MMP-7 derived peptides with MHC class-I binding motifs from canine mammary tumor tissue elicit strong antigen-specific T-cell responses in BALB/c mice.

Matrix Metalloproteinases (MMPs)-induced altered proteolysis of extracellular matrix proteins and basement membrane holds the key for tumor progression and metastasis. Matrix metalloproteinases-7 (Matrilysin), the smallest member of the MMP family also performs quite alike; thus serves as a potential candidate for anti-tumor immunotherapy. Conversely, being an endogenous tumor-associated antigen (TAA), targeting MMP-7 for immunization is challenging. But MMP-7-based xenovaccine can surmount the obstacle of poor immunogenicity and immunological tolerance, often encountered in TAA-based conventional vaccine for anti-tumor immunotherapy. This paves the way for investigating the potential of MMP-7-derived major histocompatibility complex (MHC)-binding peptides to elicit precise epitope-specific T-cell responses towards their possible inclusion in anti-tumor vaccine formulations. Perhaps it also ushers the path of achieving multiple epitope-based broad and universal cellular immunity. In current experiment, an immunoinformatics approach has been employed to identify the putative canine matrix matelloproteinases-7 (cMMP-7)-derived peptides with MHC class-I-binding motifs which can elicit potent antigen-specific immune responses in BALB/c mice. Immunization with the cMMP-7 DNA vaccine induced a strong CD8+ cytotoxic T lymphocytes (CTLs) and Th1- type response, with high level of gamma interferon (IFN-γ) production in BALB/c mice. The two identified putative MHC-I-binding nonameric peptides (Peptide32-40 and Peptide175-183) from cMMP-7 induced significant lymphocyte proliferation along with the production of IFN-γ from CD8+ T-cells in mice immunized with cMMP-7 DNA vaccine. The current observation has depicted the immunogenic potential of the two cMMP-7-derived nonapeptides for their possible exploitation in xenovaccine-mediated anti-tumor immunotherapy in mouse model.

IL-18 immunoadjuvanted xenogeneic canine MMP-7 DNA vaccine overcomes immune tolerance and supresses the growth of murine mammary tumor.

The development of the tumorigenesis and angiogenesis through proteolytic cleavage of extracellular matrix protein and basement membranes is promoted by Matrix metelloproteinases-7 (MMP-7). Consequently, MMP-7 is presumed as potential target for mammary cancer immunotherapy. However, MMP-7 is an endogenous tumor associated antigen (TAA); therefore, immunization is challenging. In current study, a potent anti-tumor immune response has been elicited through recombinant bivalent plasmid pVIVO2.IL18.cMMP7 which subside the highly metastatic 4 T1 cell line induced mammary tumors and efficiently negate the existing challenge of using MMP-7 as immunotherapeutic target. Balb/c mice were immunized with canine MMP-7 (cMMP-7) using interleukine-18 (IL-18), as an immunoadjuvant, to explore the potential of the combination regarding elicitation of a potent anti-tumor immune response. Mice vaccinated with pVIVO2.IL18.cMMP7 DNA plasmid reduced the tumor growth significantly along with augmentation of the immune response to fight against tumor antigen as depicted by substantial enrichment of CD4+ and CD8+ population in splenocytes, infiltration of immune system cells in tumor tissue and enhanced survival time of mice. Further, splenocyte supernatant examination of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were remarkably up-regulated demonstrating the stimulation of cell-mediated immune response. Thus the current observations vividly portray that administration of xenogeneic MMP-7 DNA vaccine bypasses the tolerance barrier.

Monoclonal antibody resistant mutant of Peste des petits ruminants vaccine virus.

