Patent Application
20100239605
An influenza pandemic occurs when a new influenza virus subtype appears, against which the global population has little or no immunity. During the 20th century, influenza pandemics caused millions of deaths, social disruption, and profound economic losses worldwide. Influenza experts agree that another pandemic is likely to happen, but it is unknown when. The level of global preparedness at the moment when a pandemic strikes will determine the public health and economic impact of the disease. As of today, the World Health Organization (WHO) estimates that there will be at least several hundred million outpatient visits, more than 25 million hospital admissions, and several million deaths globally, within a very short period. These concerns were highlighted in 2003, when the avian H5N1 virus reached epizootic levels in domestic fowl in a number of Asian countries, and then spread to Europe and Africa. Fortunately, its transmission to humans has so far been limited, with 246 documented infections, which were associated with high mortality accounting for 144 deaths (Sep. 14, 2006; World Health Organization (WHO) Web site).
Conventional influenza vaccines are designed to elicit neutralizing antibody responses against influenza virus hemagglutinin protein (HA). Due to the constant antigenic drift in the HA protein, the vaccine composition must be changed each year to match anticipated circulating viral strains. Such a vaccine approach is unacceptable in the face of a pandemic, because of the long time required for the isolation and identification of a pandemic strain, and construction and manufacture of an appropriate vaccine. A more effective approach to control or prevention of an influenza pandemic contemplates development of a “universal” vaccine capable of eliciting protective immunity against recently identified, highly conserved influenza virus immunological determinants. Such a vaccine should provide broad protection across influenza A virus strains. Further, such a vaccine could be manufactured throughout the year, stockpiled, and/or administered throughout the year.
The influenza matrix protein M2 has been demonstrated to serve as an effective target for vaccine development (DeFilette et al., Virology 337:149-161, 2005). M2 is a 97-amino-acid transmembrane protein of influenza type A virus (Lamb et al., Proc. Natl. Acad. Sci. U.S.A 78:4170-4174, 1981; Lamb et al., Cell 40:627-633, 1985). The mature protein forms homotetramers (Holsinger et al., Virology 183:32-43, 1991; Sugrue et al., Virology 180:617-624, 1991) that have pH-inducible ion channel activity (Pinto et al., Cell 69:517-528, 1992; Sugrue et al., Virology 180:617-624, 1991). M2-tetramers are expressed at high density in the plasma membrane of infected cells and are also incorporated at low frequency into the membranes of mature virus particles (Takeda et al., Proc. Natl. Acad. Sci. U.S.A. 100:14610-14617, 2003; Zebedee et al., J. Virol. 62:2762-2772, 1998). The M2 N-terminal 24-amino-acid ectodomain (M2e) is highly conserved among type A influenza viruses (Piers et al., Virus Res. 103:173-176, 2004). The high degree of conservation of M2e can be explained by constraints resulting from its genetic relationship with M1, the most conserved protein of the virus (Ito et al., J. Virol. 65:5491-5498, 1991), and the absence of M2e specific antibodies during natural infection (Black et al., J. Gen. Virol. 74 (Pt. 1):143-146, 1993).
As shown in the alignment below, obtained using sequences from the NCBI influenza database (http://www.ncbi.nlm.nih.gov/genomes/FLU/Database/multiple.cgi), avian H5N1 influenza virus M2e appears to be evolving toward the consensus sequence found in typical human H1, H2, and H3 viruses, suggesting that broad protection, including from new avian viruses, using the “human” influenza M2e epitope may be a possibility:
Human H1N1 (SEQ ID NO: 1)
MSLLTEVETPIRNEWGCRCNDSSD
..........T....E...S....
.........LT..G.....S....
.........LT..G.....S....
The phenomenon of evolution of the H5N1 M2e towards the H1N1 M2e sequence was recently reported based on the analysis of sequences of 800 H5H1 strains isolated from humans and birds in Indonesia and Vietnam (Smith et al., Virology 350:258-268, 2006). The evolved avian M2e peptide EVETPTRN (SEQ ID NO:2), but not its “predecessor” EVETLTRN (SEQ ID NO:3), was efficiently recognized by an anti-human M2e monoclonal antibody (Mab)(Liu et al., Microbes. Infect. 7:171-177, 2005). This is important, because some “bird-flu-like” changes have been shown previously to reduce the effectiveness of protection provided by human M2e specific Mabs. Interestingly, some “bird-flu-like” amino acid changes in M2e reduced pathogenicity of human H1N1 viruses in mice (Zharikova et al., J. Virol. 79:6644-6654, 2005).
The WHO has emphasized the possibility of “simultaneous occurrence of events with pandemic potential with different threat levels in different countries, as was the case in 2004 with poultry outbreaks of H7N3 in Canada and H5N1 in Asia” (http://www.who.int/en/). As is shown in the alignment below, M2e H7N7 differs at only one amino acid from the “humanized” variant of H5N1. The H7N7 subtype has demonstrated the ability to be transmissible between species (Koopmans et al., Lancet 363:587-593, 2004) and can be lethal for people (Fouchier et al., Proc. Natl. Acad. Sci. U.S.A 101:1356-1361, 2004). The other strains (H9N2) were also shown to be able to infect poultry and spread to people (Cameron et al., Virology 278:36-41, 2000; Li et al., J. Virol. 77:6988-6994, 2003; Wong et al., Chest 129:156-168, 2006).
