Patent Grant
11576965
A virus like particle (“VLP”) vaccine system or platform for eliciting immune responses against one or more different pathogens and the methods for making and using the novel system or platform.
Various vaccine systems or platforms have been proposed. Because these vaccine systems or platforms are not optimal, there is a need in the field for improved systems or platforms, including systems or platforms that can be effective against multiple pathogens.
Described herein is a novel VLP comprised of bovine immunodeficiency virus gag protein (“Bgag”) expressing or co-expressing one or more different target pathogen proteins. In certain embodiments, the target pathogen protein is a transmembrane protein localized to the VLP membrane. In certain embodiments, the target pathogen protein is selected from one or more different viral pathogens, including Orthomyxoviruses, Filoviruses, Coronaviruses, and Retroviruses.
In certain embodiments where the target Influenza viruses are Influenza A viruses, the transmembrane proteins are comprised of one or more different HA subtypes 1-18 (“H1”-“H18”) and/or NA subtypes 1-11 (“N1”-“N11”). For example, in certain embodiments, the transmembrane proteins comprise four different subtypes of HA and one subtype of NA. As another example, in certain other embodiments, the transmembrane proteins are comprised of three different subtypes of HA and one subtype of NA, and so on. As a further example, in certain embodiments the four HAs co-expressing in the novel Bgag VLPs are H5, H7, H9 and H10.
In certain embodiments, one or more of the different Influenza A transmembrane proteins expressed in the novel Bgag VLPs can bind only to avian or human host cells, while in certain other embodiments, one or more of the different transmembrane proteins bind to both avian and human host cells.
In certain embodiments, the Influenza A transmembrane proteins co-expressed in the novel Bgag VLPs can form homotrimers, while in certain other embodiments, the transmembrane proteins form heterotrimers or a mixture of homotrimers and heterotrimers.
In any of the VLPs described herein, the target pathogen protein can be genetically modified. For example, in certain embodiments, the variable regions of the target pathogen protein can be genetically removed. As another example, in certain embodiments, the C-terminus of the target pathogen protein can be modified to increase binding efficiency to the Bgag. As a further example, in certain embodiments, the target pathogen protein can be a chimera. One example of such a chimera is a chimera prepared by genetically modifying the C-terminus of the target Ebola glycoprotein to include the transmembrane and/or C-terminus region of Influenza HA. The Bgag can also be genetically modified. For example, in certain embodiments, the Bgag is modified to increase binding efficiency to the target pathogen protein.
In certain embodiments, the novel Bgag VLPs are capable of protecting or eliciting immune response against one or more different pathogens in a subject. In certain embodiments, the novel Bgag VLPs are capable of protecting or eliciting an immune response in a subject against one or more different types and/or subtypes of viruses, including different Influenza types and subtypes. In certain embodiments, the novel Bgag VLPs are capable of protecting or eliciting an immune response in a subject against Influenza subtypes comprising H5, H7, H9 and/or H10.
In certain embodiments, the novel Bgag VLPs have a diameter of greater than about 100 nm, and preferably from about 150 nm to about 200 nm, such as, for example, about 155 nm, about 160 nm, about 165 nm, about, 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, combinations thereof and the like.
In certain embodiments, each Bgag VLP comprises of on average of more than 375 protein spikes on the VLP membrane, preferably from about 375 to about 800 spikes, and further preferably about 800 spikes. In other embodiments, the number of spikes can be about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, combinations thereof and the like.
In certain embodiments, each Bgag VLP comprises of a large surface area, preferably a surface area that is greater than the pathogen that one of the target pathogen proteins is derived from.
Also described, is a novel vaccine comprising of the Bgag VLP capable of protecting or eliciting an immune response in a subject against one or more different pathogen. In certain embodiments, the novel vaccine comprising of the Bgag VLP is capable of protecting or eliciting an immune response in a subject against one or more different types and/or subtypes of viruses, including different Influenza subtypes. In certain embodiments, the novel vaccine comprising of the Bgag VLPs is capable of protecting or eliciting an immune response in a subject against Influenza subtypes comprising H5, H7, H9 and/or H10.
Also described, is a novel DNA vector for making the novel Bgag VLPs. In certain embodiments, the DNA vector is expressed in a carrier virus to make the novel Bgag VLPs, and preferably the carrier virus is a recombinant baculovirus (“rBV”). In certain embodiments, the rBV comprising the DNA vector is expressed in eukaryotic cells, preferably Spodoptera frugiperda (“Sf9”) cells. In certain embodiments, each target pathogen protein gene is controlled by an individual promoter, and preferably an individual polyhedrin promoter.
Also described, is a method of making the novel Bgag VLPs or vaccines using a DNA vector comprising a Bgag gene and at least one gene from one or more different target pathogens, all organized in tandem and each under the control of a promoter, preferably a polyhedrin promoter; a carrier virus, preferably the rBV; and an eukaryotic cell, preferably the Sf9.
Also described, is a method distinguishing Bgag VLP vaccinated subjects from non-vaccinated subjects.
Unless otherwise indicated, this description employs conventional chemical, biochemical, molecular biology, immunology and pharmacology methods and terms that have their ordinary meaning to persons of skill in this field (unless otherwise defined/described herein). All publications, references, patents and patent applications cited herein are hereby incorporated by reference in their entireties.
As used in this specification and the appended claims, the following general rules apply. Singular forms “a,” “an” and “the” include plural references unless the content clearly indicates otherwise. General nomenclature rules for genes and proteins also apply. That is, genes are italicized or underlined (e.g.: Bgag or Bgag), but proteins are in standard font, not italicized or underlined (e.g.: Bgag). General nomenclature rules for organism classification also apply. That is order, family, genus and species names are italicized.
As used herein, the following terms shall have the specified meaning. The term “about” takes on its plain and ordinary meaning of “approximately” as a person of skill in the art would understand. The term “comprise,” “comprising,” “contain,” “containing,” “include,” “including,” “include but not limited to,” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements.
As used herein, the following terms shall have the specified meaning.
“AIDS” means acquired immunodeficiency syndrome.
A “Bgag” means a bovine immunodeficiency virus gag protein.
A “Bgag VLP” means a Bgag based VLP, that is a virus like particle with the inner core protein Bgag.
A “BIV” means a bovine immunodeficiency virus.
“CDC” means Center for Disease Control and Prevention.
“DIVA” means differentiation of infected from vaccinated animals.
An “EboMay GP” means an Ebola glycoprotein belonging to the Mayinga strain, an example of which is shown in
An “EboMay GP-TMCT” means a chimeric EboMay GP with Influenza HA transmembrane domain and C Terminus, an example of which is shown in
An “ENV” means an HIV envelope protein.
“Fxns” means fractionations.
“H1”-“H18” means Influenza HA types 1-18.
An “HA” means an Influenza transmembrane glycoprotein hemagglutinin.
An “HIV” means a human immunodeficiency virus.
An “HK09” virus means an H9N2 Influenza A/Hong Kong/33982/2009 virus.
A “HPAI” means a highly pathogenic avian Influenza.
An “IECC Fxn 1” means an EboMay GP-TMCT VLP fraction 1 from a peak fraction collected from the ion exchange chromatography column.
An “IECC Fxn 3” means an EboMay GP-TMCT VLP fraction 3 from a peak fraction collected from the ion exchange chromatography column.
“i.n.” means intranasal, as directed to administration of vaccines.
“i.m.” means intramuscular, as directed to administration of vaccines.
An “IN/5” virus means an H5N1 Influenza A/Indonesia/5/2005 virus.
A “JX/13” virus means an H10N8 Influenza A/Jiangxi/IPB13a/2013 virus.
A “M1” means an Influenza inner core protein matrix 1.
A “M1 VLP” means a M1 based VLP, that is a virus like particle with the inner core protein M1.
“MERS” means Middle East respiratory syndrome.
“MOI” means multiplicity of infection.
“N1”-“N11” means Influenza NA types 1-11.
A “NA” means an Influenza transmembrane glycoprotein neuraminidase.
“p.c.” means post-challenge, as directed to the period after the subject has been challenged with the target pathogen(s).
“PBS” means phosphate buffered saline.
A “PEDV” means a porcine epidemic diarrhea virus.
A “PR8” virus means an H1N1 Influenza A/Puerto Rico/8/1934 virus.
An “rBV” means a recombinant baculovirus.
“RFU” means relative fluorescent units.
“SARS” means Severe Acute Respiratory Syndrome.
An “Sf9” means a Spodoptera frugiperda cell.
An “SH/13” means an H7N9 Influenza A/Shanghai/2/2013 virus.
A “subject” of the present invention is preferably an animal, such as a mouse, ferret, chicken, pig etc., and is preferably a mammal or bird, and most preferably a human.
A “target pathogen protein” includes any protein or peptide from any pathogen. The pathogen may be a virus, bacterium, prion, eukaryote, fungus, or any other micro-organism. The pathogen may also be a parasite, such as a parasitic protozoan. The pathogen may also be a multi-cellular organism, including humans. An example of a human target pathogen protein is a protein or peptide cancer marker.
A “VN/04” means an H5N1 Influenza A/VietNam/1203/2004 virus.
A “VLP” means a recombinant virus like particle.
“WHO” means World Health Organization;
Vaccines Against Pathogens
Vaccination is the most effective strategy for preventing or minimizing/treating epidemics caused by pathogens. The seasonal Influenza epidemic, for example, severely affects five (5) million people worldwide. More recently, the West African Ebola outbreaks have crippled tens of thousands of people in the continent.
