultured in the hydrogels. We also examined the potential application of MDPs as scaffolds for T cell activation and expansion and the effect of MDP encapsulation on T cell phenotype. We found high cell viability when T cells were encapsulated in noncationic MDPs, O5 and D2, and superior retention of antigen specificity and tumor-reactivity were preserved in the anionic MDP, D2. Maintenance of antigen recognition by T cells in D2 hydrogels was confirmed by quantifying immune synapses of T Cells engaged with antigen-presenting cancer cells. When 3D cultured in anionic MDP D2 coloaded with anti-CD3, anti-CD28, IL2, IL7, and IL15, we observed successful T cell proliferation evidenced by upregulation of CD27 and CD107a. This study is the first to investigate the potential of self-assembling peptide-based hydrogels as 3D scaffolds for human T cell applications and demonstrates that MDP hydrogels are a viable platform for enabling T cell in vitro activation, expansion, and maintenance of antigen specificity and therefore a promising tool for future T cell-based therapies