Protective Efficacy Evaluation of NPt-NPt-VP1₁₋₁₀₀Protein as a Candidate Vaccine Against Enterovirus 71 Infections in Mouse Model

Enterovirus 71 (EV71) is a type of human virus belonging to the Enterovirus genus within the Picornaviridae family. The virus mainly causes hand, foot and mouth disease in children which sometimes lead to severe neurological complications. Outbreaks of EV71 infections are serious health threats since effective antiviral drugs or vaccines are not currently available. Therefore, development of an effective vaccine is ideal for the prevention and control of EV71 disease outbreak. The use of recombinant EV71 viral protein offers an alternative to the more risky method of using whole live attenuated or inactivated virus. Our previous study using truncated VP1 protein (VP11-100) of EV71 virus fused to a carrier protein showed strong immune response in adult rabbits. The study however, did not address the issue of its effectiveness in young animals. This factor is important since EV71 mostly infected children younger than 5 year-old. In the present study, we investigated the protectiveefficacy of NPt-VP11-100 protein against EV71 infection in a recently-developed newborn mouse model system. Prior to investigation in the newborn mouse model, we evaluated the type of immune responses developed by adult mice against NPt-VP11-100 protein. In adult mice, the protein induced high levels of anti-VP1 IgG production. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from the immunized mice. They also produced high levels of IFN-γ. Following determination of immune responses towards NPt-VP11-100 protein in adult mice, we performed immunization and virus challenge study in newborn mice model. Since the mice was only susceptible to EV71 infection before they are 14 day-old, only two doses of immunization were carried out. Even though the IgG produced lacked neutralization properties, immunized newborn mice were still partially protected from EV71 viral challenge. They showed high (47.4%) survival rate as compared to the control group and importantly, 50% of them fully recovered from paralysis symptoms at the end of the study. Histological analysis of all the surviving mice revealed a complete clearance of EV71 viral antigens from their brains and spinal cords. In hind limb muscles, the level of antigens detected correlated directly with tissue damage and their paralysis symptoms. We also initiated a similar study in a hamster model which had longer EV71 susceptibility period. In hamster, the NPt-VP11-100 protein was also found to be highly immunogenic. Findings from the study showed that immunization with NPt-VP11-100 protein in newborn mice model confer them a partial protection against EV71 infection. NPt-VP11-100 protein therefore offers a great promise towards finding a vaccine for EV71 infections

Immunogenicity of a truncated enterovirus 71 VP1 protein fused to a Newcastle disease virus nucleocapsid protein fragment in mice.

Enterovirus 71 (EV71) is one of the viruses that cause hand, foot and mouth disease. Its viral capsid protein 1 (VP1), which contains many neutralization epitopes, is an ideal target for vaccine development. Recently, we reported the induction of a strong immune response in rabbits to a truncated VP1 fragment (Nt-VP1t) displayed on a recombinant Newcastle disease virus (NDV) capsid protein. Protective efficacy of this vaccine, however, can only be tested in mice, since all EV71 animal models thus far were developed in mouse systems. In this study, we evaluated the type of immune responses against the protein developed by adult BALB/c mice. Nt-VP1t protein induced high levels of VP1 IgG antibody production in mice. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from these mice. They also produced significant levels of IFN-γ, a Th1-related cytokine. Taken together, Nt-VP1t protein is a potent immunogen in adult mice and our findings provide the data needed for testing of its protective efficacy in mouse models of EV71 infections

The oncolytic activity of Newcastle disease virus in clear cell renal carcinoma cells in normoxic and hypoxic conditions: the interplay between von Hippel-Lindau and interferon-β signaling

Viral-mediated oncolysis is a promising cancer therapeutic approach offering an increased efficacy with less toxicity than the current therapies. The complexity of solid tumor microenvironments includes regions of hypoxia. In these regions, the transcription factor, hypoxia inducible factor (HIF), is active and regulates expression of many genes that contribute to aggressive malignancy, radio-, and chemo-resistance. To investigate the oncolytic efficacy of a highly virulent (velogenic) Newcastle disease virus (NDV) in the presence or absence of HIF-2α, renal cell carcinoma (RCC) cell lines with defective or reconstituted wild-type (wt) von Hippel-Lindau (VHL) activity were used. We show that these RCC cells responded to NDV by producing only interferon (IFN)-β, but not IFN-α, and are associated with increased STAT1 phosphorylation. Restoration of wt VHL expression enhanced NDV-induced IFN-β production, leading to prolonged STAT1 phosphorylation and increased cell death. Hypoxia augmented NDV oncolytic activity regardless of the cells' HIF-2α levels. These results highlight the potential of oncolytic NDV as a potent therapeutic agent in the killing of hypoxic cancer cells

Partial protection against enterovirus 71 (EV71) infection in a mouse model immunized with recombinant newcastle disease virus capsids displaying the EV71 VP1 fragment.