Unlike drug therapy, which treats patients who have been infected with the pathogen, vaccines protect patients against future infections by eliciting a robust immune response in the patient. Traditional vaccine developments have been narrow and specific, focusing on eliciting immune responses against a single pathogen. However, there is a growing need to develop multi-specific broad spectrum vaccines capable of eliciting robust immune responses against multiple pathogens. Here, the inventors have described, for the first time, a novel vaccine system or platform offering immune protection against one or more different pathogens.
Influenza viruses are a common viral pathogen that can be fatal to people and is a serious threat to public health (Morens and Fauci, 2012; Palese, 2006; Yen and Webster, 2009). Influenza virus is an enveloped virus containing eight segmented double-stranded negative-sense RNA genomes and belongs to the Orthomyxovirideae family. Type A, B and C Influenza viruses exist, of which Influenza A virus, and in particular avian Influenza A, has been closely monitored by the World Health Organization (“WHO”) and Center for Disease Control and Prevention (“CDC”) for its potential epidemic and pandemic outbreaks.
The Influenza A virus has, among others, two transmembrane glycoproteins hemagglutinin (“HA”) and neuraminidase (“NA”) as well as an inner core protein matrix 1 protein (“M1”). The Influenza A virus has 18 known types of HA and 11 known types of NA, resulting in a total of 198 different combinations of Influenza A subtypes. It is believed that a majority of these Influenza A subtypes can infect birds, while only some can infect humans. Since avian Influenza subtypes are capable of reassortment and frequent genetic changes (Morens and Fauci, 2012; Palese, 2004), several avian Influenza A viral subtypes have been known to start to cross the human-avian barrier.
The crossing of the human-avian barrier has created a worldwide concern that highly pathogenic avian Influenza (“HPAI”) viruses will cause a pandemic (Kang et al., 2009; Morens and Fauci, 2012), much like the 1918 H1N1 pandemic that had claimed between 40-100 million lives worldwide. Today, the H5N1 Influenza is an example of HPAI virus; however, others exist. These include, for example, the H7N9 Influenza virus that caused the 2013 outbreak and claimed the lives of many people (Chen et al., 2015; Gao et al., 2013); the H9N2 Influenza virus with an avian origin, but is known to be a human pathogen (Blanco et al., 2013; Pushko et al., 2005; Yen and Webster, 2009); and the H10N8 Influenza virus, which was only recently discovered to have gained the ability to infect humans (Garcia-Sastre and Schmolke, 2014; To et al., 2014).
Currently, vaccines for seasonal Influenza exist. However, they consist of H1N1, H3N2 and Influenza B viruses separately grown in embryonated chicken eggs and later mixed as a trivalent vaccine. Vaccine production using eggs has significant limitations. These include no broad spectrum protection; ineffective against emerging HPAI viruses; inefficient production; low yield; and scale-up issues. Because many pathogens, such as the Influenza, go through annual antigenic drift, vaccine must be continuously developed and changed in response to changes to the circulating seasonal viruses. In addition, because each of the three monovalent vaccines in Influenza must be prepared separately, the production of the trivalent blended vaccine greatly increases the vaccine production cost, increases the response times to the Influenza threat, and increases the chance for adverse reactions post administrations. Accordingly, there is a strong need to develop novel broad spectrum vaccine systems.
The Ebola virus is a Risk Group 4 pathogen, causes an extremely high mortality rate and has devastated many West African countries in recent years. The Ebola virus is an enveloped virus containing a single-stranded negative-sense RNA and belongs to the Filoviridae family. The Ebola virus has a transmembrane glycoprotein (“GP”) as well as inner core proteins. Currently, no Ebola vaccine has been approved for general use in humans.
The Middle East Respiratory Syndrome (“MERS”) coronavirus is an emerging virus identified in 2012, and like the Severe Acute Respiratory Syndrome (“SARS”) coronavirus, belongs to the Coronaviridae family. The MERS coronavirus is an enveloped virus containing a single-stranded positive-sense RNA. Also, like other coronaviruses such as Porcine Epidemic Diarrhea Virus (“PEDV”), the MERS coronavirus has transmembrane glycoproteins. Currently, very little is known about the MERS coronavirus.
The human immunodeficiency virus (“HIV”) is widely known for causing acquired immunodeficiency syndrome (“AIDS”). HIV is an enveloped virus with a single-stranded positive-sense RNA that belongs to the Retroviridae family. The HIV envelope protein (“ENV”) has been targeted for vaccine research. However, currently no HIV vaccine has been approved for clinical use in humans.
The list of pathogens harmful to animals and humans is long and growing. There is a strong need to develop a robust, broad spectrum vaccine system/platform capable of eliciting immune responses against multiple pathogens. Here, the inventors have described, for the first time, a novel vaccine platform using a novel VLP based on Bgag for eliciting specific or broad spectrum immune response against one or multiple (e.g., a plurality of) different pathogens.
Using VLPs as a Vaccine Candidate
Recombinant virus like particles (“VLPs”) are promising vaccine candidates. They are highly immunogenic, morphologically and antigenically similar to native viral particles, yet are replication incompetent. Immune protective effects of VLPs have been demonstrated in pre-clinical and clinical trials (Pushko et al., 2011). In addition, unlike live or inactivated vaccines, VLPs do not involve the production of the target pathogen, but can elicit robust immune responses against the target pathogen through the antigens of the target pathogen expressed and presented by the VLPs. Accordingly, VLPs are safe and effective vaccine candidates for many pathogens, including Influenza (Kushnir et al., 2012; Pushko et al., 2013).
Recently, VLPs have been shown to be promising vaccines for avian Influenza (Bright et al., 2007; Galarza et al., 2005; Kang et al., 2009; Perrone et al., 2009; Pushko et al., 2005; Quan et al.; Ross et al., 2009). We have shown, for example, that VLPs comprised of Influenza HA, NA, and M1 proteins (Pushko et al., 2005) elicited highly efficient protective immune responses, which in some cases exceeded immune responses elicited by traditional Influenza vaccines (Bright et al., 2007; Pushko et al., 2007). The observed high immunogenicity of Influenza VLP vaccines has been attributed to the organization of the HA protein into regular patterns resembling the Influenza viral structures, thereby favoring an activation of the host immune system (Kang et al., 2009; Pushko et al., 2013).
Unlike egg-based technology, VLPs are produced in cell culture and are engineered using methods of molecular biology. However, even today, many VLPs vaccine candidates, like the egg-dependent trivalent seasonal Influenza vaccines, are still developed as strain-specific VLPs that are individually synthesized and produced, and later mixed or blended. Moreover, many of these VLPs were prepared by using homologous HA, NA, and M1 proteins; that is, HA, NA and M1 that are derived from the same virus (Perrone et al., 2009; Pushko et al., 2010; Pushko et al., 2005; Pushko et al., 2007). This approach is problematic, because very often, the sequence data of emerging viruses, are not always readily available. For example, the M1 sequence of emerging Influenza viruses is typically not available during early stage vaccine developments of emerging Influenza vaccines. This makes developing vaccines using VLPs with homologous HA, NA and M1 proteins against, for example, newly emerging viruses particularly problematic.
In response, some have developed VLPs using HA and NA derived from a target Influenza virus and a M1 derived from a different Influenza virus (Liu et al., 2015). Others have also tried to replace M1 with the murine leukemia virus gag protein (Haynes, 2009; Haynes et al., 2009) or with the simian/human immunodeficiency virus gag protein (Guo et al., 2003). Recently, we have made and described several different M1 VLPs with an NA and three different HA each derived from a different Influenza subtype (Pushko et al., 2011; Tretyakova et al., 2013). We have further shown by electron microscopy that all three HA subtypes co-localize to the same M1 VLP (Pushko et al., 2011; Tretyakova et al., 2013). We have further shown that the M1 VLP induced highly protective immune responses against all three strains of Influenza and is an effective trivalent Influenza vaccine (Pushko et al., 2011; Tretyakova et al., 2013). That is, the M1 VLP vaccine derived from a single VLP can protect against all three Influenza viruses.
However, the M1 VLP is not the ideal candidate for vaccines, and in particular, human vaccines. For example, M1 proteins of some Influenza viruses are expressed at lower levels. Moreover, as discussed above, often the sequence for M1 or M1 is not readily available for emerging Influenza viruses. Furthermore, most people have been exposed to M1; thus, there is likely a pre-existing host immunity against the VLP expressing M1 among people. Accordingly, this would greatly compromise the efficacy of an M1 VLP in inducing specific immune responses in humans.
A Novel M1 Free Bgag VLP
The inventors, have made and described here, for the first time, a M1 free VLP using the bovine immunodeficiency virus (“BIV”) gag protein (“Bgag”), referred herein as a Bgag VLP, which expresses one or more different target pathogen proteins (see for example, Example 1). The novel Bgag VLP disclosed herein uses Bgag as its inner core protein. The BIV, which belongs to the Lentivirus genus of the Retroviridae family of viruses, has been previously used in vaccine development research and the use of BIV vectors has been previously described (Luo, 2012). However, prior to this disclosure, no one has disclosed using a Bgag VLP to express or co-express one or more different target pathogen proteins, such as the various Influenza proteins; using Bgag VLP as a vaccine candidate; or using Bgag or Bgag as a diagnostic tool.
