Invented by Peter Palese, Adolfo Garcia-Sastre, Thomas Muster, Icahn School of Medicine at Mount Sinai
The Icahn School of Medicine at Mount Sinai invention works as followsThe invention also relates to the development and use of IFN-deficient systems for selection of these attenuated virus. The invention also relates the development and the use of IFN deficient systems for selecting such attenuated virus. The mutant viruses are able to replicate in vivo, but have a reduced pathogenicity. They can therefore be used for pharmaceutical formulations and live virus vaccines.
Background for Attenuated Negative Strand Viruses with Alternated Interferon Antagonist Activity for Use as Vaccines and Pharmaceuticals
2.1 The Influenza Virus
The Virus Families with Segmented Genomes (Orthomyxoviridae Bunyaviridae Arenaviridae) are divided into two groups: those that have non-segmented (Paramyxoviridae), and those that have segmented (Rhabdoviridae Filoviridae Borna Disease Virus). Orthomyxoviridae, which is described below and used as an example, contains influenza viruses types A, B, and C, as well the Thogoto, Dhori, and infectious salmonanemia viruses.
The influenza virus consists of a core ribonucleoprotein (a helical Nucleocapsid), which contains the single-stranded genome, and a lipoprotein outer envelope that is lined by a matrix (M1) protein. The segmented influenza A virus genome consists of 8 molecules of negative polarity linear single-stranded RNAs (seven in influenza C), which encode ten different polypeptides. These include: the RNA dependent RNA polymerase (PB2, PB1, and PA) as well as the nucleoproteins (NP), which form the nucleocapsid, the matrix membrane protein (M1, M2), two surface glycoproteins that project from the lipid-containing envelope, hemagglu The nucleus is where transcription and replication take place, and the assembly of the genome occurs by budding at the plasma membrane. “Viruses can reassort their genes when they are infected with other viruses.
Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. After endocytosis, the HA molecule undergoes a conformational shift within the endosome, which promotes membrane fusion and triggers uncoating. The nucleocapsid migrates into the nucleus, where viral mRNA transcription occurs. Viral mRNA transcription is achieved by a unique method in which the viral endonuclease removes the 5?-cap from heterologous cell mRNAs. These mRNAs then act as primers to allow the viral transcriptase to transcribe viral RNA templates. Transcripts are terminated at sites between 15 and 22 bases away from their template ends, where oligo (U) sequences serve as signals to add poly(A). Six of the eight viral molecules are monocistronic and are translated into the viral polymerase protein PB2, PA, PB1, and PB2. The two other transcripts are spliced, resulting in two mRNAs that are translated into different reading frames. These mRNAs produce M1, NEP, M2, and NS1. The eight viral RNA segment code for ten different proteins, nine structural and one notstructural. In Table I, we show a summary of the genes and protein products of the influenza virus.
TABLE 1\nINFLUENZA VIRUS GENOME RNA SEGMENTS\nAND CODING ASSIGNMENTSa\nLengthb Encoded Lengthd Molecules\nSeg- (Nucle- Poly- (Amino Per\nment otides) peptidec Acids) Virion Comments\n1 2341 PB2 759 30-60 RNA\ntranscriptase\ncomponent; host\ncell RNA cap\nbinding\n2 2341 PB1 757 30-60 RNA\ntranscriptase\ncomponent;\ninitiation of\ntranscription\n3 2233 PA 716 30-60 RNA\ntranscriptase\ncomponent\n4 1778 HA 566 500 Hemagglutinin;\ntrimer; envelope\nglycoprotein;\nmediates\nattachment to\ncells\n5 1565 NP 498 1000 Nucleoprotein;\nassociated with\nRNA; structural\ncomponent of RNA\ntranscriptase\n6 1413 NA 454 100 Neuraminidase;\ntetramer;\nenvelope\nglycoprotein\n7 1027 M1 252 3000 Matrix protein;\nlines inside of\nenvelope\nM2 96 ? Structural\nprotein in\nplasma membrane;\nspliced mRNA\n8 890 NS1 230 Nonstructural\nprotein;\nfunction unknown\nNEP 121 ? Nuclear export\nprotein; spliced\nmRNA\naAdapted from R. A. Lamb and P. W. Choppin (1983), Annual Review of Biochemistry, Volume 52, 467-506.\nbFor A/PR/8/34 strain\ncDetermined by biochemical and genetic approaches\ndDetermined by nucleotide sequence analysis and protein sequencing
The influenza A virus genome consists of eight segments of negative-polarity single-stranded DNA, which code for one structural and nine nonstructural proteins. The nonstructural NS1 protein is found in abundance in influenza virus-infected cells but not in virions. The NS1 phosphoprotein is found in the virus’ nucleus during early infection, and in the cytoplasm later in the cycle (King, et. al., 1975, Virol. 64: 378). Studies with temperature-sensitive (ts) influenza mutants carrying lesions in the NS gene suggested that the NS1 protein is a transcriptional and post-transcriptional regulator of mechanisms by which the virus is able to inhibit host cell gene expression and to stimulate viral protein synthesis. The NS1 protein, like many proteins that regulate transcriptional processes and post-transcriptional processes interacts with specific RNA structures and sequences. The NS1 has been shown to bind different RNA species, including: vRNAs, poly-As, U6 snRNAs, and 5? Untranslated region of viral mRNAs, and ds RNA. (Qiu et. al. 1995, RNA1: 304. Qiu et. al. 1994, J. 68: 2425; Hatada Fukuda 1992, J Gen Virol. 73:3325-9. The expression of the NS1 from cDNA has been associated with a number of effects, including: inhibition of nucleocytoplasmic transport, inhibition pre-mRNA splicing and inhibition of host mRNA polyadenylation, as well as stimulation of viral mRNA translation (Fortes, et. al., 1994, EMBO Journal). 13: 704; Enami, et al, 1994, J. Virol. 68: 1432; de la Luna, et al., 1995, J. Virol. 69:2427; Lu, et al., 1994, Genes Dev. 8:1817; Park, et al., 1995, J. Biol. Chem. 270, 28433; Nemeroff et al., 1998, Mol. Cell. 1:1991; Chen, et al., 1994, EMBO J. 18:2273-83).