The available vaccines for control of Peste des petits ruminants do not favour differentiation of infected and vaccinated animals (DIVA). Hence, the present study was aimed to isolate and characterize monoclonal antibody resistant mutant of an Indian strain of vaccine virus "PPRV-Sungri/96" under selection pressure of virus neutralizing monoclonal antibody '4B11' specific to haemagglutinin (H) protein. We successfully isolated five monoclonal antibody resistant (mAr) mutants (PPRV-RM5, PPRV-RM6, PPRV-RM7, PPRV- E6 and PPRV- E7). The mAr mutants did not react with the anti-H mAb 4B11 whereas reacted with control anti-nucleoprotein mAb 4G6, similar to the parent vaccine virus "PPRV-Sungri/96" in indirect ELISA, cell ELISA and indirect immunofluorescence test. Cytometry analysis of mAr mutants revealed loss of binding to mAb 4B11 while maintaining binding to mAb 4G6, more or less similar to "PPRV-Sungri/96". The sequence analysis of the H-protein gene of the mAr mutants resulted in identification of two nucleotide changes leading to amino acid substitutions at position 263 and 502 (L263P and R502P) of the H protein indicating that the epitope of mAb 4B11 could be conformational in nature. Though, mAr mutant grew to a similar titre as parent vaccine virus (PPRV-Sungri/96), the in vivo work in goats to study the mAr mutant as possible negative marker vaccine candidate could not be successfully proved with mAb 4B11 based competitive ELISA. However, one of the nucleotide change (T-C) at position 788, unique to mAr mutant virus resulted in abolition of a restriction enzyme recognition site (BglII). This could be used to differentiate mAr mutant vaccine virus from other available vaccine and field strains using restriction fragment length polymorphism. However, the mAr mutant PPRV-E6 cannot be used as a candidate strain for DIVA vaccine as immune response against it cannot be differentiated based on serology.

HN protein of Newcastle disease virus sensitizes HeLa cells to TNF-α-induced apoptosis by downregulating NF-κB expression.

Hemagglutinin neuraminidase (HN) is a membrane protein of Newcastle disease virus (NDV) with the ability to induce apoptosis in many transformed cell lines. TNF-α is a multi-factorial protein that regulates cell survival, differentiation and apoptosis. In a previous study, we reported that HN protein induces apoptosis by downregulating NF-κB expression. Further, we speculated that downregulation of NF-κB expression might sensitize HeLa cells to TNF-α-mediated apoptosis. Therefore, the present study was undertaken to investigate if HN protein could sensitize HeLa cells to TNF-α and to examine the apoptotic potential of the HN protein and TNF-α in combination. The results revealed that the pro-apoptotic effects were more pronounced with the combination of HN and TNF-α than with HN or TNF-α alone, which indicates that the HN protein indeed sensitized the HeLa cells to TNF-α-induced cell death. The results of the study provide a mechanistic insight into the apoptotic action of HN protein along with TNF-α, which could be valuable in treating tumor types that are naturally resistant to TNF-α.

Poly (I:C) enhances the anti-tumor activity of canine parvovirus NS1 protein by inducing a potent anti-tumor immune response.

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.

Combined administration of the apoptin gene and poly (I:C) induces potent anti-tumor immune response and inhibits growth of mouse mammary tumors.

BACKGROUND: Many viral proteins exhibit selective cytotoxicity for tumor cells without affecting the normal diploid cells. The apoptin protein of chicken infectious anemia virus is one of such proteins, which has been shown to kill tumor cells specifically. However, an effective cancer treatment strategy also requires assistance from the immune system. Recently, poly (I:C) has been shown to be an effective cancer vaccine adjuvant. AIM: In this study, we assessed the anti-tumor potential of apoptin gene transfer alone and in combination with poly (I:C) in a 4T1 mouse mammary tumor model. METHODS: 4T1 cells were used to induce mammary tumor in Balb/c mice. Mice bearing tumors were divided into 6 groups, and each group received six intratumoral injections during a period of one month. After the last immunization, the animals were sacrificed, and peripheral blood, spleen, lungs, liver, heart, kidney and tumor tissues were collected for immunological, molecular and pathological analysis. RESULTS: We report that intratumoral administration of apoptin plasmid along with poly (I:C) not only significantly inhibited the growth of mammary tumor, but also induced a potent anti-tumor immune response as indicated by the increase in blood CD4+, CD8+ cells and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed increased secretion of Th1 cytokines (IFN-γ and IL-2). CONCLUSIONS: The results of our study demonstrate that the inclusion of poly (I:C) significantly enhanced the anti-tumor activity of apoptin mainly by inducing a potent anti-tumor immune response. Therefore, we report the use of apoptin and poly (I:C) combination as a novel and powerful strategy for cancer immunotherapy.