As disclosed herein, the inventors, have among other things, prepared for the first time, a Bgag VLP expressing and presenting one or more different target pathogen proteins (see for example, Example 1 and Example 5 to Example 12); shown for the first time that the Bgag VLP can express and present one or more different functional Influenza proteins, including simultaneously co-express and co-present functional HA belonging to four different Influenza subtypes (see for example, Example 5 to Example 7 and Example 11 to Example 12); shown for the first time that the Bgag VLP can express and present non-Influenza pathogen proteins (see for example, Example 9); and shown for the first time that the Bgag VLP can express and present genetically modified, chimeric pathogen proteins (see for example, Example 10). In addition, the inventors have described, for the first time, a method of preparing a novel Bgag VLP and Bgag VLP mediated specific (uni-target) or broad spectrum (multi-target) vaccine (see for example, Example 1 to Example 3 and Example 5 to Example 12). Further, the inventors have described benefits associated with using the Bgag VLP, including its benefits as a superior vaccine. Even further, the inventors have described the benefits of using the Bgag VLP, including as a broad spectrum vaccine candidate. And, in addition, the inventors have described multiple diagnostic methods of using the Bgag protein in connection with the Bgag VLP system.
Bgag VLPs can be Used for a Broad Spectrum of Target Pathogen Protein
The types of target pathogen proteins that can be expressed and presented by the Bgag VLP platform is very broad. For example, it can include any peptide or protein from any virus, bacterium, prion, eukaryote, fungus, parasite, or any other unicellular or multicellular organism. Moreover, when the Bgag VLP expressing one or more of these target pathogen proteins is injected or administered into a subject, the Bgag VLP can elicit an immune response against one or more of these pathogens. Accordingly, the Bgag VLP system or platform can be used as, among others, a viral vaccine, bacterium vaccine, prion vaccine, fungus vaccine and even parasite vaccine, to treat, inhibit and/or prevent pathogenic effects of these pathogens. Using the principles and methods disclosed herein, the Bgag VLP system or platform can also be used as a vaccine against one or more different virus, bacterium, prion, eukaryote, fungus, parasite, any other micro- or macro organism, combinations thereof and the like and has the advantage of offering tailored vaccine or treatment depending on the vulnerability or susceptibility of the subject. For example, a subject that is susceptible to two particular viruses, one particular bacterium and one particular fungus, can be administered a tailored vaccine to inhibit/prevent and/or treat the onset of the viruses, bacterium and fungus.
The novel Bgag VLP platform can also be used to express one or more cancer markers. For example, one or more cancer marker peptides or proteins can be used as a target pathogen protein. As a vaccine, the Bgag VLP can be used to boost a subject's immune system and increase surveillance against developing cancer cells to effectively inhibit/prevent, treat or control cancer.
Bgag VLP containing target pathogen proteins can elicit immune response and serve as a vaccine. For example, Bgag VLPs containing target pathogen proteins can be injected or otherwise administered to a subject with the purpose to induce the immune response against the target pathogen proteins. The vaccinated subject's immune response against the target pathogen is then determined by standard assays. For example, in about 2 to about 4 weeks, the blood of the vaccinated subject is drawn, and anti-target pathogen antibody is determined by ELISA, immunofluorescence antibody assay, or other antibody detection assays, and compared to the antibody profile of non-vaccinated subjects. The presence of anti-target pathogen antibody in the vaccinated subject indicates that the Bgag VLP has elicited an immune response in the subject and thus, is immunogenic in vivo. Standard challenge studies can also demonstrate the efficacy of the Bgag VLP in protecting the subject against the target pathogen.
Bgag VLPs with Consensus Target Pathogen Protein or Conserved Epitopes
In certain embodiments, the target pathogen protein of the Bgag VLP can be designed to broaden the immune protection. For example, the target pathogen protein can be derived from a consensus HA sequence, determined using advanced genetic analysis techniques (Denis et al., 2008; Ebrahimi et al.; Pica and Palese, 2013; Rao et al., 2010; Schotsaert et al., 2009; Wang and Palese, 2009; Wei et al., 2010). As another example, the target pathogen protein can be derived from conserved Influenza epitopes, for example, the ectodomain of the Influenza M2 ion channel protein (Denis et al., 2008; Ebrahimi et al.; Pica and Palese, 2013; Rao et al., 2010; Schotsaert et al., 2009; Wang and Palese, 2009; Wei et al., 2010). The Influenza Bgag VLP resulting from these designs can offer broad spectrum immune protection for a plurality or even all of Influenza subtypes.
Bgag VLPs with Genetically Modified Target Pathogen Proteins
The inventors have shown that a genetically modified target pathogen protein can be expressed and presented by the Bgag VLP (see for example, Example 8 and Example 10). For example, in certain embodiments, a portion of the target pathogen protein is expressed and presented by the Bgag VLP. In certain embodiments, some of the target pathogen protein can have certain variable regions removed. For example, Bgag VLPs can be made by using a “headless” HA, in which the most variable epitopes of HA responsible for virus neutralization are removed. An advantage of this approach is that the headless HA presented by the Bgag VLPs can induce immune response to the HA epitopes that are normally hidden in the standard VLP containing the full-length HA. As another example, Bgag VLPs can be made by using the “stem” region of HA. The immune response to the headless HA or stem HA can result in a broadly protective or universal Influenza vaccine capable of protecting against multiple strains and subtypes of Influenza virus. In these embodiments, the Bgag VLPs can present mostly the conserved regions of the target pathogen protein in an effort to elicit broad spectrum immune response.
Transmembrane proteins from target pathogens can serve as excellent target pathogen proteins and become expressed and presented by the Bgag VLP. However, cytoplasmic protein and peptide as well as secreted protein and peptide from target pathogens can also serve as target pathogen proteins through genetic engineering. For example, cytoplasmic and/or secreted protein or peptides of virus, bacterium, prion, eukaryote, fungus, parasite, parasitic protozoan or human origin (such as cancer markers) can be engineered into a transmembrane protein that can be presented by the Bgag VLP. One such example is to genetically engineer the transmembrane domain of Influenza HA onto these non-transmembrane proteins and/or peptides using standard genetically engineering methods or methods essentially described herein to create the chimeric EboMay GP-TMCT. For example, the transmembrane domain of Influenza HA can be added to the C-terminus of the prostate-specific antigen, a protein cancer marker which is normally secreted by prostate cells. Using the principles and methods essentially described herein, the genetically engineered PSA protein can be expressed and presented by Bgag VLP and used in subjects to inhibit, prevent, treat, monitor, and/or control cancer progression.
In certain embodiments, the genetic modification involves modifying the target pathogen protein to increase binding efficiency to the Bgag (see for example, Example 10). For example, modifications can be made to the C-terminus of the target pathogen to optimize binding efficiency to Bgag.
In certain other embodiments, the genetic modification involves creating a chimeric target pathogen protein. One such chimera is the EboMay GP-TMCT protein (see
In certain other embodiments, the target pathogen protein can be rationally designed and/or redesigned to improve its expression and presentation by the Bgag VLPs. For example, it has been shown that a rational design of the HIV Env protein can increase the protein presentation by 10 times (Wang et al., 2007).
In all cases, using the same principles essentially described above, the Bgag protein can also be genetically modified and improved. For example, the certain regions of the Bgag can be modified to increase binding efficiency to the target pathogen protein.
Like the other Bgag VLPs, the Bgag VLP containing genetically engineered target pathogen proteins can elicit immune response and serve as a vaccine, for example, using the principles and methods described herein.
The Novel Bgag VLP can Express and Present One or More Different Types of Target Pathogen Proteins
The inventors have made and described a novel Bgag VLP system for the expression/co-expression and presenting/co-presenting of one or more different types of target pathogen proteins (see, for example, Example 5 to Example 12). In certain embodiments, the target pathogen protein is localized to the membrane of the VLP (see for example,
The inventors have also shown that the Bgag VLPs can present target pathogen proteins just as well as the M1 VLPs. For example, the inventors have shown that the Bgag VLPs present the Influenza HA and NA proteins just as well as the M1 VLPs (see for example,
In certain embodiments, the novel Bgag VLPs have a diameter of greater than about 100 nm and up to 200 nm, and preferably from about 120 nm to about 200 nm, more preferably about 150 nm to about 200 nm, more preferably about 160 nm to about 200 nm, more preferably about 170 nm to about 200 nm, more preferably about 180 nm to about 200 nm, more preferably about 190 nm to about 200 nm. The Bgag VLP is substantially larger as compared to some innate pathogens. For example, the average diameter of an Influenza virus is approximately 100 nm; the average diameter of an HIV is about 120 nm; and the average diameter of a SARS coronavirus is about 80-90 nm. The Bgag VLP is also substantially larger than the M1 VLP. For example, the average diameter of an Influenza Bgag VLPs is from about 150 to about 180 nm, while the average diameter of an Influenza M1 VLPs is from approximately 120 to approximately 150 nm. Despite the larger size, the Bgag VLPs have general morphology that is similar to M1 VLPs as well as the Influenza virus (see for example,
Novel Bgag VLP is a versatile system that can present one or more different types of target pathogen proteins (see, for example, Example 5 to Example 12). In certain embodiments, Bgag VLP comprises of target pathogen protein selected from one pathogen (see, for example, Example 5 to Example 10), while in certain other embodiments, the Bgag VLP comprises of a target of multiple (e.g., a plurality of) pathogen proteins selected from more than one type of or subtype of pathogen (see, for example, Example 11 to Example 12). In certain embodiments, one of the pathogens selected is a virus. In certain embodiments, the vial pathogen is selected from a combination of Orthomyxoviruses, preferably Influenza virus, and further preferably Influenza A virus; Filoviruses, preferably Ebola viruses; Coronaviruses, preferably MERS viruses; and Retrovirus, preferably HIV.
