Patent Application
20220202929
Influenza is a disease caused by influenza virus infection of the respiratory tract epithelium that has a global impact, causing a high percentage of morbidity and mortality every year. Influenza pandemics in human populations due to rapid viral evolution can spread globally within months or even weeks at unpredictable intervals. Vaccine development that is initiated upon emergence of a pandemic is not sufficient to prevent or mitigate the first pandemic wave.
The present disclosure provides, in some aspects, methods of identifying influenza virus variants likely to evolve during human transmission, under the selective pressure of anti-influenza drug therapies. Being able to predict the emergence of such variants would allow the development and stockpiling of effective vaccines and other immunogenic compositions for preventing and/or treating otherwise drug-resistant strains of influenza virus. This early development and stockpiling should enable early prevention and/or containment of influenza virus infection by newly emerging variant strains, thus preventing an influenza pandemic.
Some aspects of the present disclosure provide methods comprising (a) evolving a parent strain of influenza viral particles in cell culture comprising human airway (e.g., lung) cells in the presence of an anti-influenza drug, and (b) isolating drug-resistant progeny influenza viral particles released from the human airway cells. In some embodiments, the evolving step comprises culturing human airway cells that comprise a drug-sensitive parent strain of influenza viral particles in cell culture comprising an anti-influenza drug for a period of time sufficient to inhibit viral replication and/or viral spread of at least 70% of the influenza viral particles (to reduce the influenza viral titer by at least 70%, relative to baseline (prior to expose to the drug)), and/or culturing human airway cells that comprise progeny of the influenza viral particles in cell culture comprising the anti-influenza drug.
Other aspects of the present disclosure provide methods comprising (a) culturing human airway (e.g., lung) cells that comprise a drug-sensitive parent strain of influenza viral particles in cell culture that comprises an anti-influenza drug for a period of time sufficient to inhibit viral replication and/or viral spread of a subset of the influenza viral particles (to reduce the influenza viral titer), (b) culturing human airway cells that comprise progeny of the influenza viral particles in cell culture that comprises the anti-influenza drug, and (c) isolating drug-resistant progeny influenza viral particles released from the human airway cells.
In some embodiments, the methods further comprises sequencing viral RNA obtained from the drug-resistant progeny influenza viral particles to identify a drug-resistant strain of influenza virus comprising a mutation in its genome, relative to the parent strain of influenza virus.
Also provided herein, in some aspects, are immunogenic compositions comprising an influenza virus matrix 2 (M2) antigen variant that comprises an amino acid substitution at position 31 and an amino acid substitution at position 34, relative to a H1N1 influenza virus M2 antigen, wherein the H1N1 influenza virus M2 antigen comprises the amino acid sequence of SEQ ID NO: 3. Other aspects provide immunogenic compositions comprising an influenza virus matrix 2 (M2) antigen variant that comprises an amino acid substitution at position 31 and an amino acid substitution at position 46, relative to a H1N1 influenza virus M2 antigen, wherein the H1N1 influenza virus M2 antigen comprises the amino acid sequence of SEQ ID NO: 3.
In some embodiments, the amino acid substitution at position 31 is S31N. In some embodiments, the amino acid substitution at position 34 is G34E. In some embodiments, the amino acid substitution at position 46 is L46P. In some embodiments, the influenza virus M2 antigen variant comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza virus M2 antigen variant comprises an amino acid sequence that shares at least 95% identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza virus M2 antigen variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the influenza virus M2 antigen variant comprises an amino acid sequence that shares at least 95% identity with the amino acid sequence of SEQ ID NO: 2.
Further provided herein, in some embodiments, are methods comprising administering to a subject the immunogenic composition of any one of the embodiments of the present disclosure in an effective amount to induce in the subject an antigen-specific immune response (to influenza).
