Enhancement of Antibody Responses in Chickens Vaccinated with a Plasmid DNA Construct of Avian Influenza Virus H5 Gene Infused with Hsp70 of Mycobacterium Tuberculosis

Currently, this region is battling against highly pathogenic avian influenza (HPAI) virus H5N1 and the virus has been isolated in non-poultry birds in various countries in Middle East as well as in the European and African continents. These developments have ignited global fears of an imminent influenza pandemic. The adoption of a vaccination policy, targeted either to control or to prevent infection in poultry, is generally discouraged. Nevertheless, the need to boost eradication efforts in order to limit further spread of infection and avoid heavy economic losses, and advances in modern vaccine technologies, have prompted a re-evaluation of the potential use of vaccination in poultry as an additional tool in comprehensive disease control strategies. Hence, several types of vaccines are available and some of them have been tested experimentally and/or used in commercial farms. DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the low immune intensity in clinical trials limits the application of DNA vaccines. Heat shock proteins (HSPs) or stress proteins are highly conserved molecules that act as chaperons. Among the HSPs, HSP70 family is well characterized protein that showed potent adjuvant effects on the innate and adaptive immune responses. In this study, we developed DNA vaccine based on H5 gene, and enhanced the DNA vaccine potency with Mycobacterium tuberculosis heat shock proteins 70 (HSP70) as adjuvant. Hence, a series of DNA plasmids encoding H5 and NP from Malaysian H5N1 (A/Ck/Malaysia/5858/2004) were constructed and then fused with HSP70. The H5, NP, H5-HSP70 and NP-HSP70 recombinant proteins were expressed in Vero cells. We further investigated the ability of the pcDNA3.1/H5 and pcDNA3.1/H5-HSP70 constructs in inducing H5 specific antibody responses in SPF chickens. pcDNA3.1/H5 and pcDNA3.1/H5-HSP70 were administered to 10 days old SPF chickens in three doses of 100 μg by the intramuscular route, two weeks apart. Chickens were bled every week and H5 specific antibody was measured using hemagglutination inhibition (HI) test. The ability of the constructed plasmids in inducing the expression of H5 and H5-HSP70, respectively, in chickens was examined by RT-PCR. In vivo expression was confirmed based on detection of H5 RNA transcripts in muscle and spleen of chickens inoculated with the constructed DNA vaccines. The HI test was carried out using H5 antigen from a low pathogenic avian influenza virus (LPAIV), A/Duck/Malaysia/8443/2004 (H5N2). Sequence analysis of H5 genes of H5N1 and H5N2, respectively, that was used in this study showed nucleotide and amino acid identity of more than 87%. In addition, all the chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5-HSP70 showed HI titer in week three after the first immunization. The HI titer was more prominent from first booster onwards in the chickens immunized with pcDNA3.1/H5-HSP70. This study demonstrated that chickens immunized with HSP70 based H5 DNA vaccine developed higher antibody titer compared to chickens immunized with H5 alone. However, the increase in HI antibody titer was not significantly different (P > 0.05). As expected, the control chickens inoculated with pcDNA3.1/HSP70 and pcDNA3.1 showed no evidence of HI antibody responses. In conclusion, we have demonstrated for the first time that HSP70-based H5 DNA can improve the induction of humoral immune response in chickens and is a promising candidate of DNA vaccine for AIV infection. Further studies are required to explore the role of HSP70 as genetic adjuvant for DNA vaccine in chickens

Evolution of adaptive immune response from acute to chronic phase of hepatitis C virus infection

