Patent Application
20180000925
The present disclosure relates generally to the fields of molecular biology and virology. More particularly, it concerns methods and compositions to treat inflammatory conditions, in particular those resulting from pathologic T-cell activation. It also relates to improved vaccine formulations and gene therapy methods.
Hepatitis C virus (HCV) infects more than 120 million people worldwide and is a leading cause of liver disease (Lavanchy 2009 and Rehermann 2009). Spontaneous clearance of HCV infection occurs in 20% to 40% of infected individuals, and is clearance is associated with sustained CD4+ and CD8+ T cell responses (Shoukry et al., 2003, Grakoui et al., 2003, Thimme et al., 2002, Cox et al., 2005, Thimme et al., 2001 and Lauer et al., 2004). Nevertheless, approximately 70% of infected individuals develop chronic viremia that can lead to cirrhosis and hepatocellular carcinoma (Rehermann 2009 and Li and Lemon 2013). One of the hallmarks of chronic HCV infection is an impaired HCV-specific T cell response and delayed onset of HCV-specific humoral and cellular immune responses (Cox et al., 2005, Netski et al., 2005, Chen et al., 1999, Park et al., 2013, Bowen and Walker 2005, Wedemeyer et al., 2002 and Gruener et al., 2001). HCV-specific intrahepatic T cells are associated with a low viral load and are functionally impaired during chronic HCV infection (Spangenberg et al., 2005 and Freeman et al., 2003). In addition, HCV infection is associated with reduced T cell activation (Rehermann 2009, Park et al., 2013 and Rehermann 2007), although the underlying mechanisms for this effect are not clear.
One important component for an effective adaptive immune response is the activation of CD4+ and CD8+ T cells (Medzhitov and Janeway 1998). Numerous factors appear to contribute to the blunted T cell response during HCV infection, including persistent and high antigenic viral load leading to expression of co-inhibitory receptors that result in T cell exhaustion (Radziewicz 2007 and Urbani et al., 2008), expansion of regulatory T cells, the selection of immune escape mutants (Cox et al., 2005, Netski et al., 2005 and Soderholm and Sallberg 2006), and the expression of immune-suppressive cytokines (Terilli and Cox, 2013; Liang, 2013).
HCV replicates primarily in hepatocytes and infection leads to a high plasma viral load (median value approximately 3.5×105 genome copies/mL) (Schijman et al., 2004 and Matthewsw-Greer et al., 2001). Infected hepatocytes secrete viral particles and extracellular vesicles containing viral RNA and E2 protein (Ramakrishnaiah et al., 2013, Cosset and Dreux 2014 and Masciopinto et al., 2004) which are capable of interacting with and modulating immune cell function (Dreux et al., 2012, Serti et al., 2011 and Tu et al., 2013). In addition, viral RNA is found in T and B lymphocytes of infected individuals (Schmidt et al., 1997, Wang et al., 1992, Fornasieri et al., 2000, Zignego et al., 2007), and the high concentration of HCV RNA containing particles in plasma result in abundant interactions between HCV RNA and proteins with lymphocytes.
It has been reported that the human Pegivirus (HPgV; formally called hepatitis G virus/GB virus C) is associated with a reduction in activation and proliferation of T cells in vivo (Bhattarai et al., 2012, Stapleton et al., 2011, Berzsenyi et al., 2011, Stapleton et al., 2009, Maidana-Giret et al., 2009, Nattermann et al., 2003 and Schwarze-Zander et al., 2010). HPgV particles inhibit T cell activation in vitro by inhibiting lymphocyte-specific tyrosine kinase (Lck), one important and proximal component of the T cell receptor (TCR) signaling pathway (Bhattarai et al., 2013, Bhattarai and Stapleton 2012 and Bhattrarai et al., 2012). A conserved motif within GBV-C envelope protein (E2) that is a predicted Lck substrate site is sufficient for inhibition, and mutation of the conserved tyrosine to alanine reverse the inhibition of T cell activation (Bhattarai et al., 2013, Bhattarai and Stapleton 2012 and Bhattrarai et al., 2012). Since HPgV is a closely related human virus to HCV, the HCV E2 protein sequence was examined and conserved motifs predicted to be Lck substrates were found.
HIV infection is associated with chronic immunoactivation that contributes to HIV mediated immune dysfunction, and immune activation facilitates HIV replication and pathogenesis (Grossman et al., 2006; Hazenberg et al., 2003). Although combination antiretroviral therapy (cART) suppresses HIV plasma viral load (VL), the level of immune activation markers generally do not return to levels observed in HIV-uninfected individuals (Hunt et al., 2008; Vinikoor et al., 2013). In addition, persistent immune activation observed in HIV-treated individuals has been reported to be associated with a reduced response to HIV therapy (Deeks et al., 2004; Hunt et al., 2003). Among HIV-infected subjects, HPgV coinfection is associated reduced immune activation independent of HIV VL or cART (Bhattarai et al., 2012b; Maidana-Giret et al., 2009; Stapleton et al., 2012), suggesting that GBV-C infection alters immune activation pathways. Since HPgV replication in vitro is reduced by T cell activation (Rydze et al., 2012), the development of mechanisms to inhibit immune activation is beneficial for the virus. Understanding mechanisms by which HPgV reduces chronic immune activation in HIV-infected subjects can provide approaches to treat HIV infection and HIV associated chronic immune activation. Indeed, by interfering with T cell activation pathways, many viruses increase the likelihood that it will cause persistent infection. Furthermore, by interfering with antigen presentation this impairs the ability to elicit memory T and B cell responses or high titers of antibodies.
Thus, in accordance with the present disclosure, there is provided a method of inhibiting immune cell activation comprising administering to a mammalian subject in need thereof a viral RNA segment comprising a T cell immune-inhibitory domain. The viral RNA segment may comprise about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 75, 100, 150, 175, 200, 225, 250, 300, 400 or 500 consecutive bases of the T cell immune-inhibitory domain. The viral RNA segment may encode HCV E2 sequences, GBV-C E2 sequences, YFV envelope protein or HIV gp41 or gp120/160 sequences. The viral RNA segment may further encode non-HCV E2 sequences, non-GBV-C E2 sequences, non-YFV env sequences or non-HIV gp41 sequences. The T cell may be a helper T cell suppressor T cell, or a killer T cell. The subject may be a human or a non-human mammal. The immune cell activation may in particular be T cell activation or T cell receptor signaling.
Administering may comprise intravenous, intra-arterial, oral, subcutaneous, topical or intraperitoneal administration. The method may further comprise administering a second anti-inflammatory agent, such as a steroid or a COX-2 inhibitor. The second anti-inflammatory agent may be contacted prior to or after said viral RNA segment, or at the same time as said viral RNA segment. The viral RNA segment may be provided in combination with gene therapy. The viral RNA segment may comprise at least one non-natural base. The viral RNA segment may comprise a Dicer substrate.
The viral RNA segment may be administered at 0.1-500 mg/kg/d. The viral RNA segment may be administered daily or weekly, such as daily for 7 days, 2 weeks, 3 weeks, 4 weeks, one month, 6 weeks, 8 weeks, two months, 12 weeks, or 3 months, or weekly for 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks. The viral RNA segment may be derived from Human Immunodeficiency Virus envelope gp41 or gp120/160, Yellow Fever Virus envelope protein, Bovine Viral Diarrhea Virus envelope protein, Classical Swine Fever Virus envelope protein, influenza envelope protein, Dengue Virus envelope protein, West Nile Virus envelope protein, and Japanese Encephalitis Virus envelope protein.
In another embodiment, there is provided a composition comprising a viral RNA segment comprising a T cell immune-inhibitory domain, formulated with a pharmaceutically acceptable carrier buffer or diluent. The viral RNA segment may comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, 100 consecutive bases of the native viral genome from which it is derived. The viral RNA segment may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400 or 500 bases in length. The viral RNA segment may be fused to a non-viral sequence. The composition may be formulated for pharmaceutical administration, such as topical, cutaneous, subcutaneous, alimentrary or parenteral administration.
In yet another embodiment, there is provided a method of inducing an immune response in a mammalian subject comprising administering to said subject a viral RNA segment wherein said viral RNA segment comprises one or more modified T cell immune-inhibitory domains. The modified site may comprise Dicer substrate. The RNA virus may be from the Reoviridae, Atroviridae, Caliciviridae, Hepeviridae, Picornaviridae, Togaviridae, Flaviviridae, Coronaviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Paramyxoviridae, Filoviridae, Rabdoviridae, or Retroviridae family. The RNA virus may be GBV-C, Hepatitis C Virus, Human Immunodeficiency Virus, influenza virus, Dengue Virus, West Nile Virus, Japanese Encephalitis Virus, Bovine Viral Diarrhea Virus, Classical Swine Fever Virus or Yellow Fever Virus. The viral RNA segment may be free from other viral sequences.
The viral RNA segment may be delivered via expression from an expression vector, such as a viral expression vector. The viral RNA segment may be comprised in lipid vehicle or nanoparticle. The viral RNA segment may be administered with a second viral RNA segment from a distinct serotype or strain of said virus. The viral RNA segment may be administered more than once. The viral RNA segment may be formulated with an adjuvant. The viral RNA segment may comprise a modification to an immunomodulatory domain in a viral glycoprotein. The viral RNA segment may be from a HCV E2-coding region, and HIV gp41- or gp120/160-coding region or a GBV-C E2-coding region. The site comprises a dicer substrate. The subject may be a human subject or a non-human animal subject.
Also provided is a vaccine comprising a viral RNA segment having a modification in a T cell immune-inhibitory domain. The modification may comprise a deleted segment or a mutated segment. The viral RNA segment may comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, or 100 consecutive residues of the native viral genome from which it is derived. The viral RNA segment may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, 100, 125, 150, 175, 200, 219 or 250 bases in length. The viral RNA segment may be fused to a non-viral sequence. The vaccine may be formulated with an adjuvant. The viral RNA segment may be from an HCV E2-, YFV Env, HIV 41-, HIV pg120/160 or GBV-C E2-coding region.
Another embodiment comprises a method of performing gene transfer into a subject comprising administering to said subject an expression cassette comprising a heterologous gene segment under the control of a promoter operable in cells of said subject, wherein said expression cassette further comprises a viral RNA segment comprising a T cell immune-inhibitory domain. The viral RNA segment may comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, 100 consecutive bases of the native viral genome from which it is derived. The viral RNA segment may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400 or 500 bases in length. The viral RNA segment may be fused to a non-viral sequence. The composition may be formulated for pharmaceutical administration, such as topical, cutaneous, subcutaneous, alimentrary or parenteral administration.