One of the greatest challenges for prevention and treatment of influenza virus is the rapid rate at which the virus evolves as it spreads through human populations. The accumulation of mutations in the viral genome is responsible for influenza antigenic shift over time, which results in the emergence of new influenza virus strains, limiting the effectiveness of current anti-influenza drugs and vaccines. Thus, inhibiting the ability of influenza virus to rapidly change is a major challenge for the design of novel anti-influenza drugs and new vaccines. The World Health Organization analyzes a large amount of data relating to the antigenic and genetic characteristics of influenza virus every year, predicts the possibly emerging influenza virus strains, and provides recommendations regarding the antigens to be used to create influenza vaccines for the following influenza season. Based on this recommendation, pharmaceutical and vaccine regulatory agencies develop, produce, and license influenza virus vaccines under a greatly accelerated and highly expensive time frame. Nonetheless, there is a lag behind the evolution of influenza virus strains, and it has not yet been possible to develop a new anti-influenza drug or vaccine fast enough to combat a new virus strain immediately as it emerges.
In some aspects, the present disclosure provides methods for identifying and/or predicting the emergence of drug-resistant influenza viruses. There are two main types of influenza (flu) virus: types A and B. The influenza A and B viruses that routinely spread in people (human influenza viruses) are responsible for seasonal flu epidemics each year. Influenza A viruses can be broken down into sub-types depending on the genes that make up the surface proteins. Over the course of a flu season, different types (A & B) and subtypes (e.g., influenza A) of influenza circulate and cause illness.
There are four types of influenza viruses: A, B, C and D. Human influenza A and B viruses cause seasonal epidemics of disease almost every winter in the United States. The emergence of a new and very different influenza A virus to infect people can cause an influenza pandemic. Influenza type C infections generally cause a mild respiratory illness and are not thought to cause epidemics. Influenza D viruses primarily affect cattle and are not known to infect or cause illness in people. Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus: the hemagglutinin (H) and the neuraminidase (N). There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes. (H1 through H18 and N1 through N11 respectively.) Influenza A viruses can be further broken down into different strains. Current subtypes of influenza A viruses found in people are influenza A (H1N1) and influenza A (H3N2) viruses. In the spring of 2009, a new influenza A (H1N1) virus (CDC 2009 H1N1 Flu website) emerged to cause illness in people. This virus was very different from the human influenza A (H1N1) viruses circulating at that time. The new virus caused the first influenza pandemic in more than 40 years. That virus (often called “2009 H1N1”) has now replaced the H1N1 virus that was previously circulating in humans. Herein, “H1N1” refers to any H1N1 virus circulating in humans. Influenza B viruses are not divided into subtypes, but can be further broken down into lineages and strains. Currently circulating influenza B viruses belong to one of two lineages: B/Yamagata and B/Victoria. See, e.g., cdc.gov/flu/about/viruses/types.htm (Centers for Disease Control and Prevention website).
Some methods of the present disclosure comprise evolving and/or culturing a parent strain of influenza viral particles in cell culture comprising human airway (e.g., lung) cells in the presence of an anti-influenza drug. Other methods of the present disclosure comprise culturing a drug-sensitive parent strain of influenza viral particles in cell culture comprising human airway cells in the presence of an anti-influenza drug.
The parent strains of influenza virus (and/or the progeny) may be any one of the four types of influenza viruses, although in preferred embodiments, the parent strain of influenza virus is an influenza type A virus, an influenza type B virus, or an influenza type C virus.
In some embodiments, an influenza A strains is selected from the following subtypes: H1N1, H1N2, H1N3, H1N8, H1N9, H2N2, H2N3, H2N8, H3N1, H3N2, H3N8, H4N2, H4N4, H4N6, H4N8, H5N1, H5N2, H5N3, H5N6, H5N8, H5N9, H6N1, H6N2, H6N4, H6N5, H6N6, H6N8, H7N1, H7N2, H7N3, H7N7, H7N8, H7N9, H8N4, H9N1, H9N2, H9N5, H9N8, H10N3, H10N4, H10N7, H10N8, H10N9, H11N2, H11N6, H11N9, H12N1, H12N3, H12N5, H13N6, H13N8, H14N5, H15N2, H15N8, H16N3, H17N10, and H18N11. In some embodiments, the strain of influenza virus is an influenza A (H1N1) strain. In some embodiments, the strain of influenza virus is an influenza A (H3N2) strain. In some embodiments, the strain of influenza virus is an influenza A (H5N1) strain. Non-limiting examples of particular strains of influenza virus include influenza A/WSN/33 (H1N1), influenza A/Hong Kong/8/68 (H3N2), and influenza A/Avian Influenza (H5N1), influenza A/Netherlands/602/2009 (H1N1), and influenza A/Panama/2007/99 (H3N2).