2.2 Attenuated Viral Infections
Inactivated viruses vaccines are prepared through a process of?killing’ The viral pathogen is killed by formalin or heat treatment. Inactivated vaccinations are of limited use because they don’t provide long-lasting immunity, and therefore offer limited protection. Attenuated virus vaccines are another alternative for the production of virus vaccines. Attenuated virus vaccines are capable of replicating but not pathogenic. They provide longer lasting immunity as well as greater protection. The conventional methods of producing attenuated virus involve the isolation of host range mutations that are often temperature sensitive. For example, the virus may be passed through unnatural hosts and the progeny virus selected for its immunogenicity, but not pathogenicity.
Theoretically, recombinant DNA and genetic engineering techniques would allow for a superior method of producing an attenuated viral strain, since specific mutations can be engineered into its genome. The genetic changes required to attenuate viruses are unknown or unpredictable. In general, attempts to use recombinant technology to engineer viral vaccinations have mainly been directed towards the production of subunits vaccines, which contain only the subunits of pathogens involved in the immunological response expressed in recombinant virus vectors such vaccinia or baculovirus. Recombinant DNA technology has been used to create deletion mutants of herpes viruses or polioviruses that mimic naturally attenuated viruses or host range mutants. Negative strand RNA virus manipulation was not possible until 1990.
The attenuated influenza virus produced so far may not suppress the interferon reaction in the host where they are replicated. These viruses, while beneficial as they are not pathogenic and are immunogenic, are difficult to propagate on conventional substrates in order to make vaccines. Attenuated virus may have virulence traits that are so mild, they do not allow for the host to mount a sufficient immune response to face subsequent challenges.
The present invention is a novel method of using attenuated negative-strand RNA virus formulations that have a reduced ability to inhibit the IFN response in cells, as well as the use such viruses for vaccines and pharmaceutical formulations. Mutant viruses with impaired IFN antagonist activities are attenuated. They are infectious, they can replicate in vivo and provide subclinical infection levels, but are not pathogenic. They are therefore ideal candidates for vaccines that contain live viruses. The attenuated virus can also induce a strong IFN response in vivo that has many biological effects, including protection from subsequent infections and/or antitumor responses. The attenuated virus can be used in pharmaceuticals for the treatment or prevention of other infectious diseases.
The invention includes both segmented viruses and non-segmented ones. Preferred embodiments include influenza virus, respiratory virus, Newcastle disease virus, vesicular somatitis virus and parainfluenza viruses. The invention can use naturally occurring viruses, mutants or strains. They may also be mutagenized (e.g. by repeated passages, exposure to mutagens and/or passage on non-permissive host), reassortants, genetically engineered (e.g. The reverse genetics technique can be used to create the desired phenotype. The reverse genetics technique is used to create a virus with the desired phenotype, i.e. an impaired ability of the cell IFN response. Selecting mutants or genetically modified viruses can be based on their differential growth in IFN-deficient systems and IFN-competent systems. “For example, viruses that grow in IFN-deficient systems but not IFN-competent systems (or grow less well in IFN-competent system) can also be selected.