In yet another embodiment, there is provided a method of inducing an immune response in a mammalian subject comprising administering to said subject a viral RNA segment wherein said viral RNA segment comprises one or more modified T cell immune-inhibitory domains. The modified site may comprise Dicer substrate. The RNA virus may be from the Reoviridae, Atroviridae, Caliciviridae, Hepeviridae, Picornaviridae, Togaviridae, Flaviviridae, Coronaviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Paramyxoviridae, Filoviridae, Rabdoviridae, or Retroviridae family. The RNA virus may be GBV-C, Hepatitis C Virus, Human Immunodeficiency Virus, influenza virus, Dengue Virus, West Nile Virus, Japanese Encephalitis Virus, Bovine Viral Diarrhea Virus, Classical Swine Fever Virus or Yellow Fever Virus. The viral RNA segment may be free from other viral sequences. The viral RNA segment may comprise a Dicer substrate.
In yet another embodiment, the target of the HCV RNA has been identified as a novel T cell immune modulator. This target is a phosphatase (protein tyrosine phosphatase receptor type E or epsilon, abbreviated PTPRE). PTPRE is reduced by both HCV RNA envelope E2 and YFV envelope RNA, and complete knockdown of this gene renders the cells non-viable. However, HCV and YFV RNA does not have complete complementarity, and thus merely reduces PTPRE protein expression, which in turn reduces T cell activation and functional responses. Since this partial T cell inhibition does not lead to profound immune deficiency, but does impair T cell responses, PTPRE is identified as a novel target for immune suppressant therapy. Threus, partial inhibition using nucleic acid-based methodologies (miRNA, siRNA, shRNA, antisense, etc.) can be achieved using only routine experimentation, i.e., starting with molecules having complete or high complementarity, and making sequential changes to reduce complementarity, thereby adjusting the level of inhibition such that immune function is blunted, but not completely eliminated (i.e., rendering the cells non-viable). It is expected that in contexts involving inhibitory RNAs that rely on a “seed sequence” for recognition, the seed sequence will remain completely or highly complementary, with other sequences being reduced in complementarity. For example, seed sequence complementarity might be greater than about 80%, while other regions would be 20-60% complementary.
Also provided are pharmaceutical compositions comprising a nucleic acid having the sequence:
The pharmaceutical composition may also comprise a nucleic acid sequence selected from GCATTATCC (SEQ ID NO: 38), GCAUUAUCC (SEQ ID NO: 39), GCATTACCCC (SEQ ID NO: 40), GCAUUACCCC (SEQ ID NO: 41), CUUUACAAAA (SEQ ID NO: 42), or CTTTACAAA (SEQ ID NO: 43), wherein said nucleic acid consists essentially of at least about 20 base pairs and no more than about 55 base pairs. Specific lengths include 22, 27 and 51 nucleotides.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Furthermore, where multiple steps of a method of process are recited, it is understood that the steps are not required to be performed in the particular order recited unless one of skill in the art is not be able to practice the method in a different order.
Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Previously, potential mechanisms by which GBV-C and other RNA viruses reduce immunoactivation was examined. A novel viral mechanism that inhibits T cell receptor (TCR) signaling via competition for the lymphocyte-specific protein tyrosine kinase (Lck) mediated by the persistent human Pegivirus GBV-C envelope glycoprotein E2 was found. Additional data showing that hepatitis C virus (HCV) and yellow fever virus (YFV, 17D strain) similarly inhibit T cell activation, and both the envelope glycoprotein and the envelope coding region of both of these viruses interfere with T cell activation was provided. Highly conserved RNA sequences in both viruses have been identified that are predicted to be processed into a microRNA. These sequences target PTPRE, or Receptor-type tyrosine-protein phosphatase epsilon, an enzyme that in humans is encoded by the PTPRE gene. The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Two alternatively spliced transcript variants of this gene have been reported, one of which encodes a receptor-type PTP that possesses a short extracellular domain, a single transmembrane region, and two tandem intracytoplasmic catalytic domains; another one encodes a PTP that contains a distinct hydrophilic N-terminus, and thus represents a nonreceptor-type isoform of this PTP. Studies of the similar gene in mice suggested the regulatory roles of this PTP in RAS related signal transduction pathways, cytokines induced SATA signaling, as well as the activation of voltage-gated K+ channels.
While the Lck kinase is involved in these studies of GBV-C and HCV, additional T cell inhibitory signaling molecules are also involved for HCV. Furthermore, bioinformatic predictions for other human pathogens are provided showing that they share this immunomodulatory feature, including west Nile virus (WNV), dengue viruses (DENV), Japanese encephalitis virus (JEV), Influenza A and B, and HIV. A major problem with subunit vaccines for many of these viruses is that they are poor immunogens and elicit low levels of antibody and poor memory responses. Thus, it has been posited that by identification of the T cell interacting domains of the envelope proteins, followed by mutation of important amino acids needed to interfere with T and B cell responses, they can generate more potent vaccines with improved longevity of protection.
GBV-C and the related HCV are cytoplasmic human RNA viruses that cause persistent infection. GBV-C modulates global T cell activation as determined by measurement of surface markers upregulated on CD4+ and CD8+ T cells following activation (Nattermann et al., 2003; Maidana et al., 2009; Xiang et al., 2004; Xiang et al., 2006; Schwarze-Zander et al., 2010; Stapleton et al., 2012). The effect is modest, and GBV-C infected humans are not characterized by side effects of immunosuppression (reviewed in Bhattarai & Stapleton, 2012). In contrast, HCV has been reported to be associated with an increased susceptibility to other infections, particularly HBV, bacterial infections, and schistosomiasis (reviewed in Hahn, 2003). Although anti-HCV envelope antibodies can protect chimpanzees from infection (Farci et al., 1996), immune responses to HCV envelope are weak (Fournillier et al., 2001; Cerny and Chisari, 1999). Several reasons for this have been proposed including virion or E2 association with lipids, heavy glycosylation, and marked antigenic variation (Fournillier et al., 2001).
Numerous clinical studies find an association between GBV-C infection and reduced levels of T and B cell activation (Bowen, and Walker, 2005; Lauer and Walker, 2001; Kanto et al., 1999; Krishnada et al., 2010; Kobayashi et al., 1998; Semmo et al., 2005; Eckels et al., 1999; Serti et al., 2011; Doganiuc et al., 2003; Tomova et al., 2009; Masciopinto et al., 2004). Expression of the GBV-C E2 protein in a CD4+ T cell line resulted in a block in IL-2 release, and upregulation of activation markers CD69 and CD25 following stimulation through the T cell receptor (TCR) (Bhattarai et al., 2012b). Furthermore, addition of recombinant E2 to primary human CD4 and CD8 cells blocked these three measures of TCR signaling (Bhattarai et al., 2012b).
While GBV-C replicates in T and B lymphocytes (Xiang et al., 2000; George et al., 2006), a very low proportion of lymphocytes in peripheral blood are infected (on average, <1%). Thus, infection alone is unlikely to cause the global reduction in TCR-mediated activation. It has been found that serum microvesicles obtained from GBV-C-infected people block T cell activation compared to serum microvesicles from GBV-C uninfected. It has further been reported that CD4+ T cell lines expressing E2 protein produce exosomes containing E2 which reduced T cell activation (Bhattarai et al., 2013). Previous reports indicate that HCV produces exosomes, and that E2 is incorporated in these via its interactions with the E2 receptor CD81 (Masciopinto et al., 2004), a common component of exosomes.
It is disclosed herein that HCV particles directly interfere with T cell receptor (TCR) signaling in human T cells, even in absence of viral replication. HCV particles obtained from either serum of infected humans or cell-culture inhibited TCR signaling. The inhibition of TCR signaling is mediated at least in part by the HCV envelope (E2) coding RNA and protein. HCV E2 RNA inhibited proximal TCR signaling by reducing activation of lymphocyte-specific tyrosine kinase (Lck). HCV E2 protein inhibited distal TCR signaling by reducing nuclear translocation of activated NFAT. Conserved nucleotide sequences in the RNA region coding for HCV E2 were involved in proximal TCR signaling inhibition, while inhibition of distal TCR signaling involved Lck mediated phosphorylation of a conserved tyrosine in HCV E2 protein (Y613). Both proximal and distal TCR signaling defects were reversed by mutation of the nucleotides in E2 RNA or Y613 in E2 protein. These data indicate that HCV particles can directly interfere with TCR signaling.
GBV-C envelope glycoprotein contains binding sites and substrate sites that compete with lymphocyte kinases leading to impaired activation. Hepatitis C virus (HCV) and yellow fever virus (YFV) envelopes similarly impair lymphocyte activation. It suggested that this may explain the poor immunogenicity and memory responses to immunization with recombinant envelope proteins. Using these sites as immunosuppressive agents is therefore proposed. Further, by identification and mutation of these immunomodulatory sites, envelope glycoproteins will be more immunogenic and will induce improved memory T and B cell responses.
As such, the disclosure involves two aspects, both stemming from the identification of viral envelope sequences that inhibit T cell activation. These sequences can be used to reduce host immune responses in situations where such is desired, or they can be altered and then used in the context of improved vaccination to prevent, control or limit viral infection.
This will apply for all human and animal RNA viruses including vertebrate dsRNA viruses of the family Reoviridae, and ssRNA viruses of the families Atroviridae, Caliciviridae, HEV, Picornaviridae, Togaviridae, Flaviviridae, Coronaviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Paramyxoviridae, Filoviridae, Rabdoviridae, and Retroviridae.
A. Hepatitis C Virus
HCV primarily replicates in the hepatocyte (Major et al., 1997), but is also found in association with a variety of peripheral blood cells (PBC's) (Major et al., 1997; Schmidt et al., 1997). Although controversial, it appears that HCV replicates to some extent in PBCs, and inefficient in vitro cultivation can be achieved in T- and B-cell lines (Major et al., 1997; Bartenschlager et al., 2000).
The mechanism by which HCV attaches and enters cells has not been clear. Two cellular surface receptors have been shown to interact with HCV or the HCV envelope glycoprotein E2 in vitro, leading to speculation that either may represent the HCV cellular receptor (Pileri et al., 1998; Monazahian et al., 1999; Agnello et al., 1999; Flint et al., 1999; Wuenschmann et al., 2000). It has been reported that recombinant HCV E2 binds to human CD81 (Pileri et al., 1998; Flint et al., 1999; Flint and Maidens et al., 1999; Hadlock et al., 2000; Owsianka et al., 2001; Flint and McKeating, 2000; Petracca et al., 2000; Patel et al., 2000). CD81 is a member of the tetraspanin superfamily of cell surface molecules, and is expressed on virtually all nucleated cells (Levy and Maecker, 1998). Initial studies suggested that E2 binding to CD81 may be responsible for the binding of HCV to target cells in vivo. However, although E2 has repeatedly been reported to bind CD81, only two studies presented evidence that HCV particles derived from human serum bind to this surface molecule (Pileri et al., 1998; Hadlock et al., 2000).