An influenza virion is roughly spherical and the basic structure includes a lipid bilayer outer membrane, which harbors glycoproteins HA (hemagglutinin) and NA (neuraminidase), the proteins that determine the subtype of influenza virus, and the ion channel M2. Beneath the lipid bilayer is a matrix protein (M1), which forms a shell, giving strength and rigidity to the outer membrane. Within the interior of the virion are viral RNAs, referred to as RNA segments, that code for one or two proteins. Each RNA segment includes RNA joined with several proteins, including B1, PB2, PA, NP. These RNA segments are the genes of influenza virus. The interior of the virion also contains another protein referred to as NEP.
When an influenza virus infects a cell, the individual RNA segments of the influenza virus are replicated in the nucleus. The replicated RNA segments are exported to the cytoplasm, and are incorporated into new viral particles that bud from the cell. If a cell is infected with multiple different influenza virus strains, replicated RNA segments from one virus strain can be incorporated into viral particles with replicated RNA segments from another virus strain to form a reassorted influenza virus. Reassortment refers to influenza viruses containing RNA segments from more than influenza virus strain.
Reassortment of influenza virus in vivo gives rise to new influenza virus strains. These new influenza virus strains can rapidly spread through a naïve population and can lead to an influenza outbreak. A naïve population has never encountered an antigen (e.g., influenza virus antigens) and thus has no immunity against the antigen. Methods of predicting the reassortment of influenza virus can be used to predict new influenza virus strains that can lead to outbreaks. Thus, the present disclosure also provides methods for predicting influenza gene reassortment.
A drug-sensitive influenza virus is an influenza virus that, when contacted with (exposed to, cultured in the presence of) one or more anti-influenza drug (e.g., cultured in the presence of the drug or otherwise exposed to the drug in vitro or in vivo) no longer enters into host cells, no longer replicates (multiplies) in host cells, no longer releases from host cells, and/or no longer spreads throughout the host—the virus is inhibited. While a particular influenza virus strain may be considered drug-sensitive (e.g. sensitive to oseltamivir), there may be a certain percentage (e.g., less than 30%, less than 20%, or less than 10%) of viral particles among a particular population of influenza viral particles of a particular strain that are not drug sensitive. These viral particles that are not sensitive to the drug—that continue to replicate and/or spread in the presence of the drug—are considered drug resistant. Thus, a drug-resistant influenza virus is an influenza virus that, when contacted with one or more anti-influenza drug (e.g., cultured in the presence of the drug or otherwise exposed to the drug in vitro or in vivo) continues to replicate—viral replication is not inhibited.
An anti-influenza drug is a drug that inhibits (e.g., prevents/inactivates) activity or expression of an influenza viral protein. In some embodiments, an anti-influenza virus drug inhibits influenza virus M1 protein, M2 protein, HA protein, NA protein, or a viral polymerase (e.g., subunit PB1, PA, and/or P3). Non-limiting examples of anti-influenza drugs (drugs that inhibit replication and/or spread of an influenza virus) include oseltamivir (TAMIFLU®), peramivir (RAPIVAB®), zanamivir (RELENZA®), amantadine (SYMMETREL®), rimantadine (FLUMADINE®), and baloxavir marboxil (XOFLUZA®). In some embodiments, the anti-influenza drug is oseltamivir (TAMIFLU®). In some embodiments, the anti-influenza drug is peramivir (RAPIVAB®). In some embodiments, the anti-influenza drug is zanamivir (RELENZA®). In some embodiments, the anti-influenza drug is amantadine (SYMMETREL®). In some embodiments, the anti-influenza drug is rimantadine (FLUMADINE®). In some embodiments, the anti-influenza drug is baloxavir marboxil (XOFLUZA®).
In some embodiments, the anti-influenza drug inhibits the matrix 2 (M2) protein on the surface of the influenza virus. Anti-influenza drugs that inhibit the M2 protein decrease the replication of the influenza viral particle. In some embodiments, the anti-influenza drug that inhibits the M2 protein of the influenza virus is amantadine or rimantadine.