The CD8+ T cell responses play a pivotal role in controlling viral replication during HCV infection. HCV evades the immune system by rapid viral evolution affording escape from immune selection pressure including at MHC-I restricted epitopes. However, some CTL epitopes remain conserved well past the time of establishment of chronic infection, implying additional mechanisms immune failure exists. CD8+ T cells exhibiting an exhausted phenotype have been extensively reported during the chronic stage of illness for chronic viral infections, such as HCV and HIV. Additionally, impaired differentiation and trafficking of CD8+ T cells is known to be associated with immune escape and exhaustion of CTLs, but the timing and mechanisms and expression patterns of inhibitory receptors as wells as impairments in differentiation during primary HCV infection remains unclear. HCV-specific CD8+ T cell responses against the transmitted founder virus identified via ELISpot. Immune escape was observed in the NGS data set in ~33% of all ELISpot identified epitopes. The majority of HCV-specific CD8+ responses identified via IFN- ELSPOT in chronic progressors were also characterised by a dominant population of terminally differentiated effector memory cells (CCR7lowCD45ROhighKLRG1highCD127low), and elevated expression of co-inhibitory markers (PD-1 and 2B4) targeting both conserved as well as escaped HCV variants at the peak of immune response (as early as 70-90 days post infection). However, evidence of long-term central memory subpopulations with moderate IFN-γ production was identified in a subset of responses. There was an association of viral escape with the magnitude (IFN- production) of the response, suggesting ongoing evolution of CTLs in response to prolonged viral exposure. Analysis of T-bet expression revealed that T-bet expression on HCV-specific CD8+ T cell was not associated with clearance. Immuno-phenotyping of liver showed that, liver was enriched with T cells expressing the chemokine receptors CCR2, CCR5, CXCR3, and CXCR6. Additionally, the studies revealed preferential expression of CXCR3 on HCV-specific CD8+ T cells in both chronic and acute HCV infection suggesting a key role for CXCR3 in regulation of HCV-specific CD8+ T cell trafficking to the site of infection in the liver. Taken together the studies in this thesis provide both consistent findings with more limited studies in HCV and comparable contexts in HIV, and clear contrasts with previous reports in murine LCMV models. The findings offer novel insights into our understanding of the immunopathgenesis of primary HCV and into HCV vaccine design

Immune system regulation and response during infectious bursal disease virus and Newcastle disease virus infections in chickens

Infectious bursal disease (IBD) is caused by IBD virus (IBDV), a highly infectious lymphotropic virus that induces cytocidal effect on B lymphocytes of bursa Fabricius. Hence, the disease has been considered to have the most impact due to its immunosuppressive effects in young birds. However, the effects of the virus on non B lymphocytes functions and secretion of cytokines and chemokines are poorly characterized. On the other hand, Newcastle disease virus (NDV) is a highly infectious virus, which contributed to the major causes of economic losses in poultry industry. The virus can be different into several genotypes, however, velogenic NDV are of genotypes V, VI, VII, VIII and X. Hence, understanding IBDV and NDV immunoregulation on the host immune system will provide valuable information to define the immunopathology of the respective viruses. In this study, immunophenotyping of lymphocytes and productions of cytokines and chemokines expression were analysed by using flow cytometry and GeXP/real-time PCR assays, respectively, in order to understand the roles of B, T cells and macrophages during IBDV and NDV infections. Based on in vitro study, very virulent IBDV strain UPM0081 was detected in monocytes-macrophage cell line, HD11 cells as early as 6 hours post-infection. On the other hand, ConA-C1-Vick, a chicken CD4+ and CD8+ T cell line was not responding against IBDV infection in vitro. In vitro cytocidal effect of IBDV towards HD11 cell line showed evidence of apoptosis where 6% of cells undergo early apoptosis at 24 hours followed by 11% of cells undergo late apoptosis. Up-regulation of pro-inflammatory related cytokines/chemokines and other related genes such as CXCLi1, CXCLi2, CCL4, IL12α, IL-18, IL-1β, iNOS, TLR-3 and MHCI were detected in IBDV infected HD11 cell line. In the in vivo study, vvIBDV (UPM0081) was detected in both spleen and bursa as early as day 2 post-infection in specific-pathogen-free (SPF) chickens based on PCR detection. However, infiltration of Kul1+ macrophages population in spleen and bursa was different. Expressions of cytokines, chemokines and other immunerelated genes in both spleen and bursa were compared in this study to understand the pathogenesis of vvIBDV infection. IL-10, an anti-inflammatory cytokine that commonly expressed by macrophage, was down-regulated in HD11 cells but no significant changes were detected in the bursa and spleen of the infected chicken throughout the study. Unlike constant increase of IL-6, IL-12α, and iNOS in bursa, highest level of IL-6 and IL-12α were found in spleen at day 2 days post-infections while iNOS was recorded with highest expression in spleen at day 3 post-infection. Added to this, IL-8 (CXCLi2) and IL-18 recorded the highest level of expression in day 3 and day 2 post-infection, respectively, in both bursa and spleen. In the case of NDV, immunoregulation of velogenic NDV genotype VII (IBS002) and VIII (AF2240) on chicken macrophages, B and T lymphocytes during the acute stage of the respective virus infection in SPF chickens was characterized. Both NDV genotypes induced drastic reduction of CD4+ and CD8+ T lymphocyte and associated with infiltration of macrophage in spleen at day 3 post-infection. The depletion of the T lymphocytes is probably through the process of apoptosis since 37% and 39% of ConA-C1-Vick cells undergo apoptosis at 24 and 48 hours post-infection, respectively. In addition, gene expression profiles showed an up-regulation of CCL4, CXCLi1, CXCLi2, IFN-γ, IL12α, IL-18, IL-1β, IL-6, iNOS, TLR-7, MHCI, IL-17F and TNFSF13β (p<0.05). However, both genotypes show different expression patterns where IBS002 caused a more rapid up-regulation of CXCLi2, IFN-γ, IL12α, IL-18, IL-1β, iNOS and IL-10 at 3 days post-infection (DPI), meanwhile the expression of CCL4, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 compared to IBS002 at 4 DPI. In addition, the expression of IL-10 was significantly higher in IBS002 infected chickens at 3 and 4 DPI compared to AF2240 infected chickens. Hence, infection with velogenic genotype VII and VIII NDV induce cytokines and chemokines associated with inflammatory reactions. Both the expressions of IFN-γ and CXCLi2 transcripts were up-regulated in CD4+ T cells of AF2240 and IBS002 infected chickens. However, IBS002 showed significantly higher up-regulation of CXCLi2 at day 1 and 3 postinfection compared to AF2240. Furthermore, the up-regulation of IL-18 was readily detectable in IBS002 infected CD4+ T cells at day 1 post-infection. In conclusion, the current study demonstrated the differences in the immunophenotyping of B, T and macrophage populations as well as the expressions of cytokines, chemokines and immune-related genes expression in chickens infected with different genotypes of velogenic NDV strains and vvIBDV. The findings from this study are of valuable information for future study in understanding the immunopathology of the respective virus infection and vaccine –induced immunity