Human H1N1 (SEQ ID NO: 1)
MSLLTEVETPIRNEWGCRCNDSSD
..........T..G.E...S....
..........T..G.E.K.S....
.........HT..G.....S....
.........LT..G.E.K.S....
M2e-based recombinant protein vaccines have been shown to elicit protective immune responses against both homologous and heterologous influenza A virus challenge (Fiers et al., Virus Res. 103:173-176, 2004; Slepushkin et al., Vaccine 13:1399-1402, 1995). More recent studies using an M2e peptide conjugated to keyhole limpet hemocyanin and N. meningitides outer membrane protein illustrated good immune responses not only in mice, but also in ferrets and rhesus monkeys (Fan et al., Vaccine 22:2993-3003, 2004). Protection against H1, H5, H6, and H9 influenza A viruses with a liposomal M2e vaccine was demonstrated in mice recently (Fan et al., Vaccine 22:2993-3003, 2004).
Development of delivery systems for influenza antigens is important for the development of vaccines against influenza virus infection, such as pandemic vaccines.
The invention provides, in a first aspect, rhinovirus vectors that include antigens, as described herein, such as influenza virus antigens (e.g., M2e peptides). The vectors can be non-pathogenic in humans (e.g., Human Rhinovirus 14 (HRV14). The antigens can be inserted into the vectors of the invention at, for example, the site of a neutralizing immunogen selected from the group consisting of Neutralizing Immunogen I (NimI), Neutralizing Immunogen II (NimII) (e.g., between amino acids 158 and 160 of NimII), Neutralizing Immunogen III (NimIII), and Neutralizing Immunogen IV (NimIV), or a combination thereof. The antigen (e.g., influenza virus antigen) optionally can be flanked by linker sequences on one or both ends. The rhinovirus vectors of the invention can be live or inactivated.
In a second aspect, the invention provides pharmaceutical compositions that include the rhinovirus vectors described herein and one or more pharmaceutically acceptable carriers or diluents. Optionally, such pharmaceutical compositions can further include an adjuvant (e.g., aluminum or chitin-based adjuvants), and/or one or more additional active ingredients (e.g., a Hepatitis B core protein fused with an antigen sequence, such as an M2e sequence).
In a third aspect, the invention provides methods of inducing an immune response to an antigen (e.g., an influenza virus antigen) in a subject (e.g., a human subject), involving administering to the subject a pharmaceutical composition as described herein. In one example, the subject does not have but is at risk of developing an infection, such as an influenza virus infection. In another example, the subject has an infection to which the vector induces immunity, such as an influenza virus infection. In various examples, the pharmaceutical composition is administered to the subject intranasally.
In a fourth aspect, the invention provides methods of making pharmaceutical compositions as described herein, involving admixing a rhinovirus vector as described herein and one or more pharmaceutically acceptable carriers or diluents. Optionally, these methods can involve addition of adjuvants, reconstitution of lyophilized materials, and/or admixture with other active ingredients.
In a fifth aspect, the invention provides nucleic acid molecules encoding or corresponding to the genome of the rhinovirus vectors described herein.
In a sixth aspect, the invention provides Nimll peptides including one or more heterologous antigen sequences, such as an inserted influenza virus antigen sequence (e.g., an M2e sequence).
In a seventh aspect, the invention provides methods of generating rhinovirus vectors as described herein, including an antigen, such as an influenza virus antigen (e.g., influenza virus M2e). These methods can include the steps of (i) generating a library of recombinant rhinovirus vectors based on an infectious cDNA clone that includes inserted antigen sequences (e.g., influenza virus antigen sequences), and (ii) selecting from the library recombinant viruses that (a) maintain inserted sequences upon passage, and (b) are neutralized with antibodies against the inserted sequence. In one example of these methods; the rhinovirus vector is human rhinovirus 14 (HRV14). In other examples, the inserted antigen sequence is inserted at a position selected from the group consisting of NimI, NimII, NimIII, and NimIV Optionally, the inserted antigen sequence is flanked on one or both ends with random linker sequences, as described herein.
In an eighth aspect, the invention provides methods of cultivating rhinovirus vectors including inserted antigen (e.g., influenza virus antigen) sequences. These methods involve the passaging the vectors in HeLa or MRC-5 cells.