In some embodiments, the anti-influenza drug inhibits the neuraminidase (NA) protein on the surface of the influenza virus. Anti-influenza drugs that inhibit the NA protein decrease the secretion of influenza viral particles and thus inhibit influenza virus spread. In some embodiments, the anti-influenza drug that inhibits the NA protein of the influenza virus is oseltamivir, peramivir, or zanamivir.
More than one drug may be used in the methods described herein. In some embodiments, a cell culture includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anti-influenza drugs. In some embodiments, a cell culture includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 anti-influenza drugs. In some embodiments, a cell culture includes one anti-influenza drug. In some embodiments, a cell culture includes two anti-influenza drugs.
The concentration of anti-influenza drug used herein may vary. In some embodiments, the anti-influenza drug(s) is present in the cell culture at a concentration of 0.5 μM to 10 μM. For example, the anti-influenza drug(s) may be present in the cell culture at a concentration of 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, or 10 3 μM. In some embodiments, the anti-influenza drug(s) is present in the cell culture at a concentration of 0.5-10 μM, 0.5-9 μM, 0.5-8 μM, 0.5-7 μM, 0.5-6 μM, 0.5-5 μM, 0.5-4 μM, 0.5-3 μM, 0.5-2 μM, or 0.5-1 μM.
Some methods herein comprise (a) evolving a (one or more) parent strain of influenza viral particles in cell culture comprising human airway (e.g., lung cells) in the presence of an anti-influenza drug, and (b) isolating drug-resistant progeny influenza viral particles released from the human airway cells.
“Evolving” an influenza virus comprises, in some embodiments, culturing the influenza virus under conditions that result in the emergence of a viral mutation that confers a survival benefit to the influenza virus. For example, evolving a parent strain of influenza viral particles may comprise culturing human airway cells that comprise a drug-sensitive parent strain of influenza viral particles in cell culture that includes an anti-influenza drug for a period of time sufficient to inhibit viral replication and/or viral spread of a subset of the influenza viral particles, and then culturing human airway cells that comprise progeny of the influenza viral particles in cell culture that comprises the anti-influenza drug. In some embodiments, the methods comprises culturing human airway cells that comprise a drug-sensitive parent strain of influenza viral particles in cell culture that includes an anti-influenza drug for a period of time sufficient to reduce influenza viral titer (e.g., by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, relative to baseline), and then culturing human airway cells that comprise progeny of the influenza viral particles in cell culture that comprises the anti-influenza drug.
Culturing refers to maintaining infected airway cells in vitro in conditions that promote growth and proliferation. In some embodiments, culturing includes to changing the media (passaging) in which infected airway cells are maintained. In some embodiments, infected cells are cultured for up to 4 weeks in the presence of an anti-influenza drug. In some embodiments, infected cells are cultured for up to 3 weeks in the presence of an anti-influenza drug. In some embodiments, infected cells are cultured for up to 2 weeks in the presence of an anti-influenza drug. In some embodiments, infected cells are cultured for up to 4 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, or 4 weeks in the presence of an anti-influenza drug.
In some embodiments, human airway cells comprising a drug-sensitive parent strain of influenza viral particles are cultured in the presence of an anti-influenza drug for a period of time sufficient to inhibit viral replication and/or viral spread (secretion from a host cell, e.g., a human airway cell) of at least 50% of the influenza viral particles. In some embodiments, the drug-sensitive parent strain of influenza viral particles are cultured for a period of time sufficient to inhibit viral replication and/or viral spread of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the influenza viral particles. In some embodiments, human airway cells comprising a drug-sensitive parent strain of influenza viral particles are cultured in the presence of an anti-influenza drug for a period of time sufficient to reduce influenza viral titer by at least 50%, relative to baseline. In some embodiments, human airway cells comprising a drug-sensitive parent strain of influenza viral particles are cultured in the presence of an anti-influenza drug for a period of time sufficient to reduce influenza viral titer by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, relative to baseline. In some embodiments, the drug-sensitive parent strain of influenza viral particles is cultured in the presence of an anti-influenza drug (e.g., oseltamivir (TAMIFLU®), peramivir (RAPIVAB®), zanamivir (RELENZA®), amantadine (SYMMETREL®), rimantadine (FLUMADINE®), and baloxavir marboxil (XOFLUZA®)) for a period of time sufficient to inhibit viral replication and/or viral spread of at least 90% of the influenza viral particles.