DNA Vaccination in Chickens.

Robust and sustainable development of poultry industry requires prevention of deadly infectious diseases. Vigorous vaccination of the birds is a routine practice; however, the live and inactivated vaccines that are used have inherent disadvantages. New-generation vaccines such as DNA vaccines offer several advantages over conventional vaccines. DNA vaccines, which encode an antigen of interest or multiple antigens in the target host, are stable, easy to produce and administer, do not require cold chain maintenance, and are not affected by the maternal antibodies. In addition, DNA vaccines can also be administered in ovo, and thus, mass vaccination and early induction of immune response can effectively be achieved. In this chapter, we focus on the development of DNA vaccines against important infectious viral as well as parasitic diseases of poultry.

Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.

Canine parvovirus NS1 induced apoptosis involves mitochondria, accumulation of reactive oxygen species and activation of caspases.

The non-structural protein (NS1) of parvoviruses plays an important role in viral replication and is thought to be responsible for inducing cell death. However, the detailed mechanism and the pathways involved in canine parvovirus type 2 NS1 (CPV2.NS1) induced apoptosis are not yet known. In the present study, we report that expression of CPV2.NS1 in HeLa cells arrests cells in G1 phase of the cell cycle and the apoptosis is mitochondria mediated as indicated by mitochondrial depolarization, release of cytochrome-c and activation of caspase 9. Treatment of cells with caspase 9 inhibitor Z-LEHD-FMK reduced the induction of apoptosis significantly. We also report that expression of CPV2.NS1 causes accumulation of reactive oxygen species (ROS) and treatment with an antioxidant reduces the ROS levels and the extent of apoptosis. Our results provide an insight into the mechanism of CPV2.NS1 induced apoptosis, which might prove valuable in developing NS1 protein as an oncolytic agent.

Development and evaluation of a Salmonella typhimurium flagellin based chimeric DNA vaccine against infectious bursal disease of poultry.

Infectious bursal disease (IBD) is an acute immunosuppressive disease of young chicks, caused by a double-stranded RNA virus. VP2 being the major capsid protein of the virus is an ideal vaccine candidate possessing the neutralizing epitopes. The present study involves the use of flagellin (fliC) as a genetic adjuvant to improve the immune response of VP2 based DNA vaccine against IBD. Our findings revealed that birds immunized with plasmid pCIVP2fliC showed robust immune response than pCIVP2 immunized groups. Further, challenge study proved that genetic fusion of fliC and VP2 can provide a comparatively higher level of protection against vvIBDV challenge in chickens than VP2 alone. These results thus indicate that Salmonella flagellin could enhance the immune responses and protection efficacy of a DNA vaccine candidate against IBDV infection in chickens, highlighting the potential of flagellin as a genetic adjuvant in the prevention of vvIBDV infection.

HN Protein of Newcastle Disease Virus Induces Apoptosis Through SAPK/JNK Pathway.

Many viral proteins are responsible for causing induction of apoptosis in the target cells. Hemagglutinin neuraminidase (HN), a multifunctional protein of Newcastle disease virus (NDV), is one of such proteins. The present study was undertaken to determine the apoptotic potential of the HN gene in cultured human cervical cancer cell line (HeLa cell) and to elucidate the molecular mechanisms involved. The results of the study indicate that HN protein causes apoptosis in HeLa cells, as observed by the translocation of Phosphatidylserine, activation of caspases, cleavage of poly (ADP-ribose) polymerase (PARP), and DNA fragmentation. Further, we report that expression of HN protein upregulates the SAPK/JNK pathway leading to transactivation of c-Jun which in turn activates apoptosis signaling. The results of our study provide an insight into the mechanism through which HN induces apoptosis.

Administration of IκB-kinase inhibitor PS1145 enhances apoptosis in DMBA-induced tumor in male Wistar rats.