The invention provides several advantages. For example, use of a live vector system to deliver antigens such as M2e provides advantages including: (i) the ability to elicit very strong and long-lasting antibody responses with as little as a single dose of vaccine, and (ii) greater scalability of manufacturing (i.e., more doses at a lower cost) when compared with subunit or killed vaccines. Thus, in a pandemic situation, many more people could be immunized in a relatively short period of time with a live vaccine. In addition, the HRV vectors of the invention can be delivered intranasally, resulting in both systemic and mucosal immune responses. Use of HRV14 provides additional advantages, as it is nonpathogenic and is infrequently observed in human populations (Andries et al., J. Virol. 64:1117-1123, 1990; Lee et al., Virus Genes 9:177-181, 1995), which reduces the probability of preexisting anti-vector immunity in vaccine recipient. Further, the amount of HRV needed to infect humans is very small (one tissue culture infectious dose (TCID50) (Savolainen-Kopra, “Molecular Epidemiology of Human Rhinoviruses,” Publications of the National Public Health Institute 2/2006, Helsinki, Finland, 2006)), which is a favorable feature in terms of cost-effectiveness of HRV-based vaccine manufacturing.
Other features and advantages of the invention will be apparent from the following Detailed Description, the Drawings, and the Claims.
The invention provides universal (pandemic) influenza vaccines, which are based on the use of human rhinoviruses (HRV) as vectors for efficient delivery and presentation of universal influenza virus determinants. As described further below, the extracellular domain of the influenza matrix protein 2 (M2e) is a “universal” epitope that can be included in a universal influenza (influenza A) vaccine, according to the invention. This approach provides an effective influenza pandemic vaccine, which can be administered intranasally to induce local mucosal immunity. Two examples of vaccines according to the invention, HRV14-M2e (17AA) and HRV14-M2e (23AA), are schematically illustrated in
The vectors of the invention are based on human rhinoviruses, such as the non-pathogenic serotype human rhinovirus 14 (HRV14). The HRV14 virus particle and genome structure are schematically illustrated in
An example of a molecular clone of HRV14 that can be used in the invention is pWR3.26 (American Type Culture Collection: ATCC® Number: VRMC-7™). This clone is described in further detail below, as well as by Lee et al., J. Virology 67(4):2110-2122, 1993 (also see Sequence Appendix 3). Additional sources of HRV14 can also be used in the invention (e.g., ATCC Accession No. VR284; also see GenBank Accession Nos. L05355 and K02121; Stanway et al., Nucleic Acids Res. 12(20):7859-7875, 1984; and Callahan et al., Proc. Natl. Acad. Sci. U.S.A. 82(3):732-736, 1985). In addition to HRV14, other human rhinovirus serotypes can be used in the invention. As is known in the art, there are more than 100 human rhinovirus serotypes, any of which can be in the invention used upon the derivation of an infectious clone, in the same manner as HRV14. Although described herein with respect to HRV14, the invention applies to other rhinovirus serotypes as well.
Antigen sequences can be inserted into HRV vectors, according to the invention, at different sites, as described further below. In one example, the sequences are inserted into the NimII site of a serotype such as HRV14. NimII (Neutralizing Immunogen II) is an immunodominant region in HRV14 that includes amino acid 210 of VP1 and amino acids 156, 158, 159, 161, and 162 of VP2 (Savolainen-Kopra, “Molecular Epidemiology of Human Rhinoviruses,” Publications of the National Public Health Institute 2/2006, Helsinki, Finland, 2006). In a specific example described below, the sequences are inserted between amino acids 158 and 160 of VP2. Insertions can be made at other sites within the NimII epitope as well. For example, the insertion can be made at any of positions 156, 158, 159, 161, or 162 of VP2, or at position 210 of VP1, or combinations thereof.
Additional sites at which insertions can be made, alone or in combination with insertions at other sites (e.g., the NimII site), include NimI (A and B), NimIII, and NimIV. Thus, insertions can be made, for example, at positions 91 and/or 95 of VP1 (NimIA), positions 83, 85, 138, and/or 139 of VP1 (NimIB), and/or position 287 of VP1 (NimIII) (see, e.g.,
The vectors of the invention are made using standard methods of molecular biology, which are exemplified below in the case of a vector including insertions in NimII of HRV14. In addition, and as is discussed further below, the vectors of the invention can be administered in the form of live viruses or can be inactivated prior to administration by, for example, formalin inactivation or ultraviolet treatment, using methods known to those skilled in the art.
Optionally, the vectors may include linker sequences between the HRV vector sequences and the inserted influenza sequences, on the amino and/or carboxyl-terminal ends. These linker sequences can be used to provide flexibility to inserted sequences, enabling the inserted sequences to present the inserted epitope in a manner in which it can induce an immune response. Examples of such linker sequences are provided below. Identification of linker sequences to be used with a particular insert can be carried out by, for example, the library screening method of the invention as described herein. Briefly, in this method, libraries are constructed that have random sequences in a region desired for identification of effective linker sequences. Viruses generated from the library are tested for viability and immunogenicity of the inserted sequences, to identify effective linkers.
The viral vectors of the invention can be used to deliver any peptide or protein of prophylactic or therapeutic value. For example, the vectors of the invention can be used in the induction of an immune response (prophylactic or therapeutic) to any protein-based antigen that is inserted into an HRV protein.
