Oct 11, 2020 in Medicine

Alternative Medicine: Influenza viruses

Introduction

Influenza viruses pose a major threat for public health. Globally, influenza results in between 3 to 5 million serious illnesses. From the number of people who become seriously ill, approximately 500,000 die annually (Schwartzman et al., 2015). With time, influenza pandemics have emerged, which can have large global impacts due to the fact that most humans lack immunity against it. The current trend for preventing annual influenza entails developing new vaccines each year against specific circulating virus strains. Most of the vaccines do not protect against an antigenically diverse strain or new pandemic virus. As a result, there is a need for influenza vaccines that can protect against all influenza A viruses (Schwartzman et al., 2015).

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Influenza A viruses are enveloped, negative-sense, single-stranded RNA viruses with segmented genomes. The viruses, in addition to humans, infect large number of warm blooded animal hosts, including more than 100 avian species, many mammalian species with numerous species of wild aquatic birds serving as the major natural reservoirs (Schwartzman et al., 2015). Influenza A viruses provide three types of surface proteins, namely hemagglutinin (HA), neuraminidase (NA), and matrix 2 (M2). There are sixteen HA and nine NA subtypes that are continually found in avian hosts in various combinations, for example H1N1, H3N2 and H10N1. Such wild bird viruses are believed to be the ultimate cause of human pandemic influenza viruses (Schwartzman et al., 2015).

Segmentation of the genome of influenza A virus allows reassortment of the virus and helps generate novel influenza A virus (IAV) of any subtype after mixed infections in any host. It is caused by the fact that HA and NA are encoded on separate gene segments. Swapping of the entire gene by reassortment between viruses of aquatic birds, swine, as well as human population produce new type of A influenza viruses, which is known as genetic shift that can cause devastating pandemics in a world population that is continually becoming immunologically non-resistant to the viruses (Schwartzman et al., 2015). Moreover, IAVs are dynamic RNA viruses that undergo mutation frequently. The mutations change amino acids in the antigenic segments of HA and NA proteins, therefore, the strains can evade population immunity. Even with enhanced surveillance and research on host switch events, it is not easy to predict future pandemic, including when and how a pandemic virus strain can emerge, as well as its subtype or how pathogenic the outbreak will be in humans.

Even though antiviral drugs that treat influenza have been available, vaccination remains the best public health approach to use for its control. Present live attenuated and annual inactivated vaccines are meant to offer protection against IAV that circulates, as well as the strains of influenza virus B. However, they need to be marched closely with circulating strains (Schwartzman et al., 2015). Mismatches occur as a result of rapid antigenic drifts lowering protective efficacy of vaccines. The unpredictable nature of pandemic virus emergence can complicate vaccination strategies. An effective pandemic vaccine should give a wide protection against all the sub-types of IAV.

Efforts to develop vaccines that provide broad protection against IAV subtypes have been ongoing for many years and have included experimental vaccines. However, live vaccines that are based on poxviral vectors, for instance, vaccinia vectors have been considered good alternatives. It is caused by their good safety that has been recorded for a long time. They also induce T cell responses and can be administered intramuscularly or subcutaneously. In the present work, a proposal is presented for the development of a vaccine that will be specific for H10N1 virus. The proposal provides a framework for evaluating immune responses and the possibility of using influenza vaccine based on vaccinia as the vector to express protective antigens of hemagglutinin (H), and neuraminidase (N) against H10N1.

In addition, the study relates both to preparation and use of antigenic composition and recombinant virus that comprises a vaccinia virus vector along with polynucleotides, which encode influenza A genes that will cause the production of immune response, as well as influenza infection. Apparently, the antigenic and recombinant virus can be considered a very useful vaccine that can induce an immune response in a patient against the heterogeneous influenza genes that the virus expresses.

Vaccinia has been used the longest and most extensively in humans. It is considered a relatively safe agent that does not induce vigorous immune responses. Importantly, vaccinia is a vector for temporary expression of proteins. Vaccinia is advantageous due to the wide tropism, considering its varying efficiency. It infects most cell lines that originate from mammals. Furthermore, the large genome of vaccinia permits stable insertion of large fragments of DNA above the range of most of other vectors. This proposal could lead to the development of a countermeasure against H10N1 that have the potential of spreading and causing deaths in humans.