In certain embodiments, the Bgag VLPs are capable of protecting or eliciting an immune response against one or more different pathogens in a subject. Thus, vaccines prepared with an uni-target Bgag VLP can offer robust target specific immune protection, while vaccines prepared with multi-target Bgag VLPs can offer multi-target, broad spectrum immune protection. Importantly, the broad spectrum protection is achieved by a single multi-target Bgag VLP, rather than by blending different uni-target vaccines together. However, different multi-target Bgag VLPs can certainly be further mixed and/or blended to provide an even broader immune coverage. Accordingly, the multi-target Bgag VLPs can be used to elicit broad spectrum immune protection against different target pathogens and is an important addition to the strategies for pandemic preparedness.
The Novel Bgag VLP can Express and Present One or More Different Subtypes of Target Pathogen Proteins
The inventors have shown that the Bgag VLP system or platform is an accommodating platform for presenting one or more different subtypes of target pathogen proteins (see for example, Example 5 to Example 12). In certain uni-subtype Bgag VLP embodiments, the inventors have shown that Bgag VLPs comprising functional Influenza transmembrane proteins NA and HA, including the PR8 H1 and the emerging Influenza H10, can be made (see for example, Example 5 and Example 6). Prior to this disclosure, Bgag has not been previously used for production of Influenza VLPs and it was not know that Bgag could be used for the preparation of uni-subtype VLPs. In certain multi-subtype Bgag VLP embodiments, the inventors have made and described, for the first time, a multi-subtype Bgag VLP, that is a VLP that simultaneously co-expresses and co-presents different subtypes of a target pathogen protein, which makes it capable of offering simultaneous, broad spectrum immune protection against multiple different subtypes of the target virus (see, for example, Example 11 and Example 12). Prior to this disclosure, it was not known that Bgag could be used for the preparation of multi-subtype VLPs.
In certain embodiments, the Bgag VLPs are capable of protecting or eliciting an immune response in a subject against one or more different subtypes of viruses. Thus, vaccines prepared with an uni-subtype Bgag VLP can offer robust subtype specific immune protection, while vaccines prepared with a multi-subtype Bgag VLPs can offer multi-subtype, broad spectrum immune protection. Like multi-target Bgag VLPs, the broad spectrum protection is achieved by a single multi-subtype Bgag VLP, not by blending different uni-subtype vaccines together. Although, different multi-subtype Bgag VLPs can certainly be mixed or blended to afford broader immune coverage. Together with multi-target Bgag VLP designs, the Bgag VLP systems, which can be used to elicit broad-spectrum immune protection against different types and subtypes of pathogens, is an important addition to the strategies for pandemic preparedness.
As one example, the novel Bgag VLP is capable of protecting or eliciting an immune response in a subject against one or more (e.g., a plurality of) different viral subtypes, including different Influenza viral subtypes. In certain embodiments, the Influenza A transmembrane proteins co-expressed in the novel Bgag VLPs can bind only to avian or human host cells, while in certain other embodiments, the transmembrane proteins bind to both avian and human host cells. In certain embodiments, the novel Bgag VLP comprises one or more different HA types 1-18 (“H1”-“H18”) and NA types 1-11 (“N1”-“N11”) as its target pathogen protein(s), and expresses and presents the proteins. In certain other embodiments, the novel Bgag VLPs are capable of protecting or eliciting an immune response in a subject against one or more different Influenza subtypes, for example, subtypes H5, H7, H9 and H10.
Using this novel platform, the inventors have made and described, for the first time, a quadri-subtype Bgag VLP (see for example,
Avian Influenza viruses of H5, H7, H9, and H10 subtypes have been identified as pathogens of pandemic concern (Belser et al., 2008; Garcia-Sastre and Schmolke, 2014; Palese, 2004; Pappas et al., 2007; WHO, 2013). In particular, both the VN/04 (H5N1) and HK/09 (H9N2) viruses are on the list of candidate vaccines recommended by the World Health Organization (WHO) in 2012 for pandemic preparedness (WHO, 2012a). To enhance pandemic preparedness, inactivated H5N1 vaccines have been approved (O'Neill and Donis, 2009) including cell culture-derived Influenza vaccines. However, although promising experimental vaccines have been reported (Chen et al., 2014; Kong et al., 2015; Smith et al., 2013; Tretyakova et al., 2013; Wohlbold et al., 2015), currently there are no approved human vaccines for H7, H9 or H10 subtypes (WHO, 2012a).
The novel Bgag VLP expressing one or more different target pathogen proteins is an excellent vaccine platform, because it can elicit robust immune response against one or more different target pathogens. For example injecting or otherwise administering an H10 Bgag VLP containing the hemagglutinin from H10N1 virus into ferrets elicits robust anti-H10 neutralizing antibodies in the ferrets (see for example,
The novel Bgag VLP expressing one or more different target pathogen proteins is an excellent vaccine platform, because it can also provide the vaccinated subject immune protection against one or more different target pathogens. For example, injecting or otherwise administering an H10 Bgag VLP containing the hemagglutinin from H10N1 virus into ferrets can protect the ferrets in subsequent challenges by a live Influenza H10N1 virus, as indicated by a significant reduction in replicating virus titers in, for example, nasal turbinate and trachea (see for example,
The novel Bgag VLP is an excellent broad spectrum vaccine candidate that can offer specific immune protection against at least four viral subtypes or strains or clades. As presented here, a novel multi-subtype vaccine candidate or a quadri-subtype vaccine candidate mediated by this novel Influenza H5/H7/H9/10 quadri-subtype Bgag VLP are highly effective, including for use in humans. Unlike traditional blended vaccines discussed previously, the broad spectrum immune protection achieved with the Bgag VLP platform can be achieved without mixing individual vaccines.
The Novel Bgag VLP can Express and Present One or More Different Clades of the Same Subtype of Target Pathogen Proteins
In addition to expressing and presenting target pathogen proteins belong to different types and subtypes, the Bgag VLP system can also be used to express and present target pathogen proteins belonging to different clades of the same subtype. For example, a Bgag VLP can express Influenza H5 belonging to three different clades, such as H5 clade 2.3.4.4 (A/chicken/German/2014), H5 clade 2.1.3 (A/chicken/West Java/Subang/29/2007), and H5 clade 2.2.1 (A/chicken/Egypt/121/2012). The resulting Influenza H555 Bgag VLP expresses functional HA (see for example
The resulting Influenza H555 Bag VLP also shows strong immunogenicity against Influenza H5. For example, following vaccination of chickens with the H555 Bgag VLP, the vaccinated chickens show a strong immunogenicity and presence of anti-H5 antibody as demonstrated by standard hemagglutinin inhibition assay (see for example,
Method for Preparing Bgag VLPs Expressing One or More Different Target Pathogen Proteins.
The inventors have also developed novel methods of efficiently preparing Bgag VLPs expressing and presenting one or more different target pathogen proteins (see for example, Example 1 to Example 3 and Example 5 to Example 12). In certain embodiments, the DNA vector is expressed in a carrier virus to make the novel Bgag VLP, and preferably the carrier virus is a recombinant baculovirus (“rBV”). In certain embodiments, the rBV containing the DNA vector is expressed in eukaryotic cells, preferably Spodoptera frugiperda (“Sf9”) cells. In certain embodiments, each target pathogen protein gene is controlled by an individual promoter, and preferably an individual polyhedrin promoter. In certain embodiments, the method is used to prepare Bgag VLPs expressing and presenting multiple different target pathogen proteins. In certain other embodiments, the method is used to prepare Bgag VLPs expressing and presenting multi-subtype pathogen proteins. In certain other embodiments, the method includes using a DNA vector comprising a Bgag gene and gene(s) from one or more different types of target pathogens, all organized in tandem and each under the control of a promoter, preferably a polyhedrin promoter; a carrier virus, preferably the rBV; and an eukaryotic cell, preferably the Sf9 to make a Bgag VLP expressing one or more different target pathogen proteins.
An Improved Method for Vaccine Production
VLPs expressing one or more different target pathogen proteins, such as the quadri-subtype Bgag VLPs disclosed here, are superior to traditional vaccine production methods because they are egg independent and can be prepared in a single manufacturing cycle with no need for vaccine blending while still effectively inducing broad spectrum immunity against multiple target pathogen proteins. In one embodiment, the method involves cloning Influenza HA, NA, and Bgag genes into a single rBV vector for co-expression of the proteins in for Bgag VLP production. The advantage of a single rBV vector is that this results in co-expression of multiple genes in the infected Sf9 cell, which offers a more streamlined methodology and design that can be easily scaled up. Furthermore, this method allows cloning of strain-specific antigen of target Influenza proteins into a prefabricated rBV transfer vector comprising standard Bgag and NA genes, further streamlining the process of seasonal Influenza vaccine production. For example, the quadri-subtype Bgag VLP described herein is a superior vaccine candidate for seasonal Influenza strains as compared to current egg-dependent trivalent blended methods. Together, this superior design and methodology facilitates vector preparation and accelerates the VLP production and vaccine preparation.
In addition, the novel Bgag VLP described here is a more reliable vaccine production system in the event of a pandemic or emerging viruses. For example, a pandemic Influenza or a highly virulent emerging Influenza virus can place severe strains on traditional vaccine development and production. A pandemic or emerging Influenza virus can cause an epizootic in the agricultural poultry species. Thus, in addition to threatening human health, a pandemic or emerging Influenza virus can severely threaten the supply of healthy chickens and eggs, leading to a shortage of eggs suitable for vaccine production. Because the production of vaccines against pathogens other than Influenza, such as yellow fever, mumps and measles, also heavily depends on eggs, an epizootic will place additional strain on the production and availability of these egg dependent vaccines, further straining public health.