It has been reported that, although HCV E2 binds specifically to CD81 (Wuenschmann et al., 2000), the binding of HCV particles purified from plasma was not inhibited by soluble CD81, and the extent of virus binding correlated with the level of LDLr expression (Wuenschmann et al., 2000). Additional lines of evidence argue that CD81 is not the HCV receptor. HCV E2 has a higher affinity for marmoset CD81 than human CD81, yet marmosets are not susceptible to HCV. The affinity for HCV E2 to CD81 was found to be significantly lower than predicted for a true viral receptor (Petracca et al., 2000). Using an RT-PCR based detection method, plasma-derived HCV and HCV E2 bound to U937 subcloned cells that lack expression of CD81 (Hamaia and Allain, 2001). These data suggest that CD81 is not the primary cell receptor for HCV.
Nevertheless, HCV E2 does interact with CD81, and the E2 regions involved in CD81 binding are highly conserved (Pileri et al., 1998; Flint et al., 1999; Flint and Maidens et al., 1999; Hadlock et al., 2000; Owsianka et al., 2001; Flint and McKeating, 2000; Petracca et al., 2000; Patel et al., 2000)), suggesting a functional role for CD81-E2 interactions in HCV replication (Pileri et al., 1998; Flint et al., 1999; Flint and Maidens et al., 1999; Hadlock et al., 2000; Owsianka et al., 2001; Flint and McKeating, 2000). The extremely low density of HCV found in gradient centrifugation of infectious serum suggested an association with VLDL and LDL (Hijikata et al., 1993; Bradley et al., 1991; Prince et al., 1996). Infectious virus was found at the same densities as VLDL and LDL and coprecipitated with LDL (Monazahian et al., 1999; Bradley et al., 1991; Prince et al., 1996; Thomssen and Thiele, 1993; Xiang et al., 1998). Subsequent studies (Monazahian et al., 1999; Bradley et al., 1991; Prince et al. 1996; Xiang et al., 1998) reported an interaction between HCV or HCV-LDL complexes with the low density lipoprotein receptor (LDLr) (Wuenschmann et al., 2000; Prince et al., 1996; Thomssen and Thiele, 1993; Xiang et al., 1998; Thomssen et al., 1992).
HCV present in the plasma of infected people has also been reported to interact with very-low-density (VLDL) and low-density lipoproteins (LDL). The liver synthesizes VLDL which consists of triaglycerols, cholesterol, phospholipids and the apoprotein apoB-100, VLDL's released into the blood, where it acquires additional lipoproteins CII and apoE from high-density lipoproteins (HDL). VLDL is digested by Lipoprotein Lipase (LPL), an enzyme found attached to capillary endothelial cells, to form intermediate density lipoproteins (IDL) and LDL, and apoB-100 is the only remaining apoprotein in LDL. The low-density lipoprotein receptor (LDLr) recognizes both apoE and apoB-100 and can therefore bind VLDL, IDL and chylomicron remnants in addition to LDL. (Marks et al., 1996).
HCV-RNA containing material in serum, presumably virus particles, separate into very low density particles (<1.06 g/cm3) by gradient sedimentation, suggesting that HCV associates with VLDL and LDL (Monazahian et al., 1999; Thomssen et al., 1993; Xiang et al., 1998; Prince et al., 1996; Bradley et al., 1991). In addition, particles with densities of 1.11-1.18 g/cm3 have been described (Xiang et al., 1998; Prince et al., 1996; Bradley et al., 1991; Hijikata et al., 1993). Chimpanzee infectivity studies reported that the very low density HCV particles were highly infectious, whereas the particles of higher density were not infectious (Bradley, 2000). HCV and GBV-C have different particle types, and that the functional effects of E2 protein and HCV RNA may vary between particle types (Monazahian et al., 1999; Xiang et al., 1998; Prince et al., 1996; Bradley et al., 1991). Thomssen et al. (1993) showed that HCV coprecipitated with LDL and demonstrated an interaction of HCV or HCV-LDL complexes with the LDLr (Wuenschmann et al., 2000; Thomssen et al., 1993; Xiang et al., 1998; Prince et al., 1996; Thomssen et al., 1992).
Monazahian et al. (1999) reported that expression of recombinant human LDLr in murine cells lacking human CD81 confirmed binding of HCV to these cells, and Agnello et al. (1999) reported that HCV bound to and entered fibroblasts containing LDLr, but not LDLr deficient fibroblasts, using an in situ hybridization method. Using flow cytometry, it was reported that plasma-derived HCV bound to cells expressing LDLr, but not to cells lacking the LDLr (Wuenschmann et al., 2000). No interactions between viral envelope proteins (E1 or E2) and the LDL receptor have been reported (Wuenschmann et al., 2000). Monazahian et al. (1999) found that in vitro translated HCV E1 and E2 proteins, labeled with 35S-methionine co-precipitated with VLDL, LDL and HDL (Monazahian et al., 2000).
HCV E2 is the outer protein of the viral envelope and may participate in the binding of viruses to the target cells. The protein starts at amino acid 394 of the HCV polyprotein, and extends to amino acid 747. It has a hypervariable region at the amino terminus of the protein, and the carboxy terminus includes a transmembrane domain.
Due to the deficiencies in the prior art, there remains a need for more effective treatments to lower LDL levels in a subject. There also remains a need for new and useful methods of reducing, controlling or preventing HCV infection in a subject. The presently claimed disclosure overcomes the deficiencies in the prior art by disclosing new and useful methods for reducing LDL levels in a subject. The present disclosure also discloses new and useful methods of identifying HCV inhibitors and methods of treating HCV infection.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. Hepatitis C Virus (HCV) HCV is an enveloped virus containing a positive-sense single-stranded RNA genome of approximately 9.5 kb. The genomic sequence of HCV is approximately 9401 base pairs in length (SEQ ID NO: 1). The peptide sequence for HCV can be obtained from Genbank Accession No. M62321. The viral genome consists of a lengthy 5′ untranslated region (UTR), a long open reading frame encoding a polyprotein precursor of approximately 3011 amino acids (SEQ ID NO: 2) and a short 3′ UTR. The 5′ UTR is a highly conserved part of the HCV genome and is important for the initiation and control of polyprotein translation. Translation of the HCV genome is initiated by a cap-independent mechanism known as internal ribosome entry. This mechanism involves the binding of ribosomes to an RNA sequence known as the internal ribosome entry site (IRES). The polyprotein precursor is cleaved by both host and viral proteases to yield mature viral structural and non-structural proteins. Viral structural proteins include a nucleocapsid core protein and two envelope glycoproteins, E1 and E2 (U.S. Pat. No. 6,326,151).
HCV utilizes the low density lipoprotein receptor (LDLr) for cell binding and entry (Wuenschmann et al., 2000; Monazahian et al., 1999; Agello et al., 1999). It has previously been indicated that the HCV envelope glycoprotein (HCV E2 glycoprotein) binds to the lipid moiety of human lipoproteins, and the lipid-virus complex uses the natural receptor for LDL to bind to cells. The HCV E2 glycoprotein starts at amino acid 394 of the HCV polyprotein, and extends to amino acid 747. It has a hypervariable region at the amino terminus of the protein, and the carboxy terminus includes a transmembrane domain. HCV enters the cell via endocytosis using the LDL receptor. HCV E2 glycoprotein interactions with LDL result not only in CD81-independent binding to cells (Wuenschmann et al., 2000), but also to enhancement in LDL binding and uptake by the cells.
B. Other Viruses
1. Yellow Fever Virus
Yellow fever is caused by the yellow fever virus, a 40 to 50 nm wide enveloped RNA virus belonging to the family Flaviviridae. The positive sense single-stranded RNA is approximately 11,000 nucleotides long and has a single open reading frame encoding a polyprotein. Host proteases cut this polyprotein into three structural (C, prM, E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5); the enumeration corresponds to the decreased pH induces the fusion of the endosomal membrane with the virus envelope. Thus, the capsid reaches the cytosol, decays and releases the genome. Receptor binding as well as membrane fusion are catalyzed by the protein E, which changes its conformation at low pH, which causes a rearrangement of the 90 homodimers to 60 homotrimers.
After entering the host cells, the viral genome is replicated in the rough endoplasmic reticulum (ER) and in the so-called vesicle packets. At first, an immature form of the virus particle is produced inside the ER, whose M-protein is not yet cleaved to its mature form and is therefore denoted as prM (precursor M) and forms a complex with protein E. The immature particles are processed in the Golgi apparatus by the host protein furin, which cleaves prM to M. This releases E from the complex which can now take its place in the mature, infectious virion.
The yellow fever virus is mainly transmitted through the bite of the yellow fever mosquito Aedes aegypti, but other mosquitoes such as the “tiger mosquito” (Aedes albopictus) can also serve as a vector for the virus. Like other Arboviruses which are transmitted via mosquitoes, the yellow fever virus is taken up by a female mosquito which sucks the blood of an infected person or primate. Viruses reach the stomach of the mosquito, and if the virus concentration is high enough, the virions can infect epithelial cells and replicate there. From there they reach the haemocoel (the blood system of mosquitoes) and from there the salivary glands. When the mosquito next sucks blood, it injects its saliva into the wound, and thus the virus reaches the blood of the bitten person. There are also indications for transovarial and transstadial transmission of the yellow fever virus within A. aegypti, i.e., the transmission from a female mosquito to her eggs and then larvae. This infection of vectors without a previous blood meal seems to play a role in single, sudden breakouts of the disease.
2. HIV
HIV is a member of the genus Lentivirus, part of the family of Retroviridae. Lentiviruses have many morphologies and biological properties in common. Many species are infected by lentiviruses, which are characteristically responsible for long-duration illnesses with a long incubation period. Lentiviruses are transmitted as single-stranded, positive-sense, enveloped RNA viruses. Upon entry into the target cell, the viral RNA genome is converted (reverse transcribed) into double-stranded DNA by a virally encoded reverse transcriptase that is transported along with the viral genome in the virus particle. The resulting viral DNA is then imported into the cell nucleus and integrated into the cellular DNA by a virally encoded integrase and host co-factors. Once integrated, the virus may become latent, allowing the virus and its host cell to avoid detection by the immune system. Alternatively, the virus may be transcribed, producing new RNA genomes and viral proteins that are packaged and released from the cell as new virus particles that begin the replication cycle anew.
HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms including: apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells. When CD4+ T cell numbers decline below a certain level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections.
Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is the virus that was initially discovered and termed both LAV and HTLV-III. It is more virulent, more infective, and is the cause of the majority of HIV infections globally. The lower infectivity of HIV-2 compared to HIV-1 implies that fewer of those exposed to HIV-2 will be infected per exposure. Because of its relatively poor capacity for transmission, HIV-2 is largely confined to West Africa.
HIV is different in structure from other retroviruses. It is roughly spherical with a diameter of about 120 nm, around 60 times smaller than a red blood cell, yet large for a virus. It is composed of two copies of positive single-stranded RNA that codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24. The single-stranded RNA is tightly bound to nucleocapsid proteins, p7, and enzymes needed for the development of the virion such as reverse transcriptase, proteases, ribonuclease and integrase. A matrix composed of the viral protein p17 surrounds the capsid ensuring the integrity of the virion particle.