Nuclear factor kappa-B (NF-κB), a key anti-apoptotic factor, plays a critical role in tumor cell growth, metastasis, and angiogenesis. The transcriptional activity of NF-κB is normally suppressed in the cytoplasm due to its association with a natural inhibitor molecule IκB. Phosphorylation of the IκB at Ser 32 and Ser 36 by the IκB kinase complex (IKK) marks the degradation of the molecule by 26S proteasome. As NF-κB is constitutively activated in most of the tumor cells, inhibition of the activities of IKK may significantly sensitize the tumor cells to apoptosis. In the present study, we investigated the effect of IκB kinase-specific blocker PS1145 on DMBA-induced skin tumor of male Wistar rats. We examined the apoptotic effect of PS1145 on DMBA-induced tumor by various histopathological and molecular techniques. Our results demonstrate the significant expression of major pro-apoptotic genes like caspases 2, 3, 8, 9, and p53 in PS1145-treated tumor bearing group at mRNA levels as well as significant (P < 0.05) down regulation in the expression levels of NF-κB and VEGF, the major pro-inflammatory and pro-angiogenic factors, respectively. The histopathological examination showed that the tumor progression, mitotic, AgNOR, and PCNA indices were significantly reduced in PS1145 treatment groups as compared to PBS control on day 28 of post-treatment. Furthermore, significant increase in TUNEL positive nuclei and observation of peculiar apoptotic nuclei in transmission electron microscopy were seen in PS1145 treatment group. We conclude that intravenous application of PS1145 promotes direct apoptosis in DMBA-induced skin tumor in male Wistar rats by blocking NF-κB and VEGF activities.

Comparative screening of single nucleotide polymorphisms in ß-casein and κ-casein gene in different livestock breeds of India.

The most polymorphic milk protein gene is ß-casein; 13 protein variants are known in cattle. Milk protein genetic polymorphism has received considerable research interest in recent years because of possible associations between milk protein and economically important traits in livestock. The present study was undertaken to explore the genetic polymorphisms in exon 7 of ß-casein and exon 4 of κ-casein genes in Arunachali yaks (Bos grunniens), Sahiwal (Bos indicus) cattle, malpura sheep (Ovis aries) and Gaddi goat (Capra hircus). Results of the study revealed presence of 11 SNP variants in all livestock species. Four SNPs were observed in Bos indicus; two SNPs in Bos grunniens; three SNPs in Ovis aries and three SNPs in Capra hircus. These variations are found to be synonymous in nature as these variations do not result in their corresponding amino acids. A total of five polymorphic sites have been described at the κ-casein (CSN3) locus in the Indian domestic Gaddi goat (Capra hircus) when compared with exotic goat (X60763) while sequence analysis of κ-casein gene in sheep showed three novel nucleotide changes in malpura sheep when compared with the exotic sheep (AY237637). These results highlight the importance of taking into consideration the CSN3 SNPs when performing selection for milk composition in dairy livestock breeds.

In-vitro characterization and evaluation of apoptotic potential of bicistronic plasmid encoding HN gene of Newcastle disease virus and human TNF-α.

The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-α, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-α of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 ± 1.21; 5.22 ± 0.60%, and TNF-α gene expression was found to be 15.44 ± 0.42; 6.51 ± 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-α act synergistically in the induction of apoptosis in HeLa cells.

Viral genes as oncolytic agents for cancer therapy.

Many viruses have the ability to modulate the apoptosis, and to accomplish it; viruses encode proteins which specifically interact with the cellular signaling pathways. While some viruses encode proteins, which inhibit the apoptosis or death of the infected cells, there are viruses whose encoded proteins can kill the infected cells by multiple mechanisms, including apoptosis. A particular class of these viruses has specific gene(s) in their genomes which, upon ectopic expression, can kill the tumor cells selectively without affecting the normal cells. These genes and their encoded products have demonstrated great potential to be developed as novel anticancer therapeutic agents which can specifically target and kill the cancer cells leaving the normal cells unharmed. In this review, we will discuss about the viral genes having specific cancer cell killing properties, what is known about their functioning, signaling pathways and their therapeutic applications as anticancer agents.

Development of dog mammary tumor xenograft in immunosuppressed Swiss albino mice.

Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and gamma-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.