The vectors of the invention can each include a single epitope. Alternatively, multiple epitopes can be inserted into the vectors, either at a single site (e.g., as a polytope, in which the different epitopes can be separated by a flexible linker, such as a polyglycine stretch of amino acids), at different sites (e.g., the different Nim sites), or in any combination thereof. The different epitopes can be derived from a single species of pathogen, or can be derived from different species and/or different genuses. The vectors can include multiple peptides, for example, multiple copies of peptides as listed herein or combinations of peptides such as those listed herein. As an example, the vectors can include human and avian M2e peptides (and/or consensus sequences thereof).
Antigens that can be used in the invention can be derived from, for example, infectious agents such as viruses, bacteria, and parasites. A specific example of such an infectious agent is influenza viruses, including those that infect humans (e.g., A, B, and C strains), as well as avian influenza viruses. Examples of antigens from influenza viruses include those derived from M2, hemagglutinin (HA; e.g., any one of H1-H16, or subunits thereof) (or HA subunits HA 1 and HA2), neuraminidase (NA; e.g., any one of N1-N9), M1, nucleoprotein (NP), and B proteins.
Additional sequences that can be included in the vectors of the invention are influenza virus M2e sequences. Examples of such sequences are provided throughout this specification and in Sequence Appendix 1. Specific examples of such sequences include the following: MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:1); MSLLTEVETPTRNEWECRCSDSSD (SEQ ID NO:5); MSLLTEVETLTRNGWGCRCSDSSD (SEQ ID NO:6); EVETPTRN (SEQ ID NO:2); SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:7); and SLLTEVETPIRNEWGCR (SEQ ID NO:8). Additional M2e sequences that can be used in invention include sequences from the extracellular domain of BM2 protein of influenza B (consensus MLEPFQ (SEQ ID NO:9)), and the M2e peptide from the H5N1 avian flu (MSLLTEVETLTRNGWGCRCSDSSD (SEQ ID NO:6)).
The peptides included in the vectors of the invention can include the complete sequences, noted above, or fragments including epitopes capable of inducing the desired immune response. Such fragments may include, e.g., 2-20, 3-18, 4-15, 5-12, or 6-10 amino acid fragments from within these peptides. Further, additional amino and/or carboxyl terminal amino acid sequences can be included in such peptides. Thus, the peptides can include, e.g., 1-10, 2-9, 3-8, 4-7, or 5-6 such amino acids, whether of naturally occurring, contiguous sequences, or artificial linker sequences (also see below). All such possible peptide fragments of the sequences noted above are included in the invention.
Other examples of peptides that are conserved in influenza can be used in the invention and include the NBe peptide conserved for influenza B (consensus sequence MNNATFNYTNVNPISHIRGS (SEQ ID NO:10)). Further examples of influenza peptides that can be used in the invention, as well as proteins from which such peptides can be derived (e.g., by fragmentation) are described in US 2002/0165176, US 2003/0175290, US 2004/0055024, US 2004/0116664, US 2004/0219170, US 2004/0223976, US 2005/0042229, US 2005/0003349, US 2005/0009008, US 2005/0186621, U.S. Pat. No. 4,752,473, U.S. Pat. No. 5,374,717, U.S. Pat. No. 6,169,175, U.S. Pat. No. 6,720,409, U.S. Pat. No. 6,750,325, U.S. Pat. No. 6,872,395, WO 93/15763, WO 94/06468, WO 94/17826, WO 96/10631, WO 99/07839, WO 99/58658, WO 02/14478, WO 2003/102165, WO 2004/053091, WO 2005/055957, and the enclosed Sequence Appendices 1 and 2 (and references cited therein), the contents of which are incorporated herein by reference. Further, conserved immunologic/protective T and B cell epitopes of influenza can be chosen from the www.immuneepitope.org database, in which many promising cross-protective epitopes have been recently identified (Bui et al., Proc. Natl. Acad. Sci. U.S.A 104:246-251, 2007 and supplemental tables). The invention can employ any peptide from the on-line IEDB resource can be used, e.g., influenza virus epitopes including conserved B and T cell epitopes described in Bui et al., supra.
Protective epitopes from other human/veterinary pathogens, such as parasites (e.g., malaria), other pathogenic viruses (e.g., human papilloma virus (HPV), herpes simplex viruses (HSV), human immunodeficiency viruses (HIV; e.g., gag), and hepatitis C viruses (HCV)), and bacteria (e.g., Mycobacterium tuberculosis, Clostridium difficile, and Helicobacter pylori) can also be included in the vectors of the invention. Various appropriate epitopes of these and other pathogens can be easily found in the literature. For example, cross-protective epitopes/peptides from papillomavirus L2 protein inducing broadly cross-neutralizing antibodies that protect from different HPV genotypes have been identified by Schiller and co-workers, such as amino acids 1-88, amino acids 1-200, or amino acids 17-36 of L2 protein of, e.g., HPV16 virus (WO 2006/083984 A1; QLYKTCKQAGTCPPDIIPKV (SEQ ID NO:11)). Examples of additional pathogens, as well as antigens and epitopes from these pathogens, which can be used in the invention are provided in WO 2004/053091, WO 03/102165, WO 02/14478, and US 2003/0185854, the contents of which are incorporated herein by reference.