This is, in turn, surrounded by the viral envelope that is composed of two layers of fatty molecules called phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell. Embedded in the viral envelope are proteins from the host cell and about 70 copies of a complex HIV protein that protrudes through the surface of the virus particle. This protein, known as Env, consists of a cap made of three molecules called glycoprotein (gp) 120, and a stem consisting of three gp41 molecules that anchor the structure into the viral envelope. This glycoprotein complex enables the virus to attach to and fuse with target cells to initiate the infectious cycle. Both these surface proteins, especially gp120, have been considered as targets of future treatments or vaccines against HIV.
The RNA genome consists of at least seven structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS), and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev), encoding 19 proteins. Three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles. For example, env codes for a protein called gp160 that is broken down by a cellular protease to form gp120 and gp41. The six remaining genes, tat, rev, nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatory genes for proteins that control the ability of HIV to infect cells, produce new copies of virus (replicate), or cause disease.
The two Tat proteins (p16 and p14) are transcriptional trans activators for the LTR promoter acting by binding the TAR RNA element. The TAR may also be processed into microRNAs that regulate the apoptosis genes ERCC1 and IER3. The Rev protein (p19) is involved in shuttling RNAs from the nucleus and the cytoplasm by binding to the RRE RNA element. The Vif protein (p23) suppresses the action of APOBEC3G (a cell protein that deaminates DNA:RNA hybrids and/or interferes with the Pol protein). The Vpr protein (p14) arrests cell division at G2/M. The Nef protein (p27) down-regulates CD4 (the major viral receptor), as well as the MHC class I and class II molecules.
Nef also interacts with SH3 domains. The Vpu protein (p16) influences the release of new virus particles from infected cells. The ends of each strand of HIV RNA contain an RNA sequence called the long terminal repeat (LTR). Regions in the LTR act as switches to control production of new viruses and can be triggered by proteins from either HIV or the host cell. The Psi element is involved in viral genome packaging and recognized by Gag and Rev proteins. The SLIP element (TTTTTT) is involved in the frameshift in the Gag-Pol reading frame to make functional Pol.
HIV differs from many viruses in that it has very high genetic variability. This diversity is a result of its fast replication cycle, with the generation of about 1010 virions every day, coupled with a high mutation rate of approximately 3×10−5 per nucleotide base per cycle of replication and recombinogenic properties of reverse transcriptase. This complex scenario leads to the generation of many variants of HIV in a single infected patient in the course of one day. This variability is compounded when a single cell is simultaneously infected by two or more different strains of HIV. When simultaneous infection occurs, the genome of progeny virions may be composed of RNA strands from two different strains. This hybrid virion then infects a new cell where it undergoes replication. As this happens, the reverse transcriptase, by jumping back and forth between the two different RNA templates, will generate a newly synthesized retroviral DNA sequence that is a recombinant between the two parental genomes. This recombination is evident when it occurs between subtypes.
Three groups of HIV-1 have been identified on the basis of differences in the envelope (env) region: M, N, and O. Group M is the major type and is subdivided into eight subtypes (or clades), based on the whole genome, which are geographically distinct. The most prevalent are subtypes B (found mainly in North America and Europe), A and D (found mainly in Africa), and C (found mainly in Africa and Asia); these subtypes form branches in the phylogenetic tree representing the lineage of the M group of HIV-1. Coinfection with distinct subtypes gives rise to circulating recombinant forms (CRFs). In 2000, the last year in which an analysis of global subtype prevalence was made, 47.2% of infections worldwide were of subtype C, 26.7% were of subtype A/CRF02_AG, 12.3% were of subtype B, 5.3% were of subtype D, 3.2% were of CRF_AE, and the remaining 5.3% were composed of other subtypes and CRFs. Most HIV-1 research is focused on subtype B; few laboratories focus on the other subtypes. The existence of a fourth group, “P”, has been hypothesised based on a virus isolated in 2009. The strain is apparently derived from gorilla SIV (SIVgor), first isolated from western lowland gorillas in 2006. The genetic sequence of HIV-2 is only partially homologous to HIV-1 and more closely resembles that of SIVsmm.
3. Influenza
The influenza virus is an RNA virus of the family Orthomyxoviridae, which comprises five genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus and Thogotovirus. The Influenzavirus A genus has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Sometimes viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are significant virulent human pathogens among the three influenza types and cause severe disease. The influenza A virus can be subdivided into different subtypes based on the antibody response to these viruses.
Influenzaviruses A, B and C are very similar in structure. The virus particle is 80-120 nanometers in diameter and usually roughly spherical, although filamentous forms can occur. This particle is made of a viral envelope containing two main types of glycoproteins, wrapped around a central core. The central core contains the viral RNA genome and other viral proteins that package and protect this RNA. Unusually for a virus, its genome is not a single piece of nucleic acid; instead, it contains seven or eight pieces of segmented negative-sense RNA. The Influenza A genome encodes 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2(NEP), PA, PB1, PB1-F2 and PB2.
Hemagglutinin (HA) and neuraminidase (NA) are the two large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins are targets for antiviral drugs. Furthermore, they are antigens to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA. These different types of HA and NA form the basis of the H and N distinctions in, for example, H5N1.
Influenza viruses bind through hemagglutinin onto sialic acid sugars on the surfaces of epithelial cells; typically in the nose, throat and lungs of mammals and intestines of birds. The cell imports the virus by endocytosis. In the acidic endosome, part of the hemagglutinin protein fuses the viral envelope with the vacuole's membrane, releasing the viral RNA (vRNA) molecules, accessory proteins and RNA-dependent RNA polymerase into the cytoplasm. These proteins and vRNA form a complex that is transported into the cell nucleus, where the RNA-dependent RNA polymerase begins transcribing complementary positive-sense vRNA. The vRNA is either (a) exported into the cytoplasm and translated, or (b) remains in the nucleus. Newly-synthesised viral proteins are either secreted through the Golgi apparatus onto the cell surface or transported back into the nucleus to bind vRNA and form new viral genome particles. Other viral proteins have multiple actions in the host cell, including degrading cellular mRNA and using the released nucleotides for vRNA synthesis and also inhibiting translation of host-cell mRNAs.
Negative-sense vRNAs that form the genomes of future viruses, RNA-dependent RNA polymerase, and other viral proteins are assembled into a virion. Hemagglutinin and neuraminidase molecules cluster into a bulge in the cell membrane. The vRNA and viral core proteins leave the nucleus and enter this membrane protrusion. The mature virus buds off from the cell in a sphere of host phospholipid membrane, acquiring hemagglutinin and neuraminidase with this membrane coat. As before, the viruses adhere to the cell through hemagglutinin; the mature viruses detach once their neuraminidase has cleaved sialic acid residues from the host cell. After the release of new influenza viruses, the host cell dies.
Because of the absence of RNA proofreading enzymes, the RNA-dependent RNA polymerase makes a single nucleotide insertion error roughly every 10 thousand nucleotides, which is the approximate length of the influenza vRNA. Hence, the majority of newly-manufactured influenza viruses are mutants, causing “antigenic drift.” The separation of the genome into eight separate segments of vRNA allows mixing or reassortment of vRNAs if more than one viral line has infected a single cell. The resulting rapid change in viral genetics produces antigenic shifts and allows the virus to infect new host species and quickly overcome protective immunity.
4. Other Viruses
The present disclosure contemplates the use of RNA segments deriving from other envelope proteins including West Nile virus, Japanese Encephalitis virus, Dengue virus and Classical Swine Fever virus (CSFV).
In certain aspects, the disclosure is directed to viral RNA segments, such as those encoding an HCV E2 protein or its homolog from other viruses. The provision of RNA segments can be used to modulate immune function. It is contemplated that the compositions and methods disclosed herein may be utilized to express all or part of the RNA segments thereof. In certain embodiments, compositions of the disclosure may include RNAs encoding certain proteins. The RNA segments may comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 30, 35, 40, 45, 50, 51, 75, 100, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450 or 500 consecutive bases of an RNA genome, for example, from an envelope sequence. Determination of which RNA segments possess activity may be achieved using functional assays measuring T-cell activation and proliferation as well as cytokine production, which are familiar to those of skill in the art. An exemplary HCV E2 coding region (from strain H77) is shown below:
An exemplary YFV envelope encoding sequence is shown below:
In particular, a segment from this coding region is believed to be a substrate for the Dicer enzyme, and thus RNA regions having this motif are particularly contemplated. Prediction models identify a number of RNA sequences that should serve as substrates for Dicer. Using the program miR-FIND (bioinfo.51donate.com/microrna/mir-find), a search of the HCV envelope (E2)-coding RNA/YFV env-coding sequences that purportedly inhibit T cell receptor signaling identified several potential Dicer sites, differing in number between different genotypes. Conserved ribonucleotides in the genotype 2a isolate (underlined) in the HCV E2 coding region:
The intervening sequences can vary, as it has been observed that the genotype 3 virus E2 coding region, which has some sequence diversity in this region, also inhibits T cell receptor signaling and T cell activation. It is apparent that the RNA is sufficient to inhibit T cell activation, as including a frameshift insertion at the start of the coding region did not abrogate the inhibition. Furthermore, it is clear that the RNA structure is needed, as mutation to remove the conserved sequence only rescued T cell signaling when mutations were made that change the RNA structure. Restoring the structure with alternative mutations restored the T cell inhibitory effect. The lower case letters represent the putative seed sequence, with the final capitalized C possibly being included. A comparable sequence for YFV is shown below (with lower case letters representing the seed sequence):
Thus, in certain embodiments, the RNA segment comprises at least about 51 bases of the HCV E2 protein and is 100 bases or less in length and containing a T-cell inhibitory domain, i.e., the seed sequence, or at least about 27 bases of the YFV Env protein and is 100 bases or less in length and containing a T-cell inhibitory domain, i.e., the seed sequence. Certain embodiments of the disclosure include various RNA segment encoding HCV/YFV polypeptides, in particular HCV E2 protein and YFV Nnv protein. For example, all or part of an HCV E2 protein/YFV Env protein encoding RNA may be used in various embodiments of the disclosure. In certain embodiments, the RNA segment may comprise, but is not limited to about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 225, about 220 or more bases, and any range derivable therein.
It also will be understood that sequences may include additional bases, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological activity (e.g., immunosuppression). Theses sequences may be termed “heterologous.”
Embodiments of the disclosure include various viral RNA segments and derivatives thereof. RNA segment variants can be substitutional, insertional or deletion variants. Deletion variants lack one or more bases of the native sequence that are not essential for function or immunosuppressive activity. Insertional mutants typically involve the addition of material at a non-terminal point in the RNA segment. Terminal additions, sometimes called fusions, are also contemplated.