For at least these reasons, the novel uni- or multi-target Bgag VLP disclosed here is an important vaccine platform during and in advance of these dire situations. Moreover, the Bgag VLP platform can offer a fast, broad spectrum, and robust protection for humans and/or animals against dangerous pandemic, epizootic, or emerging pathogens. In the case of an outbreak involving H5, H7, H9, and/or H10 avian Influenza viruses, the H5/H7/H9/H10 quadri-subtype Influenza vaccine disclosed here would be a valuable first line of defense, particularly when alternative vaccines are not available yet.
Bgag VLP as a Superior Vaccine Candidate
In addition to the benefits described elsewhere in the specification, the novel Bgag VLP is a also superior vaccine candidate, and in particular, a superior human vaccine candidate. One reason is because the inner core protein Bgag has no considerable homology to human pathogens. The BIV is not a human pathogen, and thus, humans would typically not have pre-existing immunity against proteins from BIV, such as Bgag. Sequence analysis of Bgag using NCBI BLASTP software with the default parameters showed that the closest similarity was only approximately 29% genetic similarity with a feline immunodeficiency virus and approximately 26% genetic similarity with a equine infectious anemia virus. Further, the inventors have detected no similarity between the BIV and human retroviral gag proteins, including HIV gag proteins. Therefore, Bgag mediated VLPs have significant advantages as a candidate for human vaccine because it is capable of evading the host's pre-existing immunity and capable of eliciting stronger and more robust immune responses, than other VLPs such as the M1 VLPs.
In certain embodiments, each Bgag VLP comprises a large surface area, preferably a surface area that is greater than the pathogen in which one of the target proteins is derived from. As disclosed in this specification, the Bgag VLP with its larger size is superior to the smaller M1 VLPs. Larger surface of area can be especially important for the Bgag VLPs presenting multiple target proteins, such as the H5/7/9/10 Bgag VLPs described in Example 11, in which multiple HA subtypes co-localize to the same VLP envelope. The inventors have shown here that the novel Bgag VLPs had an average diameter of from about 150 nm to about 180 nm, as compared to an average of from about 120 nm to about 150 nm for M1 VLPs (see for example
In certain embodiments, each Bgag VLP comprises on average of more than about 375 protein spikes on the VLP membrane, preferably from about 375 spikes to about 800 spikes, more preferably from about 475 spikes to about 800 spikes, more preferably from about 575 spikes to about 800 spikes, more preferably from about 675 spikes to about 800 spikes, more preferably from about 775 spikes to about 800 spikes, and further preferably about 800 spikes. It has been estimated that a spherical virion of average diameter 120 nm has been estimated to contain approximately 375 spikes (Harris et al., 2006). Assuming an equal distribution of trimers, a VLP with a diameter of approximately 180 nm could accommodate approximately 800 spikes, more than twice the number of spikes as a VLP with only 120 nm diameter. Thus, the Bgag VLP as a vaccine candidate is superior to the M1 VLP because it can be more efficient at eliciting a host immune response and can elicit a more robust host immune response.
Using the Bgag Gene or Bgag Protein as a Diagnostic Tool
In certain embodiments, the Bgag gene or Bgag protein is used as a diagnostic tool. One of the problems with current vaccines and other VLP vaccines, such as a M1 VLP vaccine, is the problem of differentiating vaccinated subjects from nonvaccinated subjects. This would likely not be a problem for the Bgag VLP system. For example, as disclosed herein, unlike M1, the humans and certain animals are typically not exposed to the Bgag gene or Bgag protein. Accordingly, a medical professional can use the Bgag gene or Bgag protein to determine whether a subject was previously administered the Bgag VLP vaccine. This diagnostic capability would not be possible, in for example, M1 VLP mediated vaccines, because of the ubiquity of the M1 gene and M1 protein in the human and certain animal population. This can also be especially important in veterinary applications, for example, when differentiation of infected from vaccinated animals (“DIVA”) is important (Rahn et al., 2015; Suarez, 2012).
Bgag VLPs with Target Nucleic Acids
Nucleic Acid can Also be Incorporated into Bgag VLPs to Induce or Enhance the Immune Response of the Target.
Our recent data indicate that RNA can be incorporated into the Bgag VLPs (see for example,
Protein sequences of the inner core protein and the target pathogen proteins were obtained. Based on the indicated protein sequences, the genes are codon-optimized for high-level expression in Sf9 cells (Life Technologies, Carlsbad, Calif.) and synthesized biochemically (Genscript, Piscataway, N.J.). In order to generate VLPs, genes of the inner core protein Bgag or M1 and genes of one or more of the different target pathogen proteins such as Influenza HA and NA, are cloned in tandem fashion into the baculovirus transfer vector plasmid, such that each gene is within its own transcriptional cassette that included a polyhedrin promoter upstream from each gene. Four exemplary schematic diagrams are shown in
Expression and functionality profiles of Influenza VLP HA and NA proteins are shown in
The Influenza VLPs created from each of the four constructs of
The Influenza VLPs created from each of the constructs of
The Influenza VLPs created from constructs 1-2 of
The Influenza VLPs created from each of the constructs of
The VLPs comprise of the inner core protein Bgag has the same morphology as Influenza virus as viewed under the transmission electron microscope (see
The VLPs comprise of the inner core protein M1 has the same morphology as Influenza virus as viewed under the transmission electron microscope using the same conditions as
Examples of protein sequence source are as follows.
BIV R-29 gag: GenBank, accession number AAA42763.
Influenza IN/05 M1: GenBank accession number AB136004.
Influenza PR8 HA: GenBank accession numbers ABP64731,
Influenza VN/04 HA: GenBank accession numbers AAW80717,
Influenza SH/13 HA: GenBank accession numbers YP_009118475,
Influenza HK/09 HA: GenBank accession numbers AGO17847,
Influenza JX/13 HA: GenBank accession numbers AHK10762,
Influenza PR8 NA: GenBank accession number ABD77678
Influenza IN/05 NA: GenBank accession number ABW06107.
Ebola EboMay GP: sequence listed under
Ebola EboMay GP: SEQ ID No. 1 sequence listed under
Ebola EboMay GP-TMCT: SEQ ID No. 2 sequence listed under
Influenza HA gene sequences were derived from the PR8, VN/04, SH/13, HK/09, and JX/13 virus.
Protein sequences of Bgag and four different target pathogen proteins are codon-optimized and the genes of the respective proteins are cloned as in Example 1 into a single baculovirus transfer vector plasmid. For example, the four full length genes of different pandemic Influenza HA as well as Influenza IN/05 NA are cloned into the transfer vector plasmid essentially as shown in
Bacmids containing the full-length infectious baculovirus DNA with recombinant genes for Bgag VLPs are isolated from DH10Bac E. coli using a Bac-to-Bac baculovirus expression system (Life Technologies, Carlsbad, Calif.) and are used to transfect Sf9 cells to generate the rBV. Preparations of rBV are subsequently plaque-purified. The titers of rBV preparations are determined by standard plaque assay in Sf9 cells.
Sf9 cells are maintained as suspension cultures in SF90011-SFM insect serum free medium (Life Technologies, Carlsbad, Calif.) at 27° C. For production of VLPs, Sf9 cells are used at 2×106 cells/ml and infected at a multiplicity of infection (“MOI”) of 3.0 for 72 h with rBV expressing the targeted genes. VLPs are harvested from the growth medium supernatant, clarified by filtration through a 0.2μ membrane, and then concentrated and purified by using a 20% (w/v) sucrose step gradient in phosphate buffered saline (“PBS”). Alternatively, VLPs are first purified by ion exchange chromatography as described elsewhere (Liu et al., 2015) and then subsequently purified by ultracentrifugation.
The SDS-PAGE is done in 4-12% polyacrylamide gels (Life Technologies, Carlsbad, Calif.) followed by staining with GelCode Blue stain (Pierce, Rockford, Ill.).
The Western blots are done using specific primary antibodies followed by the alkaline phosphatase-conjugated goat anti-ferret IgG (H+L). Examples of primary antibodies used are: anti-HA Clade 1 Influenza A H1N1 Viruses IT-003-001M14 mouse IgG1 monoclonal antibody (MAb), clone 15B7; anti-H5(H5N1) IT-003-005M6 mouse IgG2a MAb, clone 268D8; anti-HA(H7N9)(A/Shanghai/1/2013) IT-003-0073M1 mouse IgG1 MAb, clone 9B12; anti-H9(A/Hong Kong/33982/2009)(H9N2) IT-003-0094M5 mouse IgG1 MAb, clone 17D8; and anti-H10(A/blue-winged teal/Louisiana/Sg-00073/07(H10N7)) IT-003-034 rabbit polyclonal antibody (Immune Technology, New York, N.Y.).
Protein concentration of the VLPs is determined by using Qubit 2.0 fluorometric method (Life Technologies).
Hemagglutination functional assays are generally done as follows. VLPs are serially diluted at 2-fold increments in 50 μl volume in a 96-well plate. To each VLP dilution, 50 μl of 1% turkey red blood cell (RBC) working solution is added, mixtures of VLPs and RBCs are gently agitated and the plate was incubated at room temperature for 30-60 minutes before examination. Negative hemagglutination results appeared as dots in the center of the wells. The titer is calculated as the highest dilution factor that produced a positive reading. A positive result of the Hemagglutination assay demonstrates that the VLPs express functional HA, the HA localizes to the membrane of the VLP and the HA are presented at the surface of the VLP.