Combinatorial Cytotoxic Effects of Damnacanthal and Doxorubicin Against Human Breast Cancer MCF-7 Cells in Vitro

Despite progressive research being done on drug therapy to treat breast cancer, the number of patients succumbing to the disease is still a major issue. Combinatorial treatment using different drugs and herbs to treat cancer patients is of major interest in scientists nowadays. Doxorubicin is one of the most used drugs to treat breast cancer patients. The combination of doxorubicin to other drugs such as tamoxifen has been reported. Nevertheless, the combination of doxorubicin with a natural product-derived agent has not been studied yet. Morinda citrifolia has always been sought out for its remarkable remedies. Damnacanthal, an anthraquinone that can be extracted from the roots of Morinda citrifolia is a promising compound that possesses a variety of biological properties. This study aimed to study the therapeutic effects of damnacanthal in combination with doxorubicin in breast cancer cells. Collectively, the combination of both these molecules enhanced the efficacy of induced cell death in MCF-7 as evidenced by the MTT assay, cell cycle, annexin V and expression of apoptosis-related genes and proteins. The effectiveness of doxorubicin as an anti-cancer drug was increased upon addition of damnacanthal. These results could provide a promising approach to treat breast cancer patients

Cytotoxic effect of ethanol extract of microalga, Chaetoceros calcitrans, and its mechanisms in inducing apoptosis in human breast cancer cell line

Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00±0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00±0.52 g/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug

Molecular characterization of fusion gene of recently isolated Newcastle disease virus isolates

Intensive vaccine programs have been implemented in many countries including Malaysia, but Newcastle disease virus (NDV) outbreaks have occurred, even in well-vaccinated farms. Hence, the present study was aimed to characterize five NDV isolates obtained from NDV vaccinated broiler farms in 2011 based on sequence and phylogenetic analysis of partial fusion gene. All the isolated NDV strains showed that they are categorized as velogenic NDV based on the presence of multi-basic protease cleavage sites, 112RRRKRF117. In addition, phylogenetic analysis showed that the isolates can classified under the genotype VII, subgenotype VIId

Combinatorial cytotoxic effects of damnacanthal and doxorubicin against human breast cancer MCF-7 cells in vitro

Despite progressive research being done on drug therapy to treat breast cancer, the number of patients succumbing to the disease is still a major issue. Combinatorial treatment using different drugs and herbs to treat cancer patients is of major interest in scientists nowadays. Doxorubicin is one of the most used drugs to treat breast cancer patients. The combination of doxorubicin to other drugs such as tamoxifen has been reported. Nevertheless, the combination of doxorubicin with a natural product-derived agent has not been studied yet. Morinda citrifolia has always been sought out for its remarkable remedies. Damnacanthal, an anthraquinone that can be extracted from the roots of Morinda citrifolia is a promising compound that possesses a variety of biological properties. This study aimed to study the therapeutic effects of damnacanthal in combination with doxorubicin in breast cancer cells. Collectively, the combination of both these molecules enhanced the efficacy of induced cell death in MCF-7 as evidenced by the MTT assay, cell cycle, annexin V and expression of apoptosis-related genes and proteins. The effectiveness of doxorubicin as an anti-cancer drug was increased upon addition of damnacanthal. These results could provide a promising approach to treat breast cancer patients