General Hypothesis

Vaccinia can advance influenza virus (H10N1) vaccine development strategy by acting as a vector for polynucleotides that encode influenza A genes, which will cause the production of immune response and influenza infection.

Innovation

The existence of NA is important in creating a complex protective immune response to resist the infecting virus. NA takes part in destroying the cellular receptor for HA by cutting terminal NA residues, which originate from carbohydrate moieties found on the cell surfaces of infected cells. Furthermore, NA cuts neuraminic acid residues originating from the viral proteins, thereby preventing viral aggregations. Apparently, such mechanism has been used to hypothesize proposal that NA will facilitate the release of viral progeny. NA achieves such result when it prevents the particles formed from building up along the cell surface or help in transporting the virus through mucus on the mucosal surface. Administering neuraminidase as a chemical inhibitor limits the severity, as well as spread of the infecting virus. Neuraminidase can fight influenza infection by stopping it from budding from its host cell.

Aims

1. To evaluate a novel vaccination approach using vaccinia as a vector for combating H10N1 infection.

2. To determine that NA supports viral progeny release by stopping the new particles from building up along the cell membrane.

3. To determine the effectiveness of neuraminidase in combating H10N1 by preventing the host from budding.

4. To measure the effectiveness of the vaccine after challenging it with H10N1 virus.

Proposed Plan

The proposed project will test novel vaccination strategies for using vaccinia as a vector for neuraminidase to combat H10N1 infection by preventing budding and building up along the hosts cell mebrane.

We anticipate that vaccinia will successfully act as a vector for NA, which will then prevent the virus from budding. Once the virus fails to bud, it will not be easy to infect other cells.

Statistical Analysis

Chicken embryos groups (n=9) will be analyzed to determine, if statistical significance occurs at a confidence level of 99.95 percent. The survival rates of the mice groups will be determined using the log-rank test, which will be analyzed by the GraphPad Prism Software.

Aims

Aim1: NA facilitates the release of viral progeny by preventing the new particles formed from accumulating along the cell membrane. NA allows the release of progeny virions from the hosts cell surface upon H10N1 infection. NA-specific antibodies will be induced, which will be boosted by vaccination with trivalent influenza vaccines. Anti-NA antibodies can provide intrasubtypic immunity. Nevertheless, such antibodies cannot prevent virus infection, but they hinder the release of newly formed virus particles.

NA-specific antibodies can be elicited through natural infection and also through immunization to provide intraspecific protection. Vaccination using a DNA plasmid that expresses NA provides protection against infection with a virus that has a structure similar to the influenza virus.

Aim2: Evaluating the use of vaccinia as a vector for combating H10N1 infection.Serial passage of non-replicating Modified Vaccinia Ankara (MVA) in primary and secondary chicken embryo fibroblast cultures is not expected to cause deletions in the viral genome, virulence or immune evasion factors (Altenburg et al., 2014). Non-replicating MVA is expected to allow unimpaired synthesis of viral early, intermediate and abundant late gene products, which make it a safe and efficient viral vector. We will ensure that the target gene sequences are transcribed under specific control of poxviral promoters, which are only recognized and activated by virus encoded enzymes and transcription factors. Full clearance is of recombinant virus and recombinant DNA is expected to occur only a few days after vaccine administration (Altenburg et al., 2014).

Aim 3: To determine how effective neuraminidase is in preventing the host virus from budding. HA is expected to change membrane shape, which, together with lipid-raft mediated concentration effects, allows the beginning of budding. Plasmid-based expression of HA in the 293T cells will cause alteration of the membrane curvature. When budding is complete, virus in the form of particles is released (Rossman & Lamb, 2011). There is no clear understanding of how HA takes part in single-protein budding and how strong it is dependent on additional proteins that cause budding when viral infection occurs. HA starts budding of influenza, but the assembly of viral protein blocks HA from completing the process of budding. The budding necessitates the recruitment of other viral components (Rossman & Lamb, 2011).

During the actual research, safety will be paramount. Protective gear will be used, including hood. The control for the experiment will be chicken embryo fibroblast not treated with the vector (Kulkarni, 2013).

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