In some embodiments, human airway cells comprising the parent strain of influenza viral particles are cultured for a period of time sufficient to enable multiple rounds of viral replication. For example, human airway cells comprising the parent strain of influenza viral particles may be cultured for a period of time sufficient to enable at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 rounds of viral replication.
The period of time any population of human airway cells is cultured may depend on the desired result, for example, inhibition of viral replication in a certain percentage of the population, or emergence of a certain percentage of drug-resistant progeny viral particles. In some embodiments, the period of time is at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 60 hours. In some embodiments, the period of time is 12-60 hours, 12-48 hours, 12-36 hours, 12-24 hours, 24-60 hours, 24-48 hours, 24-36 hours, 36-60 hours, 36-48 hours, or 48-60 hours.
Replication of a virus can be determined/monitored by measuring viral titer, for example. Viral titer is a measure of the quantity of virus in a given volume. Non-limiting methods of measuring viral titer include viral plaque assay, quantitative polymerase chain reaction (qPCR) of viral proteins, 50% tissue culture infectious dose assay (TCID50), and focus forming assay. A decreased viral titer is indicative of a decrease in viral replication and thus viral spread. An increased viral titer is indicative of an increase in viral replication and thus viral spread.
In some embodiments, the viral titer is reduced by at least 90% in cells cultured in the presence of anti-influenza drug compared with cells not cultured in the presence of the anti-influenza drug. In some embodiments, the viral titer is reduced by at least 50%. In some embodiments, the viral titer is reduced by at least 75%. In some embodiments, contacting the infected airway cells with the anti-influenza drug reduces viral titer by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to infected airway cells that are not contacted with the anti-influenza drug.
Human airway cells comprising progeny influenza viral particles, in some embodiments, are cultured in the presence of the anti-influenza drug until the rate of viral replication increases to greater than 50% (the rate of inhibition of viral replication decreases). For example, human airway cells comprising the progeny influenza viral particles, in some embodiments, are cultured in the presence of the anti-influenza drug until the rate of viral replication increases to greater than 60%, greater than 70%, greater than 80% or greater than 90%.
Thus, in some embodiments, methods herein comprise culturing human airway cells that comprise a drug-sensitive strain of influenza viral particles in cell culture comprising the drug until the rate of viral inhibition reaches at least 50% (at least 50% of the viral particles are inhibited), and culturing human airway cells that comprise progeny of the influenza viral particles in cell culture comprising human airway cells in the presence of the anti-influenza drug until the rate of viral replication increases to at least 50%. In some embodiments, human airway cells that comprise the drug-sensitive strain of influenza viral particles are cultured until the rate of viral inhibition reaches at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, human airway cells that comprise the progeny of the influenza viral particles are cultured until the rate of viral replication increases to at least 60%, at least 70%, at least 80%, or at least 90%.
Culturing of human airway cells that comprise the progeny influenza viral particles, in some embodiments, comprises passaging (subculturing) human airway cells comprising viral particles of the parent strain and/or of progeny of the parent strain. Passaging refers to the process of renewing the cell culture growth media, e.g., to enable further propagation of the viral particles. In some embodiments, the human airway cells are passaged at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 times, to produce the drug-resistant progeny influenza viral particles. In some embodiments, the human airway cells are passaged 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 10-50, 10-40, 10-30, 10-25, 10-20, or 20-25 times, to produce the drug-resistant progeny influenza viral particles.
Methods herein, in some embodiments, comprise isolating drug-resistant progeny influenza viral particles (e.g., released from human airway cells). Isolating refers to separating viral particles from the culture (e.g., any components in the culture, such as cells). Isolating may be by any method known or developed in the art, including viral plaque assay formation, trypan blue staining, and magnetic sorting using DynaBeads (ThermoFisher Scientific). For example, the drug-resistant progeny influenza viral particles may isolated from drug-resistant virus pools through viral plaque purification. Other isolation/purifications may be used.