The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein. Accordingly, sequences may have from about 70% to about 80%; or from about 81% to about 90%; or from about 91% to about 99%; of bases that are identical to the native sequence.
The present disclosure may employ RNA segments that comprise modified, non-natural and/or unusual bases. Certain oligonucleotide modifications can improve stability against nuclease degradation has been achieved by introducing a phosphorothioate (P═S) backbone linkage at the 3′ end for exonuclease resistance and 2′ modifications (2′-OMe, 2′-F and related) for endonuclease resistance. A motif having entirely of 2′-O-methyl and 2′-fluoro nucleotides has shown enhanced plasma stability and increased in vitro potency. Sequences containing a 4′-thioribose modification have been shown to have a stability 600 times greater than that of natural RNA. Crystal structure studies reveal that 4′-thioriboses adopt conformations very similar to the C3′-endo pucker observed for unmodified sugars in the native duplex. Stretches of 4′-thio-RNA were well tolerated in both the guide and nonguide strands.
In the boranophosphate linkage, a non-bridging phosphodiester oxygen is replaced by an isoelectronic borane (BH3) moiety. Boranophosphate siRNAs have been synthesized by enzymatic routes using T7 RNA polymerase and a boranophosphate ribonucleoside triphosphate in the transcription reaction. Boranophosphate siRNAs are more active than native siRNAs if the center of the guide strand is not modified, and they may be at least ten times more nuclease resistant than unmodified siRNAs.
Certain terminal conjugates have been reported to improve or direct cellular uptake. Chemically-stabilized siRNAs with partial phosphorothioate backbone and 2′-β-methyl sugar modifications on the sense and antisense strands (discussed above) showed significantly enhanced resistance towards degradation by exo- and endonucleases in serum and in tissue homogenates, and the conjugation of cholesterol to the 3′ end of the sense strand of an NAA by means of a pyrrolidine linker does not result in a significant loss of gene-silencing activity in cell culture. These studies demonstrate that cholesterol conjugation significantly improves in vivo pharmacological properties of NAAs.
“Isolated substantially away from other coding sequences” means that the gene of interest forms part of the coding region of the nucleic acid segment, and that the segment does not contain large portions of naturally-occurring coding nucleic acid, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the nucleic acid segment as originally isolated, and does not exclude genes or coding regions later added to the segment by human manipulation.
In particular embodiments, the disclosure concerns isolated nucleic acid segments and recombinant vectors incorporating DNA sequences encoding viral envelope polypeptides or peptides. These polypeptides/peptides include within their amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to viral envelope polypeptides. Also envisioned are variants that have modification in one or more kinase sites within these polypeptides.
The nucleic acid segments used in the present disclosure, regardless of the length of the coding sequence itself, may be combined with other DNA or RNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length being limited solely by the ease of preparation and use in the intended recombinant DNA protocol.
A. Vectors Carrying HCV E2 Sequences or Other Viral Envelope Regions
The present disclosure also encompasses the use of vectors to provide the RNA segment. The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). In particular embodiments, gene therapy or immunization vectors are contemplated. One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference.
The term “expression vector” or “expression construct” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
1. Promoters and Enhancers
A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the nucleic acid segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment. The promoter may be heterologous or exogenous, i.e., from a different source than viral sequence. In some examples, a prokaryotic promoter is employed for use with in vitro transcription of a desired sequence. Prokaryotic promoters for use with many commercially available systems include T7, T3, and Sp6.
The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse α(XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).
2. Initiation Signals
A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. For proteins, it is well known that the initiation codon needs to be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
3. Multiple Cloning Sites
Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference). “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
4. Splicing Sites
Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may include donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. See Chandler et al., 1997, herein incorporated by reference.
5. Termination Signals
The vectors or constructs of the present disclosure will generally comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.
In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, the terminator comprises a signal for the cleavage of the RNA, and in other embodiments the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.
Terminators contemplated for use in the disclosure include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
6. Polyadenylation Signals
For expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the disclosure, and/or any such sequence may be employed. Some embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
7. Origins of Replication
In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at which replication is initiated. Alternatively, an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.
8. Selectable and Screenable Markers
In certain embodiments of the disclosure, the cells containing a nucleic acid construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.
Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.
B. Host Cells
As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which refers to any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.
Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector, expression of part or all of the vector-encoded nucleic acid sequences, or production of infectious viral particles. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials. An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5α, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and S
Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either an eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
C. Introduction of Nucleic Acids into Cells
In certain embodiments, a nucleic acid may be introduced into a cell. There are a number of ways in which nucleic acid molecules such as vectors may be introduced into cells. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral transcripts stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
“Viral expression vector” is meant to include those vectors containing sequences of that virus sufficient to express a polynucleotide that has been cloned therein. A number of such viral vectors have already been thoroughly researched, including adenovirus, adeno-associated viruses, retroviruses, herpesviruses, and vaccinia viruses.
Delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious viral particle. Several non-viral methods for the transfer of expression vectors into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), liposome (Ghosh and Bachhawat, 1991; Kaneda et al., 1989) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
In certain embodiments, the nucleic acid encoding a gene or genes may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression vector is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.
Transfer of a nucleic acid molecule may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro, but it may be applied to in vivo use as well.
A. Inflammatory Conditions
The present disclosure relates to the use of viral RNA segment for the modulation of immune responses, particularly those relating to pathologic inflammation. In one embodiment, the pathologic inflammation relates to interleukin-2 (IL-2) expression. IL-2 has multiple, sometimes opposing, functions during an inflammatory response. It is a potent inducer of T cell proliferation and T-helper 1 (Th1) and Th2 effector T cell differentiation and provides T cells with a long-lasting competitive advantage resulting in the optimal survival and function of memory cells. In a regulatory role, IL-2 is important for the development, survival, and function of regulatory T cells, it enhances Fas-mediated activation-induced cell death, and it inhibits the development of inflammatory Th17 cells. Thus, in its dual and contrasting functions, IL-2 contributes to both the induction and the termination of inflammatory immune responses.
The present disclosure would therefore seek to intervene in those diseases where, for example, IL-2 is activating T cells and leading to inflammatory states. Such diseases include autoimmune diseases like multiple sclerosis, psoriasis, inflammatory bowel disorders, early arthritis, juvenile arthritis, rheumatoid arthritis, enteropathic arthritis, psoriatic arthritis, ankylosing spondylitis, familial Mediterranean fever, amyotrophic lateral sclerosis, systemic lupus erythematosus, ulcerative colitis, inflammatory bowel disease, Sjögren's syndrome, or Crohn's disease. Other inflammatory conditions include cardiovascular disease, trauma, or pancreatitis.
B. Gene Therapy
In one embodiment, the disclosure contemplates including immunosuppressive sequences from one or more of the viruses mentioned herein nucleic acid constructs used for gene therapy. One problem with gene therapy is achieving sustained expression, which generally requires multiple administrations. Whether from a single administration or multiple ones, immune reactions against the delivery vector/vehicle can result. Therefore, including immunomodulatory domains described herein is suggested herein in gene therapy vectors as a way to blunt unwanted and limiting host immune reactions against the gene therapeutic modality.
C. Pharmaceutical Formulations and Routes of Administration
Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render proteins stable. Buffers also will be employed when RNA segment are introduced into a patient. Aqueous compositions of the present disclosure comprise an effective amount of the RNA segment dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous media. Such compositions also are referred to as inocula. The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
The percentage of active compound in any pharmaceutical preparation is dependent upon both the activity of the compound. Typically, such compositions should contain at least 0.1% active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The form should be sterile and should be fluid to the extent that easy injection is possible. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention or control of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, phenylmercuric nitrate, m-cresol, and the like. In some embodiments isotonic solutions, for example, sugars or sodium chloride may be used. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the needed amount in the appropriate solvent with various other ingredients enumerated above, as necessary, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can be vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The present disclosure contemplates a viral RNA immunosuppressive segment, and nucleic acid molecules coding therefor. In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects of the present disclosure involve administering an effective amount of an aqueous composition. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Those of skill in the art are well aware of how to administer agents, as well as gene delivery to in vivo and ex vivo situations.
The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human, as appropriate. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents, can also be incorporated into the compositions.
In addition to the compounds formulated for parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.
The active compounds of the present disclosure can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, intrathoracic, sub-cutaneous, or even intraperitoneal routes. Administration by i.v. or i.m. is specifically contemplated.
The active compositions may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
In certain embodiments, it may be desirable to provide a continuous supply of compositions to the patient. For intravenous or intraarterial routes, this is accomplished by drip system. For various approaches, delayed release formulations could be used that provided limited but constant amounts of the therapeutic agent over and extended period of time. For internal application, continuous perfusion may be used. This could be accomplished by catheterization followed by continuous administration of the therapeutic agent. The time period for perfusion would be selected by the clinician for the particular patient and situation, but times could range from about 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2 weeks or longer. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the injections are administered. It is believed that higher doses may be achieved via perfusion, however.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
An effective amount of the therapeutic composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.
RNA segments may be administered in a dose that can vary from 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/kg of weight to 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg/kg of weight in one or more daily, weekly, monthly, or yearly administrations during one or various days, weeks, months, or years. The RNA segments can be administered by parenteral injection (intravenous, intraperitoneal, intramuscular, subcutaneous, intracavity or transdermic).
In many instances, it will be desirable to have multiple administrations of the RNA segments of the disclosure. The compositions of the disclosure may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations will normally be at from one to twelve week intervals, more usually from one to four week intervals.
Dosages commonly used for formulations that provide passive immunity are in the range of from 0.5 mL to 10 mL per dose, or in the range of 2 mL to 5 mL per dose. Repeated doses to deliver the appropriate amount of active compound are common. Both the age and size by weight of the recipient should be considered when determining the appropriate dosage of active ingredient and volume to administer.
Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability, and toxicity of the particular therapeutic substance.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
As used herein, the term in vitro administration refers to manipulations performed on cells removed from an animal, including, but not limited to, cells in culture. The term ex vivo administration refers to cells that have been manipulated in vitro, and are subsequently administered to a living animal. The term in vivo administration includes all manipulations performed on cells within an animal.
D. Combinations with Anti-Inflammatories
It is common in many fields of medicine to treat a disease with multiple therapeutic modalities, often called “combination therapies.” Inflammatory diseases are no exception. To treat inflammatory disorders using the methods and compositions of the present disclosure, one would generally contact a target cell or subject with a viral RNA immunosuppressive segment and at least one other therapy. These therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameter. This process may involve contacting the cells/subjects with the both agents/therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell/subject with two distinct compositions or formulations, at the same time, wherein one composition includes a viral immunosuppressive segment and the other includes the other agent.