Influenza NA enzyme functional assays are generally done as follows. The functional neuraminidase enzymatic activity is determined using a fluorescence-based NA assay (NA-Fluor, Life Technologies) with methyl umbelliferone N-acetyl neuraminic acid (MUNANA; Sigma, St Louis, Mo.) as a substrate according to manufacturer's instructions. Diluent (saline or PBS) was used as a negative control.
Transmission electron microscopy are done by absorbing VLP samples onto a freshly discharged 400 mesh carbon parlodion-coated copper grids (Poly-Sciences, Warrington, Pa.). The grids are rinsed with buffer containing 20 mM Tris, pH 7.4, and 120 mM KCl and negatively stained with 1% phosphotungstic acid, then dried by aspiration. VLPs are visualized on a Hitachi H-7600 transmission electron microscope (Hitachi High Technologies America, Schaumburg, Ill.) operating at 80 kV and digitally captured with a CCD camera at 1 k×1 k resolution (Advanced Microscopy Techniques Corp., Danvers, Mass.).
The genetic stability of rBV and stability of the expression of Bgag VLPs can be demonstrated by standard methods, such as serial passage of rBV in the carrier cell and the expression of VLPs containing target protein can be assessed. For example, expression of quadri-subtype Influenza Bgag VLP can be shown to be stable by first passing the rBV containing quadri-subtype Influenza Bgag VLP in Sf9 cells at MOI of 0.01 five times, then expressing VLPs and measuring the presence of functional HA in a Hemagglutination functional assay (see for example,
The co-localization of the target pathogen proteins in the Bgag VLPs can be demonstrated by standard methods, such as immuno-precipitation or immuno-electron microscopy. For example, H9, H10 and H7 can be shown to colocalize with the H5 on H5/H7/H9/H10 Influenza Bgag VLP by first immunoprecipitating the H5/H7/H9/H10 Influenza Bgag VLP using SureBeads magnetic beads (BioRad, Hercules, Calif.) and an anti-H5 antibody. SureBeads can first be bound to the anti-H5 antibody at 20° C. for 1 hour. Any unbound anti-H5 antibody can be washed away. Then Bgag VLPs are incubated to SureBeads with anti-H5 antibody. Any Bgag VLPs not bound to SureBeads are washed away. The Bgag VLPs captured can be eluted with Laemmli buffer at 70° C. for 10 minutes then analyzed on SDS-PAGE and/or Western blot using anti-H7, anti-H9, and/or H10 antibodies to confirm co-localization of the HA target proteins (see for example,
As shown in
The amount of each type of target pathogen protein present in the Bgag VLP can also be determined by standard methods, such as semi-quantitative Western blot. For example, the quantity of H5, H7, H9 and H10 present on the H5/H7/H9/H10 Influenza Bgag VLP can be measured by comparing the quadri-subtype Bgag VLP to purified H5, H7, H9, H9 protein standards using a Qubit 2.0 flurometric method (Life Technologies). The concentration of purified HA protein standard can be measured by comparing its densitometry on a stained SDS-PAGE to a BSA standard with known concentration. A linear standard curve for the BSA standard can be generated using NIH ImageJ Software and the amount of purified HA protein standard can be determined. To determine the concentration of each of the HA proteins in the quadri-subtype Bgag VLP, semi-quantitative Western blot can be performed, where the band intensity of quadri-subtype Bgag VLP is measured by densitometry and compared to the HA standard with known concentration (see, for example
As shown in
Based on the semi-quantitative Western blot, the distribution of HA in the quadri-subtype Bgag VLP are determined (see for example,
VLPs are prepared by using Bgag and Influenza PR8 proteins in Sf9 cell mediated recombinant baculovirus (rBV) expression system essentially as described in Example 1-Example 3. The Influenza PR8 H1 is cloned into rBV along with the full-length Bgag and NA genes essentially as shown in
All four preparations of VLPs exhibited HA activity with the turkey RBC with approximately equivalent titers, as shown in
The Influenza NA is not detected by Western blot or SDS-PAGE consistent with prior studies. However, VLPs exhibited NA enzyme activity in the functional NA assay, as shown in
Finally, the electron microscopic examination confirmed presence of the enveloped particles in the VLP preparations (
Taken together, these results confirmed formation of the Bgag VLPs expressing and presenting HA and NA, proteins.
The inventors have prepared Bgag VLPs comprising of emerging Influenza strains such as the JX/13 (H10N8) avian-origin virus using the methods and principles essentially as described in Example 1-Example 3 and Example 5. In 2013, JX/13 (H10N8) Influenza virus has caused human infection and was identified as a pathogen of pandemic concern (Garcia-Sastre and Schmolke, 2014; To et al., 2014). The JX/13 (H10N8) Influenza virus is originally isolated from the elderly patient in China, who died as the result of infection (Garcia-Sastre and Schmolke, 2014; To et al., 2014).
The rBV is configured to express three genes, the H10 gene derived from the JX/13 Influenza virus, the NA gene and the Bgag gene. An exemplary schematic diagram of an rBV construct expressing Influenza VLP comprising of HA 10 (“H10”) in Sf9 cells is shown in
The Influenza VLPs created from the constructs of
The Influenza VLPs created from the H10 construct of
Functional NA enzyme activity is also confirmed (see
The H10 VLPs created from the construct of
Preparation and assessment of Bgag VLPs can also be made using only HA or NA alone as the target pathogen protein using the methods and principles essentially described in Example 1-Example 6.
Preparation and assessment of Bgag VLPs can also be made by using a genetically modified HA and or NA using the methods and principles essentially described in Example 1-Example 7.
Using a similar principle and approach, Bgag VLPs using a genetically modified NA. For example, the Bgag VLPs can be designed to include only a partial sequences of the NA.
Preparation and assessment of Bgag VLPs can also be made by using the target protein of other non-Influenza pathogens using the methods and principles essentially described in Example 1-Example 8. For example, Bgag VLPs can be made by using Ebola virus glycoprotein (GP) such as EboMay GP, whose protein sequence is described in
As a further example, using the same methods and principles, Bgag VLPs can be made using target proteins from other filoviruses. The resulting Bgag VLP is highly useful as a pan-filovirus vaccine.
As a further example, Bgag VLPs can also be made by using MERS spike glycoprotein. The resulting Bgag VLP is highly useful as a vaccine to prevent MERS infections. As a further example, Bgag VLPs can also be made using target proteins from other coronaviruses. The resulting Bgag VLP is highly useful as a vaccine for coronaviruses.
As a further example, Bgag VLPs can also be made by using other antigens from the enveloped and non-enveloped viruses. For example, Bgag VLPs can also be made by using HIV env glycoprotein. The resulting Bgag VLP is highly useful as a vaccine against HIV infections. Bgag VLPs can also be made using target glycoproteins from other retroviruses to prepare vaccines against the retrovirus.
Preparation and assessment of Bgag VLPs can also be made using genetically modified target pathogen proteins to optimize interactions with Bgag using the methods and principles essentially described in Example 1-Example 9. For example, the glycoproteins of target enveloped viruses can be modified to create chimeric glycoproteins. The inventors have prepared an example such a chimeric target pathogen protein by genetically modifying EboMay GP to contain the transmembrane domain and the C-terminus of Influenza HA protein EboMay TMCT as shown in
EboMay GP-TMCT VLPs express relevant Ebola protein as demonstrated by Western blot (see
EboMay GP-TMCT VLPs express Bgag and relevant Ebola protein as demonstrated by SDS-PAGE gel stained with Coomassie (see
The EboMay GP-TMCT VLPs has the general, spherical envelop morphology as viewed under the transmission electron microscope (see
Like Bgag VLPs expressing other target pathogen proteins, both EboMay GP VLP and EboMay GP-TMCT Bgag VLP can induce immune response to Ebola GP epitopes and can serve as vaccine to prevent Ebola viral infections. For example, EboMay GP Bgag VLP and EboMay GP-TMCT Bgag VLP is injected or otherwise administered to a subject for the purpose to induce an immune response in the subject. The vaccinated subject's immune response against the Ebola virus is then determined by standard assays. For example, in 2 to 4 weeks, the blood of the vaccinated subject is drawn, and anti-Ebola antibody (or more specifically anti-EboMay GP antibody) is determined by ELISA, immunofluorescence antibody assay, or other antibody detection assays, and compared to the antibody profile of non-vaccinated subjects. The presence of anti-Ebola antibody in the vaccinated subject indicates that the Bgag VLP has elicited an immune response in the subject and thus, is immunogenic in vivo. Standard challenge studies can also demonstrate the efficacy of the Bgag VLP. For example, the vaccinated subjects that are seropositive are challenged with a pathogenic Ebola virus in a high-containment laboratory to evaluate the Bgag VLP vaccine's effectiveness in preventing disease causing Ebola. The survival of the vaccinated subjects post-challenge is indicative of the protective efficacy of the Bgag VLP vaccines.
The inventors have prepared and evaluated the first quadri-subtype Bgag VLPs using the methods and principles essentially described in Example 1-Example 10. The inventors have used HA proteins derived from the four avian-origin Influenza viruses, for example the H5N1, H7N9, H9N2, and H10N8 subtypes. The HPAI H5N1 virus, VN/04 of clade 1, is originally isolated from a fatal human case (Maines et al., 2005). The H7N9 virus, SH/13, is isolated from a hospitalized patient with a fatal disease (Gao et al., 2013). The H9N2 virus, HK/09 of G1 clade, is originally isolated from a nasopharyngeal aspirate of an adult female patient (Cheng, 2010). The JX/13 (H10N8) Influenza virus is originally isolated from the elderly patient in China, who died as the result of infection (Garcia-Sastre and Schmolke, 2014; To et al., 2014).