Additional examples of pathogens from which antigens can be obtained are listed in Table 1, below, and specific examples of such antigens include those listed in Table 2. In addition, specific examples of epitopes that can be inserted into the vectors of the invention are provided in Table 3. As is noted in Table 3, epitopes that are used in the vectors of the invention can be B cell epitopes (i.e., neutralizing epitopes) or T cell epitopes (i.e., T helper and cytotoxic T cell-specific epitopes).
The vectors of the invention can be used to deliver antigens in addition to pathogen-derived antigens. For example, the vectors can be used to deliver tumor-associated antigens for use in immunotherapeutic methods against cancer. Numerous tumor-associated antigens are known in the art and can be administered according to the invention. Examples of cancers (and corresponding tumor associated antigens) are as follows: melanoma (NY-ESO-1 protein (specifically CTL epitope located at amino acid positions 157-165), CAMEL, MART 1, gp100, tyrosine-related proteins TRP1 and 2, and MUC1); adenocarcinoma (ErbB2 protein); colorectal cancer (17-1A,791Tgp72, and carcinoembryonic antigen); prostate cancer (PSA1 and PSA3). Heat shock protein (hsp110) can also be used as such an antigen.
In another example of the invention, exogenous proteins that encode an epitope(s) of an allergy-inducing antigen to which an immune response is desired can be used. In addition, the vectors of the invention can include ligands that are used to target the vectors to deliver peptides, such as antigens, to particular cells (e.g., cells that include receptors for the ligands) in subjects to whom the vectors administered.
The size of the peptide or protein that is inserted into the vectors of the invention can range in length from, for example, from 3-1000 amino acids in length, for example, from 5-500, 10-100, 20-55, 25-45, or 35-40 amino acids in length, as can be determined to be appropriate by those of skill in the art. Thus, peptides in the range of 10-25, 12-22, and 15-20 amino acids in length can be used in the invention. Further, the peptides noted herein can include additional sequences or can be reduced in length, also as can be determined to be appropriate by those skilled in the art. The peptides listed herein can be present in the vectors of the invention as shown herein, or can be modified by, e.g., substitution or deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids). In addition, the peptides can be present in the vectors in the context of larger peptides. Optionally, peptides such as those described above and elsewhere herein include additional sequences on the amino and/or carboxyl terminal ends, as discussed above, whether such sequences are naturally associated with the peptide sequences (i.e., the sequences with which the peptides are contiguous in the influenza virus (or other source) genome) or not (e.g., synthetic linker sequences). The peptides can thus include, e.g., 1-25, 2-20, 3-15, 4-10, or 4-8 amino acid sequences on one or both ends. As a specific example, the peptide may include 1-3 linker sequences at amino and/or carboxyl terminal ends.
When used in immunization methods, the vectors of the invention can be administered as a primary prophylactic agent in adults or children at risk of infection by a particular pathogen, such as influenza virus. The vectors can also be used as secondary agents for treating infected patients by stimulating an immune response against the pathogen from which the peptide antigen is derived. In the context of immunization against cancer, the vaccines can be administered against subjects at risk of developing cancer or to subjects that already have cancer.
For vaccine applications, optionally, adjuvants that are known to those skilled in the art can be used. Adjuvants are selected based on the route of administration. In the case of intranasal administration, chitin microparticles (CMP) can be used (Asahi-Ozaki et al., Microbes and Infection 8:2706-2714, 2006; Ozdemir et al., Clinical and Experimental Allergy 36:960-968, 2006; Strong et al., Clinical and Experimental Allergy 32:1794-1800, 2002). Other adjuvants suitable for use in administration via the mucosal route (e.g., intranasal or oral routes) include the heat-labile toxin of E. coli (LT) or mutant derivatives thereof. In the case of inactivated virus, parenteral adjuvants can be used including, for example, aluminum compounds (e.g., an aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate compound), liposomal formulations, synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide, monophosphoryl lipid A, or polyphosphazine.
In addition, genes encoding cytokines that have adjuvant activities can be inserted into the vectors of the invention. Thus, genes encoding cytokines, such as GM-CSF, IL-2, IL-12, IL-13, or IL-5, can be inserted together with foreign antigen genes to produce a vaccine that results in enhanced immune responses, or to modulate immunity directed more specifically towards cellular, humoral, or mucosal responses. Alternatively, cytokines can be delivered, simultaneously or sequentially, separately from a recombinant vaccine virus by means that are well known (e.g., direct inoculation, naked DNA, in a viral vector, etc.).