Alternatively, the immunosuppressive RNA segment may precede or follow the other treatment by intervals ranging from minutes to weeks. One would generally ensure that a significant period of time did not expire between the time of each delivery, such that the therapies would still be able to exert an advantageously combined effect on the cell/subject. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 12 hours. In some situations, it may be desirable to extend the time period for treatment significantly; however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
It also is conceivable that more than one administration of either a viral immunosuppressive segment or the other therapy will be desired. Various combinations may be employed, where the viral RNA segment is “A,” and the other therapy is “B,” as exemplified below:
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
Other combinations are contemplated.
Agents or factors suitable for use in a combined therapy against an inflammatory disorder include steroids, glucocorticoids, non-steriodal anti-inflammatory drugs (NSAIDS; including COX-1 and COX-2 inhibitors), aspirin, ibuprofen, and naproxen. Analgesics are commonly associated with anti-inflammatory drugs but which have no anti-inflammatory effects. An example is paracetamol, called acetaminophen in the U.S. and sold under the brand name of Tylenol. As opposed to NSAIDS, which reduce pain and inflammation by inhibiting COX enzymes, paracetamol has recently been shown to block the reuptake of endocannabinoids, which only reduces pain, likely explaining why it has minimal effect on inflammation.
The skilled artisan is directed to “Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
It also should be pointed out that any of the foregoing therapies may prove useful by themselves in treating inflammation.
In an embodiment of the present disclosure, a method of inducing an enhanced immune response to the engineered viral RNA segments rather than native RNAs to prevent, control or limit viral infection is provided. Modified viral RNA segments lacking one or more immunosuppressive sites will be used in subunit or whole virus immunization. An effective amount of a vaccine composition, generally, is defined as that amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of the disease or condition or symptoms thereof. More rigorous definitions may apply, including elimination, eradication or cure of disease.
A. Administration
The compositions of the present disclosure may be used in vivo to modify or modulate an immune response, and thus constitute therapeutic and prophylactic vaccines. Thus, the compositions can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Administration by intradermal and intramuscular routes is specifically contemplated. The vaccine can also be administered by a topical route directly to the mucosa, for example by nasal drops or mist, inhalation, or by nebulizer.
Some variation in dosage and regimen will necessarily occur depending on the age and medical condition of the subject being treated, as well as the route chosen. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In many instances, it will be desirable to have multiple administrations of the vaccine. Thus, the compositions of the disclosure may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations will normally be at from one to twelve week intervals, more usually from one to six week intervals. Periodic re-administration will be desirable with recurrent exposure to the pathogen.
The administration may use various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.
B. Measuring Immune Responses
One of ordinary skill would know various assays to determine whether an immune response against a vaccine was generated. The phrase “immune response” includes both cellular and humoral immune responses. Various B lymphocyte and T lymphocyte assays are well known, such as ELISAs, cytotoxic T lymphocyte (CTL) assays, such as chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and cytokine production assays. See Benjamini et al. (1991), hereby incorporated by reference.
C. Injectable Formulations
One method for the delivery of a pharmaceutical according to the present disclosure is via injection. However, the pharmaceutical compositions disclosed herein may alternatively be administered intravenously, intradermally, intramuscularly, or even intraperitoneally as described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporated herein by reference in its entirety).
Injection may be by syringe or any other method used for injection of a solution, as long as the agent can pass through the particular gauge of needle required for injection. A novel needleless injection system has been described (U.S. Pat. No. 5,846,233) having a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery.
Solutions of the vaccine as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent or control the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). The form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
The prevention or control of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments isotonic agents, for example, sugars or sodium chloride may be used. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermolysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
Sterile injectable solutions are prepared by incorporating the active compounds in the needed amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and any of the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation can be vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.
The phrase “pharmaceutically-acceptable” or “pharmacologically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous injectable composition that contains a protein as an active ingredient is well understood in the art.
D. Inhalable or Aerosol Formulations
A particular mode of administration contemplated for the RNA segments of the present disclosure is via inhalation and/or administration to the nasal mucosa, i.e., intranasal administration. A variety of commercial vaccines (influenza, measles) are currently administered using a nasal mist formulation. The methods of the present disclosure can be carried out using a delivery similar to that used with the Flu-Mist® product, which employs the BD AccuSpray® System (Becton Dickinson). Also useful for this route are nebulizers, such as jet nebulizers and ultrasonic nebulizers.
E. Additional Vaccine Components
In other embodiments of the disclosure, the antigenic composition may comprise an additional immunostimulatory agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination.
1. Adjuvants
As also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants have been used to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and as such adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. Suitable molecule adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. Other adjuvants that may also be used include IL-1, IL-2, IL-4, IL-7, IL-12, γ-interferon, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated. MHC antigens may even be used.
In one aspect, an adjuvant effect is achieved by use of an agent, such as alum, used in about 0.05 to about 0.1% solution in phosphate buffered saline. Alternatively, the antigen is made as an admixture with synthetic polymers of sugars (CARBOPOL®) used as an about 0.25% solution. Adjuvant effect may also be made my aggregation of the antigen in the vaccine by heat treatment with temperatures ranging between about 70° C. to about 101° C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin, mixture with bacterial cell(s) such as C. parvum, an endotoxin or a lipopolysaccharide component of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles, such as mannide mono-oleate (Aracel A), or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute, also may be employed.
Some adjuvants, for example, certain organic molecules obtained from bacteria, act on the host rather than on the antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), a bacterial peptidoglycan. The effects of MDP, as with the majority of adjuvants, are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly. The effects of adjuvants, therefore, are not antigen-specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.
In certain embodiments, hemocyanins and hemoerythrins may also be used in the disclosure. Hemocyanin from keyhole limpet (KLH) can be used in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.
Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al., 1989). The doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin et al., 1989). Polyamine varieties of polysaccharides such as chitin and chitosan, including deacetylated chitin can be used.
Another group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.
U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide which is described for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses. The compounds of U.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, are contemplated for use with cellular carriers and other embodiments of the present disclosure.
BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cell wall skeleton (CWS) may also be used as adjuvants, with or without trehalose dimycolate. Trehalose dimycolate may be used itself. Trehalose dimycolate administration has been reported to correlate with augmented resistance to influenza virus infection in mice (Azuma et al., 1988). Trehalose dimycolate may be prepared as described in U.S. Pat. No. 4,579,945. BCG is an important clinical tool because of its immunostimulatory properties. BCG acts to stimulate the reticulo-endothelial system, activates natural killer cells and increases proliferation of hematopoietic stem cells. Cell wall extracts of BCG have proven to have excellent immune adjuvant activity. Molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990). Live BCG is an effective and safe vaccine used worldwide to prevent tuberculosis. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly 2 billion immunizations, BCG has a long record of safe use in man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. An exemplary BCG vaccine is TICE BCG (Organon Inc., West Orange, N.J.).
Amphipathic and surface active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the present disclosure. Nonionic block copolymer surfactants (Rabinovich et al., 1994) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al., 1988). Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present disclosure.
Another group of adjuvants are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Pat. No. 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals. Of course, the detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant-incorporated cells. For example, combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900. Combinations of CWS and trehalose dimycolate, without detoxified endotoxins, are also envisioned to be useful, as described in U.S. Pat. No. 4,520,019.
Those of skill in the art will know the different kinds of adjuvants that can be conjugated to cellular vaccines in accordance with this disclosure and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram-cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the disclosure (Takada et al., 1995).
Various adjuvants, even those that are not commonly used in humans, may still be employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.
Adjuvants may be encoded by a nucleic acid (e.g., DNA or RNA). It is contemplated that such adjuvants may be also be encoded in a nucleic acid (e.g., an expression vector) encoding the antigen, or in a separate vector or other construct. Nucleic acids encoding the adjuvants can be delivered directly, such as for example with lipids or liposomes.
2. Biological Response Modifiers
In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokines such as γ-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.
3. Chemokines
Chemokines, nucleic acids that encode for chemokines, and/or cells that express such also may be used as vaccine components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine coding sequence in combination with, for example, a cytokine coding sequence, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include, for example, RANTES, MCAF, MIP1-α, MIP1-β, IP-10 and combinations thereof. The skilled artisan will recognize that certain cytokines (e.g., IFN's) are also known to have chemoattractant effects and could also be classified under the term chemokines.
The following examples are included to demonstrate certain embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques that were determined to function well in the practice of the disclosure, and thus can be considered to constitute some modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Cells and Viruses.
Human hepatocellular carcinoma cell line (Huh-7.5; kindly provided by Dr. Charles Rice, The Rockefeller University) was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine at 37° C. in a 5% CO2. HCV positive human serum infected with genotypes (1, 1a, 1b, 2, 2b and 3) or negative control serum was prepared from blood obtained from patients or from healthy blood donors. Huh 7 cells containing replicons consisting of either the full length HCV genome or the NS2-5 region of the genomes were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg) and maintained as described (Lohmann et al., 1999 and Quinkert et al., 2005). Serum extracellular vesicles (EV) were purified from serum using the ExoQuick reagent (Systems Biosciences) according to the manufacturer's instructions. Specifically, human sera was incubated with the Exoquick reagent for 1 hr (4° C.) and centrifuged 30 min (10,000 g) as recommended. The pellet was resuspended in RPMI and stored at −20° C. until use. This reagent has been reported to yield EVs from both cell culture supernatant and human serum (Fabbri et al., 2012). Cell culture derived, infectious HCV particles (HCVcc) were obtained by transfecting Huh7.5 cells with in vitro transcribed HCV RNA from J6/JFH infectious clone (kindly provided by Dr. Takaji Wakita, Tokyo Metropolitan Institute of Neuroscience, and Dr. Charles Rice, Rockefeller University) as described by others (Lindenbach et al., 2005). Cell culture supernatant was harvested 72 hrs following transfection and concentrated. The HCV titer in the culture supernatant was 4.98×107 (copies/mL). 4.98×107 particles were added to 1×106 cells. HCV (E1-E2) pseudotyped HIV particles (HCVpp) or HIV gag particles without a viral envelope (GAGpp) were generated in HEK 293T cells using pNL4-3-Luc.R-E- (NIRRRP catalog #3417) as described (Mohr et al., 2010). HCVcc, HCVpp and GAGpp in supernatants were concentrated using Amicon 100K filter units (Millipore) and HCVpp/GAGpp were quantified using p24 ELISA (Zeptometrix Inc.).
Expression of HCV Envelope Protein.
Coding regions of HCV E2 protein from J6/JFH plasmid (aa 384-747) (Lindenbach et al., 2005) or from a genotype 3 isolate obtained from a patient from the University of Iowa were amplified and ligated into a modified pTRE2-HGY plasmid (Clontech, Inc.) as previously described (Xiang et al., 2012). HCV sequences were confirmed by sequencing plasmid DNA (University of Iowa DNA Core Facility). The modified plasmid generates a bicistronic message encoding the HCV E2 sequence followed by stop codons, the encephalomyocarditis virus (EMC) internal ribosomal entry site (IRES) directing the translation of GFP. Jurkat (tet-off) cell lines (Clontech, Inc) were transfected (Nucleofector II, Lonza Inc.) and cell lines were selected for hygromycin and G418 resistance. GFP positive cells were bulk sorted (BD FACS Aria, (University of Iowa Flow Cytometry Facility) and GFP expression was assessed by flow cytometry (BD LSR II). HCV E2 protein expression was determined by immunoblot using human monoclonal antibodies (HC33-1, kindly provided by Dr. Steven Foung, Stanford University). All cell lines were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin with hygromycin and G418 (200 μg/mL).