To prepare quadri-subtype VLPs, an rBV transfer vector plasmid is prepared as described in Example 2 to co-express the HA genes from VN/05, SH/13, HK/09, and JX/13 Influenza, NA genes from IN/05 and the Bgag gene. The H5, H7, H9, and H10 genes encode polypeptides of 568 aa, 560 aa, 560 aa, and 561 aa in length with predicted average molecular masses of 64.5 kDa, 62.1 kDa, 62.9 kDa, and 62.3 kDa, respectively. The NA and Bgag are 449 aa and 476 aa in length and has expected molecular masses of 49.1 kDa and 53.5 kDa, respectively.
An exemplary schematic diagram of an rBV construct expressing an Influenza quasi-subtype H5/7/9/10 Bgag VLPs in Sf9 cells is shown in
For preparation of the VLPs, Sf9 cells are infected with the rBV at an MOI of 3 to allow expression of H5, H7, H9, H10, NA, and gag genes, and the VLPs are harvested from culture supernatant on day 3 post infection. The growth medium from infected Sf9 cells is clarified by centrifugation and filtration through 0.2μ membrane. The VLPs are subsequently purified by ion exchange chromatography.
Influenza VLP morphology and the expression and functionality profiles the HA and NA proteins are shown in
The Influenza VLPs created from the construct of
The presence of Bgag and of HA proteins is confirmed by SDS-PAGE and Western blot using antibodies specific for each HA, as shown in
In
In
Together, these results suggested that all HA proteins, NA and Bgag proteins are present in the quadri-subtype VLPs produced by the Sf9 cells and co-purified by ultracentrifugation or chromatography.
The quadri-subtype H5/7/9/10 VLPs created from the construct of
Preparation and assessments of other quadri-subtype Bgag VLPs or multi-subtype Bgag VLPs can be done using the methods and principles essentially described in Example 1-Example 11. For example, multi-subtype Bgag VLPs comprising of H1, H2, H3 and other subtypes can be prepared.
In addition, multi-subtype Bgag VLPs comprising of Influenza B HA can also be prepared and assessed using the methods and principles essentially described in Example 1-Example 11.
In addition, multi-variant Bgag VLPs comprising of target protein variants of the same virus subtype can be prepared and assessed using the methods and principles essentially described in Example 1-Example 11. For example, distinct variants (clades) of H5N1 virus can be co-incorporated into the Bgag VLPs. For example, we have prepared multi-variant Bgag VLPs, in which three variants of H5N1 HA relevant for human vaccine, were co-expressed in the VLPs. The H5N1 HA we prepared can, for example, be derived from HPAI H5N1 viruses A/VietNam/1203/2004, A/Egypt/3300-NAMRU3/2008, and A/Hubei/1/2010, all of which are recommended by the WHO for H5N1 vaccine development. For example, we have also prepared multi-variant Bgag VLPs, in which three variants of H5N1 HA relevant for veterinary (poultry species) vaccine, were co-expressed in the VLP.
The H10 Bgag VLP is prepared for vaccination using an H10 from the JX/13 avian-origin influenza H10N8 virus essentially as described in Example 6. The JX/13 Influenza virus has caused a fatal human infection in an elderly patient in China in 2013 and has been identified as a pathogen of pandemic concern. The rBV vector is configured to co-express three genes, the H10, the NA and the Bgag essentially as described in Example 1 to Example 5. The resulting rBV is used to infect Sf9 cells to prepare the uni-subtype H10 Bgag VLP shown in for example,
The Bgag VLPs produced from Sf9 cells are then purified as essentially described in, for example, Example 3. The filtrate containing the Bgag VLPs migrates through the membrane and becomes the filtrate fraction, while the rBV vector particles are separated by the membrane. This is confirmed by anion exchange chromatography. For example, the anion exchange chromatograph shows the Bgag VLP is separated from the rBV and is present in the filtrate fraction (see for example,
Consistent with the previous observations, HA in the H10 Bgag VLP is expressed as HA0, with approximately 60-64 kDa and no detectable processing of HA1 and HA2 (see, for example,
As shown in Table 1, three lots of H10 Bgag VLPs are prepared and characterized. All H10 Bgag VLPs lots have hemagglutination activity, with a titer of 8,192 to 65,536 per 50 μl of Bgag VLPs with a concentration of approximately 3.5 mg/ml of total protein. Manufacturing or testing conditions contributes to the lot-to-lot variations.
Quadri-subtype H5/7/9/10 VLP is also prepared and purified for vaccination essentially as described in Example 11. Prior to vaccination, the Bgag VLPs can be formulated in PBS.
The expression of the H5, H7, H9 and H10 genes is evaluated by indirect immunofluorescence assay. For example, using H5, H7, H9 and H10 subtype-specific antibodies, the indirect immunofluorescence assay show the expression of each subtype is present (see for example,
The quadri-subtype H5/7/9/10 Bgag VLP is purified and analyzed by anion exchange chromatography (see for example,
Like H10 Bgag VLPs, the HAs on the quadri-subtype H5/7/9/10 Bgag VLP are uncleaved HA0 at approximately 62 to 64 kDa in size and consistent with 64.5 kDa, 62.1 kDa, 62.9 kDa, and 62.3 kDa predicted by the H5, H7, H9, and H10 genes, respectively (see for example,
As shown in Table 2, three lots of quadri-subtype H5/7/9/10 Bgag VLPs are prepared and characterized. All quadri-subtype H5/7/9/10 Bgag VLPs lots have hemagglutination activity as measured by hemagglutination function assays essentially described Example 4.
Indirect immunofluorescence assays can be performed. For example, 0.3 ml aliquots of rBV-infected Sf9 cells are seeded into eight-well Nunc LabTek slides. After 72 h incubation at 28° C., Sf9 cells are fixed with cold acetone, and indirect immunofluorescence microscopy is performed using H5, H7, H9, and H10 specific antisera. Examples of antisera used can be the same used for Western blot, essentially as described in Example 4. Cells expressing H5, H7, H9, and H10 are visualized using FITC-conjugated goat anti-mouse IgG (H+L) (KPL, Gaithersburg, Md.). Mounting medium containing propidium iodide nuclear counterstain is used in IFA to visualize cell nuclei.
As shown in
Hemagglutination inhibition assay can be performed. For example, all sera are treated with receptor-destroying enzyme from Vibrio cholerae (Denka Seiken, Tokyo, Japan) then hemagglutination inhibition assay is performed using freshly prepared 0.5% turkey or 1% horse red blood cells (RBCs) with 4 HA units of each indicated antigen.
Homologous Influenza antigens that are used to determine HI titers includes the full-length homologous recombinant antigens, rH5 derived from A/Viet Nam/1203/2004 (H5N1); rH9 from A/Hong Kong/33982/2009 (H9N2); rH7 from A/Anhui/1/2013 (H7N9), which has identical amino acid sequence as A/Shanghai/2/2013(H7N9)); and rH10 from A/Jiangxi-DongHu/346/2013 (H10N8). These reference antigens are expressed using BEVS and isolated and purified from Sf9 cells according to standard method.
Heterologous influenza antigens is also used for determination of Hemagglutination inhibition titers. They include, for example, BPL-inactivated viruses A/teal/Egypt/12908-NAMRU3/2005 (H10N1); A/California/07/2009 NYMC X-197A (H1N1); A/Switzerland/9715293/2013 (H3N2); and the BPL-inactivated H5N1 viruses A/Hong Kong/156/1997 (clade 0), A/Cambodia/X012331/2013 (clade 1.1.2), A/Indonesia/05/2005 (clade 2.1.3.2), A/bar-headed goose/Qinghai/1A/2005: PR8, (clade 2.2), and A/Bangladesh/3233/2011 (clade 2.2.2).
Bgag VLPs can be vaccinated in to subjects, such as adult male Fitch ferrets (4 to 5 months of age, Triple F Farms, Sayre, Pa.), using standard methods. For example, one group of four animals can be inoculated three times (weeks 0, 4 and 22) intramuscularly (“i.m.”) with H10 Bgag VLPs (30 μg of total HA). Two groups of five ferrets are vaccinated with quadri-subtype Bgag VLPs either i.m. or intranasally (“i.n.”) (120 μg of total HA containing 30 μg of each HA subtype). A control group of four ferrets received PBS as placebo. Prior to initial vaccination, the ferrets are confirmed to be serologically negative by hemagglutination inhibition assay for currently circulating influenza viruses. All ferrets are bled on weeks 4, 8, 12, 16, and 26 for collection of serum to assess specific antibody titers by hemagglutination inhibition. Animals are also bled after challenge with an Influenza virus, such as the A/teal/Egypt/12908-NAMRU3/2005 (H10N1) virus.
Bgag VLPs expressing H10 and Bgag VLPs expressing H5, H7, H9 and H10 are formulated in PBS for vaccinations. Ferrets vaccinated with H10 Bgag VLPs are administered at a dose of 30 μg of HA. Ferrets vaccinated with quasi-subtype H5/7/9/10 Bgag VLPs are administered at a dose of 120 μg of HA. Since H5, H7, H9, and H10 are present on the quasi-subtype H5/7/9/10 Bgag VLPs at approximately 20-30%, the 120 μg dose correspond to approximately 30 μg of each subtype. This administration regimen is consistent with that of H5N1 vaccines. For example, for the A/Vietnam/1203/04 (H5N1) vaccine, healthy adults were administered 2 i.m. injections of 2 doses of 45 μg and 90 μg resulted in detection of potentially protective neutralizing antibody (Treanor J J et al. 2006).