Toll-like receptor-based adjuvants: enhancing the immune response to vaccines against infectious diseases of chicken.

Huge productivity loss due to infectious diseases in chickens is a major problem and, hence, robust development of the poultry industry requires control of poultry health. Immunization using vaccines is routine practice; however, to combat infectious diseases, conventional vaccines as well as new-generation recombinant vaccines alone, due to relatively weak immunogenicity, may not be effective enough to provide optimum immunity. With this in mind, there is a need to incorporate better and more suitable adjuvants in the vaccines to elicit the elevated immune response in the host. Over last few decades, with the increase in the knowledge of innate immune functioning, efforts have been made to enhance vaccine potency using novel adjuvants like Toll-like receptor based adjuvant systems. In this review, we will discuss the potential use of toll-like receptor ligands as an adjuvant in vaccines against the infectious diseases of chickens.

Flagellin a toll-like receptor 5 agonist as an adjuvant in chicken vaccines.

Chicken raised under commercial conditions are vulnerable to environmental exposure to a number of pathogens. Therefore, regular vaccination of the flock is an absolute requirement to prevent the occurrence of infectious diseases. To combat infectious diseases, vaccines require inclusion of effective adjuvants that promote enhanced protection and do not cause any undesired adverse reaction when administered to birds along with the vaccine. With this perspective in mind, there is an increased need for effective better vaccine adjuvants. Efforts are being made to enhance vaccine efficacy by the use of suitable adjuvants, particularly Toll-like receptor (TLR)-based adjuvants. TLRs are among the types of pattern recognition receptors (PRRs) that recognize conserved pathogen molecules. A number of studies have documented the effectiveness of flagellin as an adjuvant as well as its ability to promote cytokine production by a range of innate immune cells. This minireview summarizes our current understanding of flagellin action, its role in inducing cytokine response in chicken cells, and the potential use of flagellin as well as its combination with other TLR ligands as an adjuvant in chicken vaccines.

Recombinant flagellin and its cross-talk with lipopolysaccharide--effect on pooled chicken peripheral blood mononuclear cells.

Toll-like receptors (TLRs) are one of the types of pattern recognition receptors (PRRs) that recognize conserved pathogen molecules. TLRs link innate and adaptive arms of immune system and are implicated in the development of defense against invading pathogens. Lipopolysaccharide (LPS) and flagellin are recognized by TLR4 and TLR5, respectively. In this study, the effect of flagellin and lipopolysaccharide alone and in combination on chicken peripheral blood mononuclear cells (PBMCs) was investigated. The FliC gene of Salmonella typhimurium was expressed in a prokaryotic expression system and the recombinant flagellin was used to stimulate the chicken PBMCs. A combination of recombinant flagellin and LPS synergistically upregulated nitric oxide production, IL-12 and IL-6 expression but antagonistically down regulated IL-4 expression in comparison to recombinant flagellin alone. The results indicate that these agonists synergistically interact and enhance macrophage function and promote Th1 immune response in chicken PBMCs.

In silico proteomic characterization of human epidermal growth factor receptor 2 (HER-2) for the mapping of high affinity antigenic determinants against breast cancer.

Multiple different approaches are being used to activate the immune system against breast cancer. Vaccine therapy in general follows the principle that injections of various substances ultimately result in the presentation of tumor peptides to the patient's immune system. We proposed a potential in silico DNA vaccine against breast cancer by integrating high affinity T cell (MHC-I and MHC-II) and B cell (continuous and discontinuous) epitopes. The matching of the HLA haplotype and antigen was performed to provide the appropriate peptide epitope suitable for majority of the patients. The immunogenic nature of the antigenic construct was also enhanced by the administration of consensus epitopes. The potency of DNA vaccines depends on the efficient expression and presentation of the encoded antigen of interest and the chances of efficient expression of our antigenic construct in host organism was also verified by in silico approaches. An attempt was made to overcome the limited potency of the DNA vaccine by targeting DNA to professional antigen-presenting cells (APCs). A higher immune response theoretically corresponds to a higher survival rate of patients. Therefore, optimization studies were also employed to enhance the immunogenicity of proposed in silico DNA vaccine.