Enterovirus 71 (EV71) infection may cause severe neurological complications, particularly in young children. Despite the risks, there are still no commercially available EV71 vaccines. Hence, a candidate vaccine construct, containing recombinant Newcastle disease virus capsids that display an EV71 VP1 fragment (NPt-VP1 1-100) protein, was evaluated in a mouse model of EV71 infection. Previously, it was shown that this protein construct provoked a strong immune response in vaccinated adult rabbits. That study, however, did not address the issue of its effectiveness against EV71 infection in young animals. In the present study, EV71 viral challenge in vaccinated newborn mice resulted in more than 40% increase in survival rate. Significantly, half of the surviving mice fully recovered from their paralysis. Histological analysis of all of the surviving mice revealed a complete clearance of EV71 viral antigens from their brains and spinal cords. In hind limb muscles, the amounts of the antigens detected correlated with the degrees of tissue damage and paralysis. Findings from this study provide evidence that immunization with the NPt-VP1 1-100 immunogen in a newborn mouse model confers partial protection against EV71 infection, and also highlights the importance of NPt-VP1 1-100 as a possible candidate vaccine for protection against EV71 infections

Human renal carcinoma cells respond to Newcastle disease virus infection through activation of the p38 MAPK/NF-κB/IκBα pathway

Purpose: Newcastle disease virus (NDV) is an oncolytic virus that is known to have a higher preference to cancer cells than to normal cells. It has been proposed that this higher preference may be due to defects in the interferon (IFN) responses of cancer cells. The exact mechanism underlying this process, however, remains to be resolved. In the present study, we examined the antiviral response towards NDV infection of clear cell renal cell carcinoma (ccRCC) cells. ccRCC is associated with mutations of the von Hippel-Lindau tumor suppressor gene VHL, whose protein product is important for eliciting cellular responses to changes in oxygen levels. The most common first line treatment strategy of ccRCC includes IFN. Unfortunately, most ccRCC cases are diagnosed at a late stage and often are resistant to IFN-based therapies. Alternative treatment approaches, including virotherapy using oncolytic viruses, are currently being investigated. The present study was designed to investigate the mechanistic pathways underlying the response of ccRCC cells to oncolytic NDV infection. Methods and results: We found that NDV induces activation of NF-κB in ccRCC cells by inducing phosphorylation and subsequent degradation of IκBα. IκBα was found to be phosphorylated as early as 1 hour post-infection and to result in rapid NF-κB nuclear translocation and activation. Importantly, p38 MAPK phosphorylation was found to occur upstream of the NDV-induced NF-κB activation. Restoration of VHL in ccRCC cells did not result in a reduction of this phosphorylation. A similar phenomenon was also observed in several other cancer-derived cell lines. Conclusion: Our data provide evidence for involvement of the p38 MAPK/NF-κB/IκBα pathway in NDV infection and subsequent induction of apoptosis in ccRCC cells

Mechanisms of oncolytic activity of Newcastle disease virus strain AF2240 in human renal carcinoma cell line

Newcastle disease virus (NDV) is an oncolytic virus that is known to selectively replicate in cancer cells compared to normal cells. It has been proposed that this preference is due to a defect in the cancer cells' interferon (IFN) responses. The exact mechanism underlying this process, however, remains unknown. In the present study, the antiviral response towards NDV infection by clear cell renal cell carcinoma (RCC) cells was examined. The most common first line treatment of RCC is using IFN. Unfortunately, most RCC cases are diagnosed at a late stage and often are resistant to IFN therapies. Alternative treatment approaches, including virotherapy, using oncolytic viruses, are currently being investigated. The present study used proteomic, molecular, immunological and biochemical techniques to investigate the mechanistic pathways that are involved in the response of RCC cells with defective or reconstituted wild type (wt) von Hippel-Lindau (VHL) gene activity to an oncolytic NDV infection. It was observed that NDV induced activation of NF-B in RCC cells by inducing phosphorylation of IB and its subsequent degradation. IB was phosphorylated as early as 1 hour post-infection and resulted in rapid NF-B nuclear translocation and activation. Importantly, p38 MAPK phosphorylation occurred upstream of the NF-B activation. These data provide evidence for the involvement of the p38 MAPK/NF-B/IB pathway in NDV infection and eventual apoptosis of RCC cells. Since the results indicated that there was a possible correlation between the pathway and IFN-β signaling, additional experiments were performed to further understand the IFN-β signalling, specifically STAT pathway, in NDV-infected RCC cells under various microenvironmental factors. The complexity of solid tumor microenvironments includes regions of hypoxia. In these regions, the transcription factor, hypoxia inducible factor (HIF), is active and regulates expression of many genes that contribute to aggressive malignancy, radioand chemo-resistance. To investigate the oncolytic efficacy of a highly virulent (velogenic) Newcastle disease virus (NDV) in the presence or absence of HIF-2α,renal cell carcinoma (RCC) cell lines with defective or reconstituted wild type (wt) von Hippel-Lindau (VHL) gene activity were used. The data showed that these RCC cells responded to NDV by producing only IFN-β, but not IFN-α and are associated with increased STAT1 phosphorylation. Restoration of wt VHL expression enhanced NDV-induced IFN-β production, leading to prolonged STAT1 phosphorylation and increased cell death. Hypoxia augmented NDV oncolytic activity regardless of the cells' HIF-2α levels. In summary, this study demonstrates IFN-β may play important role in NDV oncolysis through activation of p38 MAPK/NF-B/IB and STAT pathways in renal cell carcinoma. Altogether, these findings provide a better mechanistic understanding of NDV-mediated cell death and also highlight the potential of oncolytic local strain of NDV AF2240 as a potent therapeutic agent against normoxic and hypoxic cancer cells, especially renal cell carcinoma