The viruses of the invention can be used in combination with other vaccination approaches. For example, the viruses can be administered in combination with subunit vaccines including the same or different antigens. The combination methods of the invention can include co-administration of viruses of the invention with other forms of the antigen (e.g., subunit forms or delivery vehicles including hepatitis core protein (e.g., hepatitis B core particles containing M2e peptide on the surface produced in E. coli (HBc-M2e; Fiers et al., Virus Res. 103:173-176, 2004; WO 2005/055957; US 2003/0138769 A1; US 2004/0146524A1; US 2007/0036826 A1)), or inactivated whole or partial virus). Alternatively, the vectors of the present invention can be used in combination with other approaches (such as subunit or HBc approaches) in a prime-boost strategy, with either the vectors of the invention or the other approaches being used as the prime, followed by use of the other approach as the boost, or the reverse. Further, the invention includes prime-boost strategies employing the vectors of the present invention as both prime and boost agents. Thus, such methods can involve an initial administration of a vector according to the invention, with one or more (e.g., 1, 2, 3, or 4) follow-up administrations that may take place one or more weeks, months, or years after the initial administration.
The vectors of the invention can be administered to subjects, such as mammals (e.g., human subjects) using standard methods. In the case of intranasal administration, the vectors can be administered in the form of nose-drops or by inhalation of an aerosolized or nebulized formulation. The viruses can be in lyophilized form or dissolved in a physiologically compatible solution or buffer, such as saline or water. Standard methods of preparation and formulation can be used as described, for example, in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Further, determination of an appropriate dosage amount and regimen can readily be determined by those of skill in the art.
The vectors of the invention can be administered to subjects, such as humans, as live or killed vaccines. The live vaccines can be administered intranasally using methods known to those of skill in the art (see, e.g., Grünberg et al., Am. J. Respir. Crit. Car. Med. 156:609-616, 1997). Appropriate dosage amounts and regimens can readily be determined by those of skill in the art. As an example, the dose range can be, e.g., 103 to 108 pfu per dose. The vaccine can advantageously be administered in a single dose, however, boosting can be carried out as well, if determined to be necessary by those skilled in the art. As to inactivated vaccines, the virus can be killed with, e.g., formalin or UV treatment, and administered intranasally at about 108 pfu per dose, optionally with appropriate adjuvant (e.g., chitin or mutant LT; see above). In such approaches, it may be advantageous to administer more than one (e.g., 2-3) dose.
The invention is based, in part, on the following experimental examples.
We have constructed HRV14 NimII-M2e recombinant viruses. The viruses have been shown to express M2e on the virion surface, as demonstrated by the ability of anti-M2e Mab to neutralize the infectivity of the recombinant viruses.
Three types of HRV14-M2e constructs were created (
1. HRV14-NimII-23AA carrying the 23 AA of M2e inserted between AA159 and 160 of VP2 (NimII site);
2. HRV14-NimII-XXX23AA library. This set of constructs (plasmid library) was similar to the first construct except for the presence of a 3-AA randomized N-terminal linker fused to the peptide. This randomized linker was generated by the M2e sequence using a 5′ (direct) primer containing 9 randomized nucleotides coding for the linker amino acids; and
3. HRV14-NimII-XXX17AA library. This library was generated the same way as the first, but contained a shortened M2e peptide containing only the first 17 AA of M2e.
To facilitate the cloning process into the HRV14 infectious clone, we modified the pWR3.26 infectious clone by replacing its pUC plasmid backbone with that of the pEt vector (Novagen). Resulting plasmid pWR1 (
To monitor genetic stability of the constructed viruses, we incorporated an XhoI cleavage site in the middle of the M2e sequence by silent mutagenesis. An RT-PCR fragment obtained from virus containing mutated M2e gene is cleaved by XhoI, while the corresponding DNA product produced on wild type HRV14 remains undigested (
We carried out neutralization studies with the virus libraries with an anti-M2e Mab (14C2 MAb, Abcam, Inc. Cat# ab5416). Virus neutralization can be also used as a tool to demonstrate purity of libraries (i.e., the absence of wild type HRV14). The results of a plaque reduction neutralization test (PRNT) demonstrated extremely high specificity and neutralizing ability of Mab 14C2 against both libraries (
Both libraries were shown to be extremely susceptible to neutralization by the anti-M2e Mab (
After 4 passages in H1 HeLa cells, six individual clones from each library were plaque purified and, after an additional 4 passages, characterized by sequencing of the carried insert. Each library gave rise to one dominant and stably replicating viral clone. All viruses isolated from HRV14-NimII-XXX23AA library had the same insert sequence, GHTSLLKEVETPIRNEWGSRSNDSSD (SEQ ID NO:12) with GHT as an N-terminal linker, whereas all of the viruses from the HRV14-NimII-XXX17AA library exhibited the same sequence, QPASLLTEVETPIRNEWGSR (SEQ ID NO:13), but with QPA as the N-terminal linker. All viable clones carrying the 23 AA insert had a substitution at position amino acid 7 from a tyrosine to lysine (position 4 in the M2e foreign insert). The clones carrying the 17 AA insert all contained wild type M2e sequence. These results indicate that genetically stable recombinant HRV-M2e viruses can be isolated. In further in vivo studies, the potential of HRV14-M2e(17AA) to provide protection against PR8 strain of Influenza A was evaluated using intraperitoneal route of administration.