Two vaccination routes, the i.m. and the i.n. route are compared for the quadri-subtype Bgag VLP vaccine. The Vaccination regimen includes three vaccinations (primary vaccination and two boost vaccinations on weeks 0, 4 and 22). Hemagglutination inhibition titers against the homologous recombinant HA reference antigens rH5, rH7, rH9 and rH10 are measured. The serum of mock-vaccinated ferrets does not have HA neutralizing antibodies against any of the HA tested (see for example,
After primary vaccination of H10 Bgag VLPs via i.m., the HA neutralizing antibodies becomes detectable and increases after the first boost. Then, HA neutralizing antibody titers gradually decreased until the second boost. Because a relatively small number of ferrets (4-5/group) are used, considerable variation in responses are observed, especially in the i.n. samples.
Serum of subjects administered the H10 Bgag VLP by i.m. can have comparable HA neutralizing antibody titers as subjects administered the with the quadri-subtype Bgag VLPs (see for example
In the quadri-subtype H5/7/9/10 Bgag VLP-vaccinated groups, the lowest neutralizing antibody titers detected are against rH5, in which detectable neutralizing antibody titers required at least two vaccinations. This is consistent with the relatively low immunogenicity of the H5 subtype. In contrast, neutralizing antibody titers against rH7, rH9 and rH10 are detectable after a single vaccination in both i.m. and i.n groups, increases after a boost, and then gradually decreases before a second boost (see for example,
As shown in
To investigate cross-protective capabilities of VLP vaccinations, pre-challenge sera are tested by hemagglutinin inhibition assay with several Influenza isolates, including the H1, H3, H5 and H10 subtypes. No detectable cross-protective neutralizing antibody to H1 and H3 viruses are detected in sera from either H10 or quadri-subtype VLPs vaccinated ferrets after either i.m. or i.n. vaccinations. However, cross-reactive neutralizing antibody are detected when tested with multiple divergent isolates/clades of H5N1 viruses, as well H10N1 virus (see for example,
As shown in
Ferrets are anesthetized with an i.m. injection of a ketamine-xylazine-atropine cocktail and challenged by i.n. on week 27 with 106 EID50 of influenza A/teal/Egypt/12908-NAMRU3/2005 (H10N1) virus in a total volume of 1 ml (500 μl per nostril) diluted in PBS. Viral challenge can take place at week 27, five weeks after the final vaccination. Following the H10N1 challenge, ferrets are monitored daily for four days to observe changes in body weight and temperature, as well as clinical signs of illness. Nasal wash samples are collected at 2 and 4 days post-challenge (“p.c.”). At day 4 p.c., animals are euthanized and nasal turbinates, trachea, and lungs are collected. Virus titers in the samples from lower and upper respiratory tract are determined in eggs. The statistical significance of the differences of viral titer between vaccinated and PBS control subjects are determined by two-way ANOVA statistical analysis.
Following vaccination with either the H10 Bgag VLPs or the quadri-subtype Bgag VLPs, ferrets are challenged with a live target pathogen, for example the H10N1 Influenza virus. A heterologous challenge can be performed, for example, with the A/teal/Egypt/12908-NAMRU3/2005 (H10N1) virus, as the virus is geographically and evolutionarily distinct from the H10 antigen of the A/Jiangxi/IPB13a/2013 (H10N8) virus present in the Bgag VLP vaccines administered to the ferrets. Cross-reactive titers are confirmed by hemagglutination inhibition assay for the H10N1 virus (see for example,
Reduction of replicating virus titers is even more marked in the nasal turbinates and in the tissues of trachea and lung tissues (see for example,
As shown in
Sf9 cells are maintained as suspension cultures in SF90011-SFM insect serum free medium (Life Technologies) at 27±2° C. For production of H555 Bgag VLPs, 2 L of Sf9 cells at concentration 2×106 cells/ml are infected at a multiplicity of infection (MOI) of 3.0 with rBV expressing three indicated H5 genes, as well as NA and BIV gag genes. Bgag VLPs are harvested at 72 h postinfection from the growth medium supernatant, sterile filtered using 0.2 μm filter and concentrated by using tangential flow filtration (TFF, 500,000 MWCO) prior to purification. Bgag VLPs are purified by using ion exchange chromatography followed by ultracentrifugation. Anion exchange chromatography was used to remove major impurities such as baculovirus and host cell DNA using standard methods. Then, ultracentrifugation is used to purify and concentrate the Bgag VLPs using standard methods. Bgag VLP preparations are characterized and stored at 2-8° C. in a PBS buffer until vaccinations. Characterization of the Bgag VLPs, including by SDS-PAGE and Western blot, are performed essentially herein.
Vaccination and viral challenges Bgag VLP H555 vaccine are formulated with a commercial adjuvant (SEPPIC, Montanide 70/30, Fairfield, N.J.) to contain 1536 HA units per dose of H555 VLPs. Because all three H5 genes are expressed from the identical expression cassettes in Sf9 cells, it is expected that H5 clades are present at similar levels in the H555 VLPs. Specific Pathogen-Free (SPF) chickens (n=30) are vaccinated subcutaneously with 0.2 ml of H555 Bgag VLP at day 1 of age and 0.5 ml at day 21 of age. Control birds can receive PBS. Birds are arbitrarily placed into groups of 10 and challenged intranasally (106 EID50 per bird) with one of the following HPAI isolates at day 35: GyrF H5N8 (clade 2.3.4.4); Ck/WJ H5N1 (clade 2.1.3) or Ck/Egypt H5N1 (clade 2.2.1). After challenges, birds are monitored for clinical signs daily, serum are taken at day 0 and day 14 post challenge, and oral swabs were taken to measure virus shedding.
To determine virus shedding, oropharyngeal and cloacal swabs are collected in sterile brain heart infusion medium and kept frozen at −70° C. Viral RNA are extracted using Trizol LS reagent (Invitrogen, Calsbad, Calif.) and the MagMAX AI/ND Viral RNA Isolation Kit (Ambion, Austin, Tex.). Quantitative real time RT-PCR (qRRT-PCR) is performed using standard methods. Briefly, qRRT-PCR targeting the Influenza M gene is conducted using AgPath-ID one-step RT-PCR Kit (Ambion) and the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Calsbad, Calif.). For viral quantification, a standard curve is established with viral RNA extracted from the titrated challenge virus, GyrF H5N8, Ck/WJ H5N1 or Ck/Egypt H5N1. Results are reported as EID50/ml equivalents and the lower limit of detection being 100.9 EID50/ml for samples from chickens.
Statistical analysis Kaplan-Meier survival curves are generated with Prism 5 (GraphPad Co., San Diego, Calif.). The Mantel-Cox log-rank test is used to compare survival curves between the experimental groups (Prism 5). Statistical differences in mean and standard error between hemagglutinin inhibition titers are analyzed using ANOVA (Prism 5). Lower case letters indicate statistical significance between compared groups. The Student t-test is used for pair-wise comparison of virus titers from oral and cloacal swabs (Prism 5). All statistical tests are performed using p<0.05.
Following vaccination with Bgag VLPs and prior to challenge, antibody levels against the vaccine antigen are determined by hemagglutinin inhibition assay. As shown in
Sham and vaccinated birds are challenged with one of three HPAI viruses two weeks after vaccination. Pre-challenge and p.c. HA neutralizing antibody titers to challenge viruses are shown on
Vaccine protection is measured by survival and reduction in viral shedding in chickens following virus challenges with three H5 viruses (see for example
Virus shedding is significantly reduced against the three H5 isolates tested (see for example
Accordingly, triple-clade H555 Bgag VLP immunization offers significant protection against lethal Influenza following challenges with two distinct isolates of H5N1 virus and H5N8 HPAI virus.
The following publications, references, patents and patent applications are hereby incorporated by reference in their entireties.
This application is a National Stage of PCT/US2016/040838, filed Jul. 1, 2016, and designating the United States (published on Jan. 5, 2017, as WO 2017/004586 A1), which claims priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 62/188,084, filed Jul. 2, 2015, each hereby expressly incorporated by reference in its entirety and each assigned to the assignee hereof.
This subject matter was made in part with U.S. government support under Grant Number AI111532-02 awarded by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health and Grant Number 2011-33610-30433 awarded by the National Institute of Food and Agriculture at the United States Department of Agriculture. The U.S. government may have certain rights in the subject matter.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/040838 | 7/1/2016 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/004586 | 1/5/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 8236558 | Luo et al. | Aug 2012 | B2 |
| 20060088909 | Compans et al. | Apr 2006 | A1 |
| 20100143406 | Smith et al. | Jun 2010 | A1 |
| 20100247574 | Mahmood et al. | Sep 2010 | A1 |
| 20110159031 | Falkner et al. | Jun 2011 | A1 |
| 20110171733 | Luo et al. | Jul 2011 | A1 |
| 20110217274 | Reid | Sep 2011 | A1 |
| 20130295135 | Smith et al. | Nov 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 60335139 | Jan 2011 | DE |
| 1576143 | Sep 2005 | EP |
| 2015-15954 | Jan 2015 | JP |
| 2535365 | Dec 2014 | RU |
| 2008094200 | Aug 2008 | WO |
| 2015066715 | May 2015 | WO |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20180369364 A1 | Dec 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62188084 | Jul 2015 | US |