The attenuated viruses can be used directly as active ingredients in pharmaceutical or vaccine formulations. The attenuated viruses can also be used as a vector or “backbone” for recombinantly produced vaccines. Recombinantly made vaccines can be produced using the attenuated virus as a vector or?backbone? In order to achieve this,’reverse genetics’ can be used. In order to achieve this, the “reverse genetics” technique can be applied in order to introduce mutations or foreign epitopes within the virus that has been attenuated. This strain would then serve as the “parental” strain. strain. Vaccines can then be developed to immunize against different strain variants or, alternatively, completely different infectious agents, disease antigens or diseases. Attenuated viruses can be engineered so that they express neutralizing epitopes from other strains. The attenuated mutant can also be engineered to express epitopes from other viruses than negative-strand RNA virus (e.g. gp120 or gp41 HIV). In addition, the virus can be modified to contain epitopes from non-viral pathogens such as bacteria, parasites and fungi. Another alternative is to prepare cancer vaccines, such as e.g. By engineering tumor antigens in the attenuated virus backbone.
In a specific embodiment, using RNA viruses with segmented genes, reassortment can be used to transfer an attenuated genotype from a segmented RNA viral strain (a mutant or mutagenized strain, or even a genetically modified virus) to another virus strain (a mutant or mutagenized strain, or even a genetically-engineered strain).
The attenuated virus, which induces robust IFN responses, can also be used as a pharmaceutical formulation for the prevention or treatment of viral infections or IFN-treatable disease, such cancer. The tropism can be changed to direct the virus towards a specific organ, tissue, or cell in vivo, or ex vivo. This approach allows the IFN response to be induced at the site of the target, thereby minimizing or avoiding the side effects associated with systemic IFN treatment. In order to achieve this, attenuated viruses can be engineered so that they express a ligand specifically for a receptor on the organ, tissue, or cell of interest.
The invention is based in part on the Applicants discovery that NS1 from wild type influenza virus acts as an IFN antagonist. NS1 inhibits IFN mediated responses of virus-infected cells. The mutant viruses deficient in NS1 were potent inducers for the cellular IFN responses and showed an attenuated phenotype when tested in vivo. The mutant viruses are able to replicate in vivo but with reduced pathogenicity. The attenuated characteristics of the virus of the invention, while not intended to be bound by any theory or explanation of how the invention functions, are presumably due their ability induce a robust IFN cellular response and their impaired capacity to antagonize host IFN. The beneficial characteristics of attenuated virus of the invention are not solely due to their effects on cellular interferon responses. In fact, alterations to other activities associated with NS1 could contribute to the desired attenuated phenotype.
The mutant influenza virus with impaired IFN antagonist activities were shown to reproduce in vivo, generating titers sufficient to induce immune and cytokine response. For instance, vaccination of animals with attenuated virus decreased viral titer when they were then challenged with wild type influenza virus. Attenuated influenza virus also showed antiviral activity and antitumor properties. The attenuated virus prevented the replication of wild-type influenza viruses and superinfected eggs with other viruses, such as Sendai. Injecting animals with tumor cells and then inoculating them with attenuated influenza virus reduced the number foci. The attenuated influenza virus, which is known to trigger a CTL response (cytotoxic lymphocytes), is an attractive candidate for cancer vaccinations.
The Applicants have demonstrated that NS1 C-terminal-truncation mutants replicate to high titers in IFN deficient substrates such as 6 and 7 day old embryonated chicken eggs, as well as the allantoic membrane of 10-day-old embryonated chicken eggs. In particular, the Applicants have demonstrated that an NS1 C-terminal-truncation mutant replicates to high titers in IFN deficient substrates, such as 6 and 7-day-old embryonated chicken eggs, as well as in the allantoic membrane of 10-day-old embryonated chicken eggs, the conventional substrate for influenza virus that does not permit the growth of influenza virus mutants in which the entire NS1 gene is deleted (also referred to herein as ?knockout? mutants). NS1C mutants are less likely to replicate in embryonated egg cells older than 12 days. This method allows for the first generation and identification live negative stranded RNA viruses with altered but not eliminated IFN antagonist activity. These viruses can also grow on substrates that are suitable for vaccine preparation. This approach allows, for the first, an efficient selection system for viruses that contain mutations which confer altered but not abolished interferon antagonist activities.
The invention also relates the use of IFN-deficient systems for propagating attenuated virus strains that cannot be grown using conventional systems used in vaccine production. The term “IFN-deficient system” is used. As used herein, the term “IFN-deficient systems” refers to systems such as cells, celllines, animals (such as mice, turkeys and chickens), and animal lines, that do not produce IFN at all or only produce low amounts of IFN. They may also not respond to IFN at all or respond to IFN less efficiently, or have a deficiency in antiviral gene activity induced by IFN. In order to achieve this, Applicants have designed or identified a variety of IFN deficient systems which can be used. These include but are not limited, young embryonated egg, IFN deficient cell lines, such as VERO cell or genetically modified cell lines, such as STAT1 Knockouts. Alternatives include pre-treating embryonated cells or eggs with compounds that inhibit IFN (such as drugs, antibodies and antisense). Another embodiment is to use eggs that are deficient in IFNs. For example, eggs from STAT1-negative birds, such as fowl (including but not limited transgenic chickens and ducks), or cell lines.
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