9 week old female Balb/c mice (8 mice per group) were primed on day 0, then boosted on day 21 by intraperitoneal administration with either sucrose purified HRV14-M2e(17AA; see a note (4) to Table 4) virus at 5.0×106 pfu of HRV14-M2e(17 AA), 1.3×107 pfu of parental HRV14, or mock (PBS) as negative controls, mixed with 100 μg of adjuvant (aluminum hydroxide) in a 500 μL volume. As a gold standard, a current vaccine candidate ACAM-FluA (recombinant Hepatitis B core particles carrying 3 copies of M2e) was used. The latter was used alone or in combination with HRV14-M2e or HRV14 for prime/boost (Table 4). To demonstrate protection, all mice were subjected to challenge with 4 LD50 of influenza A/PR/8/34 (H1N1) virus on day 35. Morbidity and mortality were monitored for 21 days. To test for serum antibody against the carried peptide, mice were bled prior to inoculation (baseline) and again on day 33. M2e-specific antibody titers in sera were determined by an established ELISA performed in microtiter plates coated with synthetic M2e peptide. Titers of M2e-specific total IgG, Ig2a, and Ig2b were determined.
a. Immunogenicity
i. Total IgG in Immunized Animals
M2e-specific antibody titers were measured for each group using pooled serum samples (
Based on the assumption made by Arnold et al., 2006 (Arnold, G. F. and Arnold, E. Chimeric Virus Vaccine. Ser. No. 11/176,182 [US 2006/0088549 A1], 1-57. Apr. 27, 2006. US. Jul. 7, 2005) an immunizing dose of 109 pfu of HRV14 corresponds to approximately 10 μg of protein. We have roughly estimated that one immunizing dose of recombinant HRV-M2e virus represents 10 ng of protein. Taking into account differences in molecular mass and the multiplicity of subunits in the recombinant Hepatitis B core particles, we speculate that one immunizing dose of HBc-M2e contained approximately 10,000 times more M2e protein than that of HRV-M2e. Comparable antibody levels using HRV vectors perhaps supports a more immunogenic presentation system using a cheaper production methodology.
The level of M2e antibodies was inversely proportional to a number of doses of HRV14-M2e(17AA). Indeed three doses of HRV14-M2e(17AA) virus (group 1) elicited the lowest M2-e specific response (ET=2.700), whereas two dose regiment elicited 10 times higher (group 2; ET=24, 300) and one dose 3 times higher then two doses (group 5; ET=72,900). To verify whether this correlation is due to anti-vector immunity, we tested separately immune response of all groups to HRV14 vector (
M2e-specific ELISA of individual serum samples (
ii. IgG2a, IgG2b, and IgG1 Subtypes of Antibodies in Immunized Animals
The dominant M2-specific Ab isotype in M2e vaccinated mice was shown to be IgG2b with some IgG2a (Jegerlehner et al., J. Immunol. 172.9:5598-5605, 2004). These two isotypes have been shown to be the most important mediators of antibody-dependent cytotoxicity (ADCC) in mice (Denkers et al., J. Immunol. 135:2183, 1985), which is believed is the major mechanism for M2e-dependent protection. In this study we have tested pooled group and individual sera samples for IgG1, IgG2a, and IgG2b isotype titers.
Groups 4 (prime with HRV14-M2e (17AA)/boost with ACAM-FluA) and 7 (prime/boost with ACAM-FluA) demonstrated the highest titers of IgG1 and IgG2a antibodies among other groups (
b. Morbidity and Mortality
Mice were monitored for morbidity and mortality for 28 days after challenge with PR8 strain. As is shown in
Thus, HRV14-M2e (17 AA) virus is highly immunogenic and protective in mice. It compares responses to the traditional recombinant protein regimen and a combination of the two in a prime-boost regimen. The latter demonstrated a significantly different immune response than recombinant protein alone: two doses of recombinant HBc carrying M2e (Acam-FluA) elicited dominant IgG1 antibody subtype, whereas prime with HRV14-M2e(17AA) and boost with Acam-FluA generated IgG2a as a dominant isotype, which was shown to be important for ADCC. Moreover, the latter group demonstrated highest protection over all other groups.
It is important to note that because HRV does note replicate in mice, inoculation of HRV-M2e recombinants in this model is with a suitable parenteral adjuvant and mimics immunization with an inactivated vaccine. We propose to ultimately evaluate in humans, two options: live recombinant HRV14-M2e virus vaccine and/or inactivated vaccine (e.g., formalin-inactivated) co-administered with a licensed parenteral adjuvant such as aluminum hydroxide.
In this in vivo study, the potential of HRV14-M2e (17AA) to provide protection against non-mortal challenge with PR8 strain of Influenza A was evaluated using intranasal route of administration. Note: The HRV14-M2e (17AA) sequence was described above.