Cell Isolation and Stimulation.
Peripheral blood mononuclear cells (PBMCs) were prepared from blood obtained from healthy donors by Ficoll-gradient centrifugation. PBMCs were incubated with HCV positive or negative serum (100 μl for each unless otherwise stated) overnight. CD3+ (T) cells were enriched by positive selection using magnetic system according to manufacturer's instructions (Miltenyi Biotec, Auburn, Calif.), and purity assessed by flow cytometry. PBMCs (1×106 cells/mL) were stimulated with plate-bound anti-CD3 (100 ng/mL, OKT3 clone, eBioscience) and soluble CD28 antibody (100 ng/mL, clone CD28.2, BD Biosciences). Jurkat cells (5×106 cells/mL) were stimulated with anti-CD3 and soluble CD28 (both at 5 μg/mL) or phorbol-12-myristate-13-acetate (PMA, 50 ng/mL) and ionomycin (1 μg/mL) (P+I). Cellular receptor expression and cytokine release were measured 24 hours post-stimulation by flow cytometry and ELISA respectively. For Lck inhibition, Jurkat cells were incubated with Lck inhibitor II (EMD Millipore) at 100 μg/mL overnight before stimulating with P+I.
Flow Cytometry:
Cell surface receptor expression was measured with CD69 (APC), or CD45 (PE) antibodies (BD Biosciences) using the manufacturer's recommendations. Cells were incubated on ice for 1 hour, washed 3 times with PBS and fixed in 2% paraformaldehyde (Polysciences). Purified extracellular cellular vesicles (EV) were stained with either anti-CD63 exo-flow staining kit (Systems Biosciences) or CFSE dye (5 μM) for 15 minutes at 37° C. EVs were washed in PBS four times and concentrated using Amicon 100K filter units (Millipore). Data was acquired on BD LSR II flow cytometer using single stained CompBeads (BD Biosciences) for compensation. At least 10,000 total events were collected in each study and the FlowJo software program (Tree Star Inc.) was used for data analysis. All flow cytometry studies were repeated at least three times with consistent results.
HCV PCR.
After overnight incubation, PBMCs were incubated in trypsin for 1 minute and washed twice with RPMI. Total RNA was isolated (RNeasy Kit, Qiagen) and cDNA was made with HCV 5′ UTR specific primers or random hexamers. For first round RT-PCR, the outer primers were sense 5′CTCCACCCAATGAATCACTCCC (SEQ ID NO: 29) and antisense 5′GAGGTTTAGGATTCGTGCTC (SEQ ID NO: 30). For nested PCR, the primers were sense 5′CGTTAGTATGAGTGTCGTGC (SEQ ID NO: 31) and antisense 5′GATGCACGGTCTACGAGACC (SEQ ID NO: 32). The final product size was 250 bp. GAPDH primers used were sense 5′ATCCCATCACCATCTTCCAG (SEQ ID NO: 33) and antisense 5′CCATCACGCCACAGTTTCC (SEQ ID NO: 34) which generates a product size of 383 bp.
HCV E2 derived small RNAs were identified as follows, total RNA from Jurkat cells expressing HCV E2 was isolated (RNeasy Kit, Qiagen). RNA was ligated to a pre-adenylated DNA universal miRNA cloning linker (New England Biolabs) using T4 RNA ligase 2 (New England Biolabs) according to the manufacture's protocol. Ligated RNA was purified using RNA columns (Qiagen) and cDNA transcribed using a DNA linker primer (5′-ATTGATGGTGCCTACAG-3′ (SEQ ID NO: 35)). PCR was carried out using HCV E2 primer (5′-TCCTGATACCACTTACCTCAA-3′ (SEQ ID NO: 36)) and DNA linker primer. PCR products were cloned into TA cloning vector (Invitrogen) and DNA sequences were obtained by sequencing plasmid (University of Iowa DNA Core Facility).
ELISA and Immunoblot Analyses.
IL-2 cytokine released into cell culture supernatant was quantified using human IL-2 ELISA kit (BD Biosciences) according to the manufacturer's instructions. Jurkat cells were stimulated with anti-CD3 (5 μg/mL) for the indicated times prior to addition of cell lysis buffer (Cell Signaling). Following PMA/Ionomycin stimulation for 15 min, nuclear proteins were isolated using nuclear protein isolation kit (NEPER, Thermo Scientific) following manufacturer's instructions. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (BIORAD). Membranes were incubated in protein-free blocking buffer (Thermo Scientific) for 1 hour at room temperature followed by incubation with primary antibodies. Proteins were detected with Amersham ECL (GE Healthcare) using a Kodak Imager. Primary antibodies used were: NFAT and pLAT (Y226; BD Biosciences); total LAT (Biolegend); pZAP70 (Y319); total ZAP70; pLck (Y394/pSrcY416); total Lck (Y394); Csk; YY1 (all from Cell Signaling Technology); PTPRE (Origene, clone 4B2). Immunoblots were quantified by ImageJ.
In Vitro Kinase Assay.
Phosphorylation of HCV E2 protein by Lck was measured by incubating recombinant E2 protein (eEnzyme) with or without human Lck (R&D Systems) and CD45 (Enzo Life Sciences) as recommended by the manufacturer. Samples were subjected to immunoblot analysis as described above. Phosphorylation was determined by immunoblot analysis with phosphotyrosine antibodies (Invitrogen) and HCV E2 protein was identified using anti-HCV E2 human monoclonal antibodies described above.
Immunoprecipitation.
Jurkat cells stably expressing HCV E2 (aa 384-715 of the HCV polyprotein) with a C-terminal influenza hemagluttinin (HA) tag were stimulated with 10 μg/mL of anti-CD3 for 15 min and lysed (25 mM Tris, 150 mM NaCl, 5% Glycerol, 1 mM EDTA, 1% NP40; 1 hr on ice). Cell lysates were incubated with anti-HA agarose beads (Thermo Scientific) or NFAT antibodies conjugated to protein G beads at 4° C. overnight. Beads were pelleted and washed 3 times in lysis buffer. Bound proteins were eluted in 2× LaemmLi sample buffer prior to immunoblot analysis.
Statistics:
Statistics were performed using GraphPad software V4.0 (GraphPad Software Inc.). Two-sided Student's t test was used to compare results between test and controls. P values less than 0.05 were considered statistically significant.
Ethics Statement:
This study was approved by the University of Iowa Institutional Review Board. All subjects (healthy donors and subjects with viral infections) provided written informed consent.
HCV Particles Inhibit TCR Signaling in Primary Human T Cells.
Serum from HCV-infected subjects (genotypes 1, 1a, 1b, 2, 2b and 3; RNA concentration range 1×105 to 1×106 RNA genome equivalents [GE] per mL), or HCV uninfected controls were incubated with PBMCs obtained from healthy blood donors. Following TCR stimulation with anti-CD3/CD28, signaling was quantified by measuring IL-2 release into culture supernatants or by measuring CD69 expression on these primary CD4+ and CD8+ T lymphocytes. All HCV RNA positive sera inhibited IL-2 release (
To remove serum factors that may interfere with TCR signaling, serum extracellular vesicles (EV) were prepared using a commercial reagent (Exoquick, Systems Biosciences). This method of purification yields well characterized EV consistent with exosomes from human serum (Fabbri et al., 2012). Exosomes are reported to contain HCV envelope proteins, HCV RNA and to transmit HCV infection in vitro (Ramakrishnaiah et al., 2013; Cosset and Dreux, 2014; Masciopinto et al., 2004; Bukong et al., 2014 and Dreux et al., 2012). Purified HCV RNA-containing EV contained CD63 and CD81 but did not contain CD69 and CD25 (
To further examine EV interactions with lymphocytes, sera from HCV infected or uninfected subjects were incubated with purified T cells at 37° C. or at 4° C. Following two hour incubation and wash (at room temperature), cells were stimulated with anti-CD3/CD28. HCV positive sera significantly inhibited TCR signaling at 37° C. but not 4° C. (
Next, infectious HCV particles generated in a hepatocyte cell line (Huh7.5 cells) were incubated with PBMCs prior to TCR stimulation. Similar to serum-derived EV, cell culture infectious HCV particles (HCVcc) inhibited TCR signaling in CD4+ and CD8+ T cells (
In summary, incubation of primary human PBMCs with i) serum from HCV-infected individuals, ii) HCV RNA and CD63/CD81 containing serum-derived EV, iii) HCVcc and iv) HCVpp reduced TCR-mediated activation in primary human CD4+ and CD8+ T cells compared to controls.
HCV E2 Coding RNA Inhibits Proximal TCR Signaling Pathways.
Since HCVpp particles contain essentially only HCV E1 and E2, the major HCV envelope glycoprotein (E2) was examined for its ability to inhibit T cell activation through the TCR. Since HCV does not replicate well, if at all, in lymphocytes, Jurkat (CD4+) T cell lines stably expressing HCV E2 protein (
Following TCR stimulation, Lck phosphorylation (Y394) was reduced in Jurkat cells expressing HCV E2 protein compared to controls (
To characterize HCV E2 region(s) required for inhibition of TCR signaling, a series of Jurkat cell lines expressing truncated E2 proteins were generated (
Using kinase-specific phosphorylation substrate prediction models, the tyrosine at HCV E2 aa 613 (Y613) was predicted to be an Src/Lck substrate (
To determine if E2 protein was required for TCR inhibition, a Jurkat cell line expressing HCV E2 RNA coding sequences with a frame-shift mutation was generated. This cell line expressed HCV E2 RNA, but not E2 protein (
HCV has considerable sequence diversity among isolates, including the sequences encoding E2 aa 603-619 (world-wide-web at hcv.lanl.gov). The HCV E2 RNA and protein expression from a different HCV isolate (genotype 3; GT-3), containing 13 nt differences in the RNA coding aa 603-619 were examined (
Bioinformatics analyses predicted the conserved nucleotides within the HCV E2 603-619 coding RNA sequences are needed to generate an RNA structure that would be processed by Dicer, the cytoplasmic endoribonuclease involved in the microRNA (miRNA) pathway (
To understand the mechanism by which vd-sRNA inhibits TCR signaling, additional analyses were performed to identify potential targets for this vd-sRNA sequence. Two conserved sites complementarity to vd-sRNA were found within the 3′ untranslated region (UTR) of a protein tyrosine phosphatase type E (PTPRE;
To determine the specificity and HCV E2 coding RNA requirements for PTPRE knockdown, the PTPRE 3′UTR sequence was inserted into the 3′UTR of GFP in an expression plasmid. GFP expression in 293T cells was reduced by co-transfection of HCV E2 coding plasmid compared to GFP without PTPRE 3′UTR (
To further examine the specificity of the HCV E2 RNA for targeting cellular genes, the predicted seed sequence for PTPRE was replaced with a sequence targeting a cellular gene expressed in the Jurkat cells (CXCR4) (
Together, these data demonstrate that HCV E2 RNA expressed in vitro is processed into short RNA that inhibits PTPRE expression in human hepatocyte (Huh 7) and T (Jurkat) cells, and inhibits TCR-mediated Src (Lck) signaling. Addition of HCV RNA-containing serum to 293 cells also inhibits PTPRE expression, thus this effect is highly likely to be biologically relevant.