2. Experimental Design 9 week old female Balb/c mice (8 mice per group) were primed on day 0, then boosted on days 21 by intranasal administration with either sucrose purified HRV14-M2e(17AA) or HRV14 (see a note (3) to Table 5) virus at 108 pfu per dose (groups 3-6), mixed with 5 μg of Heat-Labile Toxin of E. coli (LT) adjuvant in a 50 μL volume. As a gold standard a vaccine comprising recombinant Hepatitis B core particles carrying 3 copies of M2e (AcamFluA) was used. The latter was used alone or in combination with HRV14-M2e or HRV14 for prime/boost (Table 5). To demonstrate protection, all mice were subjected to challenge with 4 LD50 of influenza A/PR/8/34 (H1N1) virus on day 35. Morbidity and mortality were monitored for 21 days. To test for serum antibody against the carried peptide, mice were bled prior to inoculation (baseline) and again on day 33. M2e-specific antibody titers in sera were determined by an established ELISA performed in microtiter plates coated with synthetic M2e. Titers of M2-e specific total IgG, Ig2a, and Ig2b were determined.
a. Immunogenicity
i. M2e-Specific Antibody Titers
Antibody titers were measured for each group using pooled serum samples (
As in the case of IP administration, priming with HRV14-M2e and boosting with AcamFluA generated the highest level of IgG2a (
b. Morbidity
Mice were monitored for morbidity for 17 days after none-mortal challenge with PR8 strain (
We have identified a new HRV neutralizing immunogen: Neutralizing Immunogen IV (NimIV). It can be used for the development of epitope-insertion recombinant vaccines. NimIV is highly immunogenic, inducing high virus neutralizing titers in mice. NimIV of HRVs involves a C-terminal region of the structural protein VP 1. This epitope can be exchanged between different HRV serotypes. If NimIV of one HRV is introduced into another serotype virus, it confers unto the resulting chimeric recombinant the neutralization characteristics of the donor serotype. Synthetic NimIV peptides were shown to be efficiently recognized by corresponding serotype-specific antibodies in ELISA and Western blot experiments. Specifically, an HRV14-NimIVHRV6 chimera was produced by replacing the NimIVHRV14 in HRV14 with NimIV from HRV6 virus. This virus was efficiently neutralized with anti-HRV6 polyclonal antibodies and also elicited anti-HRV6 neutralizing response in mice. The 50% neutralizing titer of sera from mice immunized with HRV14-NimIVHRV6 was ˜1:800 against HRV6 virus, and only 1:400 against HRV14 (
The protective efficacy of vaccine candidates can be tested in a mouse influenza challenge model using appropriate virus strains. The prototype influenza challenge strain used in our studies is mouse-adapted strain A/PR/8/34 (H1N1). The virus was obtained from the American Type Culture Collection (catalog number VR-1469, lot number 2013488) and adapted to in vivo growth by serial passage in Balb/c mice. For mouse passage, virus was inoculated intranasally and lung tissue homogenates were prepared 3 days later. The homogenate was blind-passaged in additional mice through passage 5. An additional passage was used to prepare aliquots of lung homogenate that serve as the challenge stock.
For challenge of mice, virus is delivered intranasally in a volume of 50 μl, The mice are anesthetized during inoculation to inhibit the gag reflex and allow passage of the virus into the lungs. Mice infected with a lethal dose of virus lose weight rapidly and most die 7-9 days after inoculation. The median lethal dose (LD50) of mouse-adapted A/PR/8/34 virus was determined to be 7.5 plaque-forming units (pfu) in adult Balb/c mice. Results for a typical protection experiment are shown in
Other influenza virus strains will be similarly adapted to growth in mouse lungs. In some cases strains may be used without in vivo adaptation or may not become sufficiently pathogenic even after serial lung passage. In this case, rather than measuring morbidity and mortality, we will measure virus replication in lung and nasal turbinate tissues. Tissues are harvested 3 days after challenge, disrupted by sonication in 1 ml of tissue culture medium and titrated for virus concentration by plaque or TCID50 assay.
Campylobacter jejuni
Helicobacter pylori
Salmonella typhi
Vibrio cholerae
Clostridium difficile
Clostridium tetani
Streptococccus pyogenes
Bordetella pertussis
Neisseria meningitides
Neisseria gonorrhoea
Legionella neumophilus
Clamydial spp.
Haemophilus spp.
Shigella spp.
Plasmodium spp.
Schistosoma spp.
Trypanosoma spp.
Toxoplasma spp.
Cryptosporidia spp.
Pneumocystis spp.
Leishmania spp.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Use of singular forms herein, such as “a” and “the,” does not exclude indication of the corresponding plural form, unless the context indicates to the contrary. Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of the invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Other embodiments are within the following claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/21102 | 10/1/2007 | WO | 00 | 5/17/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60880664 | Jan 2007 | US | |
| 60848308 | Sep 2006 | US |