Together, these data demonstrate that HCV E2 RNA expressed in vitro is processed into short RNA that inhibits PTPRE expression in human hepatocyte (Huh 7) and T (Jurkat) cells, and inhibits TCR-mediated Src (Lck) signaling. Addition of HCV RNA-containing serum to 293 cells also inhibits PTPRE expression, thus this effect is highly likely to be biologically relevant.
HCV E2 Protein Inhibits Distal TCR Signaling.
T cell activation can be initiated in vitro by stimulating downstream of TCR using phorbol-12-myristate-13-acetate (PMA) and ionomycin (P+I). To determine if HCV E2 RNA inhibited proximal and distal TCR-mediated signaling, Jurkat cells were stimulated with P+I, and cells expressing just HCV RNA did not inhibit distal signaling (
The Y613 of HCV E2 protein is a predicted Lck substrate, thus phosphorylation of this residue was tested. In vitro recombinant HCV E2 was phosphorylated by Lck and dephosphorylated by CD45 (
To determine the mechanism by which phospho-HCV E2 inhibited P+I induced IL-2 release, the activation and nuclear translocation of the nuclear factor of activated T cells (NFAT), a transcription factor required for IL-2 mRNA transcription was assessed. Upon P+I stimulation, NFAT was activated (dephosphorylated) similarly in control and HCV E2 protein expressing Jurkat cells (
Next, the effect of HCV enveloped particles on distal TCR signaling in primary human T cells was assessed. Following P+I stimulation, IL-2 release from healthy human PBMCs was inhibited by HCV particles obtained from serum (
Taken together, these data confirm that HCV RNA-containing serum, HCVcc, HCVpp, HCV E2 protein, but not HCV E2 RNA inhibit distal TCR signaling in primary human T cells and the CD4+ human T cell line. This inhibition requires Lck phosphorylation of Y613 of HCV E2 protein, and does not require viral replication, as HCVpp inhibit distal signaling.
Yellow Fever Virus (YFV) RNA and Envelope Proteins Inhibit T Cell Signaling.
A major problem with the HCV and GBV-C studies are that neither virus replicates well in vitro, and although HCV is found in lymphocytes in vivo, it does not replicate extensively in lymphocytes. We previously showed that the vaccine strains of YFV (17D) and mumps (Jeryl Lyn) strains replicate in PBMCs and CD3+ (T) cells in vitro (Xiang et al., 2009; Mohr et al., 2008). Bioinformatic analyses revealed that both viruses have conserved motifs predicted to serve as a substrate for Lck (Xue et al., 2011). To examine a potential interaction with Lck, we first studied replication of YFV and mumps in Jurkat cells with and without Lck. YFV produced less virus in the presence of Lck, while mumps replicated to higher level in Lck containing cells (
YFV infection of primary human CD3+ (T) cells prior to anti-CD3 activation reduced TCR-signaling as measured by IL-2 release (
Mice were immunized IP with either YFV or mumps virus. Initial studies were in Balb/C mice; however, studies were repeated in C57/black 6 mice with similar results. All data shown are in black 6 mice. Stock viruses were both produced in Vero cells. Experiments were controlled by immunizing with sham infected Vero cell culture supernatant. In addition, UV-inactivated YFV and mumps virus were tested. The viral titer (prior to inactivation) and protein concentrations of the different inocula were normalized. Following IP immunization with YFV, TCR-activation of splenocytes was measured longitudinally for 18 days, and TCR signaling was found to decrease by day 4, reach the nadir at day 10-12, and then increase by day 18—though not to baseline. Subsequently, after YFV immunization ovalbumin (in alum) was administered, and 7 days later a second dose of ova in alum was given. Animals were sacrificed 7 days after the boost, and splenocytes and draining lymph nodes were examined for immune responses. Mice immunized with YFV had significantly reduced IL-2, IFN-γ, and ova-specific antibodies compared to the cell culture controls (
These data indicate that viral envelope coding RNA and env proteins from several viruses interfere with TCR signaling, potentially delaying or reducing env-specific immune responses and facilitating replication. Although HCV and YFV share this phenotype, different mechanisms appear to be utilized by different viruses.
HCV establishes persistent infection through complex and incompletely understood mechanisms. A strong T cell response correlates with effective control and clearance of HCV infection; however, the majority of infected individuals fail to clear viremia (Rehermann, 2009). Chronic infection is associated with a reduction in HCV-specific intrahepatic and peripheral blood T cells, suggesting that HCV proteins, RNA, or both inhibit T cell function. Here the inventors
As disclosed herein, HCV particles directly reduced T cell activation via inhibition of the T cell receptor (TCR) signaling pathway. Serum, HCV RNA-containing EV from HCV-infected individuals and HCVcc inhibited TCR signaling in human T cells. Inhibition of TCR signaling did not require replication, as replication incompetent HCVpp inhibited TCR signaling. The relative inhibition of TCR signaling by HCV positive sera was stronger than cell culture derived HCVcc or HCVpp. This is not surprising, since HCV positive sera contain TCR inhibitory cytokines (IL-10, TGF-β) that are not present in HCVcc, purified EVs and HCVpp (Nelson et al., 1997 and Reiser et al., 1997). Nevertheless, in the absence of these serum factors, HCVcc, HCV positive EVs and HCVpp also inhibited TCR signaling in PBMCs and purified T cells, thus viral E2 protein and RNA were sufficient to alter TCR signaling.
HCV RNA encoding E2 and E2 protein itself independently inhibited TCR signaling at two distinct sites within the TCR signaling pathway. HCV E2 RNA inhibited proximal TCR signaling by reducing activation of Lck, and inhibition required highly conserved nucleotide sequences flanking the conserved E2 Y613. The RNA region contains conserved residues from a structure processed by Dicer into a vd-sRNA. Mutation of four of the conserved residues abolished the predicted RNA secondary structure and restored TCR signaling. Conserved sequences in the vd-sRNA sequence were predicted to target a PTPRE, a phosphatase involved in Src kinase signaling.
PTPRE translation was significantly reduced in lymphocyte cells expressing HCV E2 RNA and in hepatocytes expressing the HCV full-length genome. PTPRE regulates Src signaling through Grb2 (Toledano-Katchalski and Elson, 1999), and Grb2 deficient cells have impaired Lck activation (Jang et al., 2010), suggesting that HCV-derived, short RNA interferes with PTPRE translation leading to reduced TCR signaling. HCV replication in hepatocytes is enhanced by inhibition of Src kinases (Supekova et al., 2008), thus knockdown of hepatocyte PTPRE which regulates Src kinases by this HCV-derived short RNA may also facilitate viral replication in hepatocytes in addition to interfering with T cell signaling in lymphocytes. The addition of HCV RNA-containing serum to 293 cells targeted PTPRE sequences when added to GFP compared to HCV RNA negative serum, illustrating the biological relevance of this observation.
Using an ELISA based assay of PTPRE activity, it was shown that a pan-phosphatase inhibitor potently inhibits PTPRE (
A second mechanism of inhibition of TCR signaling involved E2 protein phosphorylation. Four lines of evidence in the studies herein support a role for Lck-mediated phosphorylation of HCV E2 protein at Y613. First, E2 was phosphorylated in vitro by Lck. Second, native E2 protein expressed in Jurkat cells was phosphorylated following TCR engagement yet E2 with an Y613A mutation was not. Third, an Lck inhibitor rescued the HCV E2 protein-mediated effect on NFAT translocation following P+I activation, and finally, mutation of Y613 to alanine or phenylalanine restored distal TCR signaling. Phosphorylated HCV E2 inhibited distal TCR signaling by reducing NFAT nuclear translocation. NFAT nuclear import and export is regulated by large number of cellular proteins and non-coding RNAs (Sharma et al., 2011). Phospho-E2 may interfere with any of these factors, or combinations of factors, resulting in impaired NFAT nuclear translocation.
Together, the results indicate that HCV E2 Y613 served as an Lck substrate, and that Lck-mediated phosphorylation of Y613 was required for distal TCR signaling inhibition. Although, the amount of viral RNA and E2 protein transferred to the target T cells is unlikely to be comparable to that in the Jurkat cells expressing E2, significant inhibition of T cell activation was measured in cells incubated with serum, EVs or viral particles (HCVccs and HCVpps) following stimulation with a potent TCR agonist (CD3/CD28 antibodies). Thus the small amount of E2 protein and RNA present in serum and EVs is sufficient to reduce TCR activation. Although E1 could influence T cell activation inhibition, E2 protein was sufficient to inhibit distal TCR signaling, and mutation of the Y613 residue restored TCR signaling in the context of E2 protein expression (Y613A, Y613F) or HCV particles (HCVpp; Y613F). A model illustrating HCV particle interactions and the steps of E2 RNA and protein inhibition of TCR signaling is shown in
During HCV infection, the concentration of serum HCV RNA-containing particles is high, frequently greater than one million copies of viral RNA per milliliter (Schijman et al., 2004 and Matthews-Greer, 2001). Thus there are abundant interactions between viral RNA-containing particles and lymphocytes. In addition to virions, HCV RNA is also present in extracellular vesicles (Ramakrishnaiah et al., 2013; Cosset and Dreux, 2014 and Dreux et al., 2012). Extracellular vesicles containing HCV, HPgV or hepatitis A virus RNA have been reported to deliver viral RNA into uninfected cells and initiate infection (Ramakrishnaiah et al., 2013; Cosset and Dreux, 2014; Chivero et al., 2014 and Feng et al., 2013). Although, in vitro effects of HCV sera on TCR appears to be potent and dose-dependent (
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of various embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application claims benefit of priority to U.S. Provisional Application Ser. No. 62/051,727, filed Sep. 17, 2014, the entire contents of which are hereby incorporated by reference.
This invention was made with government support under Grant No. RO1 AI-58740 awarded by the National Institutes of Allergy and Infectious Disease and Merit Review Grant I01BX000207 from the Department of Veterans Affairs. The United States Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/49167 | 9/9/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62051727 | Sep 2014 | US |