The method, in some embodiments, further comprise sequencing viral RNA obtained from the drug-resistant progeny influenza viral particles to identify a (one or more) drug-resistant strain of influenza virus comprising a mutation in its genome, relative to the parent strain of influenza virus. Any sequencing method may be used. See, e.g., Marston D A et al. BCM Genomics 2013; 14:444; Goya S et al. PLoS One 2018; 13(6): e0199714; and Keller M W et al. Scientific Reports 2018; 8(14408): 1-8, each of which is incorporated herein by reference.
The influenza viral particles herein evolved under the selective pressure of an anti-influenza drug may acquire one or more mutation (e.g., in a viral protein, such as M1 protein, M2 protein, HA protein, NA protein, and/or a viral polymerase (e.g., subunit PB1, PA, and/or P3)) that confers resistance to the anti-influenza drug. The mutation may be any mutation that results in a change in the amino acid sequence of the progeny viral particles, relative to the parent viral particles. Examples of mutations include point mutations (substitutions), insertions, and deletions. The mutation may be any one, or any combination, of the foregoing mutations. In some embodiments, the influenza viral particles acquire at least 2 mutations in an influenza viral protein. In some embodiments, the live infected airway cells comprise at least 3, 4, 5, 6, 7, 8, 9, or 10 mutations in an influenza viral protein.
Also provided herein are methods of developing (producing) a vaccine or other immunogenic composition against the drug-resistant strain of influenza virus. Methods of making influenza virus (flu) vaccines are known, including egg-based flu vaccines, cell-based flu vaccines, and recombinant flu vaccine. See, e.g., Centers for Disease Control and Prevention website (cdc.gov) and the U.S. Food and Drug Vaccine Product Approval Process, each of which is incorporated herein by reference. Non-limiting examples of vaccines that may be developed as provided herein include live-attenuated virus vaccines, inactivated viral vaccines, recombinant viral vaccines, polypeptide vaccines, DNA vaccines, RNA vaccines, and virus-like particles.
The present disclosure provides, in some embodiments, immunogenic compositions for preventing and/or treating influenza (influenza virus infection). These compositions (e.g., pharmaceutical compositions) include at least one influenza virus antigen, or nucleic acid encoding influenza virus antigen, of a variant influenza virus strain identifying using the methods of the present disclosure. Antigens are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens). An immunogenic fragment induces or is capable of inducing an immune response to influenza. It should be understood that the term “protein” encompasses polypeptides and peptides and the term “antigen” encompasses antigenic fragments.
In some embodiments, an immunogenic composition comprises an influenza virus matrix 2 (M2) antigen variant that comprises an amino acid substitution at position 31, relative to a H1N1 influenza virus M2 antigen, wherein the H1N1 influenza virus M2 antigen comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the influenza virus M2 antigen variant further comprises an amino acid substitution at position 34. In some embodiments, the influenza virus M2 antigen variant further comprises an amino acid substitution at position 46. In some embodiments, the influenza virus M2 antigen variant further comprises an amino acid substitution at position 31 and at position 34. In some embodiments, the influenza virus M2 antigen variant further comprises an amino acid substitution at position 31 and at position 46. In some embodiments, the amino acid substitution at position 31 is S31N. In some embodiments, the amino acid substitution at position 34 is G34E. In some embodiments, the amino acid substitution at position 46 is L46P. In some embodiments, the influenza virus M2 antigen variant comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza virus M2 antigen variant comprises an amino acid sequence that shares at least 90% identity or at least 95% identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the influenza virus M2 antigen variant comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the influenza virus M2 antigen variant comprises an amino acid sequence that shares at least 90% identity or at least 95% identity with the amino acid sequence of SEQ ID NO: 2.
Further provided herein, in some embodiments, is a method comprising administering to a subject (e.g., a human subject) the immunogenic composition of any one of the embodiments of the present disclosure in an effective amount to induce in the subject an antigen-specific immune response. In some embodiments, the antigen-specific immune response is a neutralizing antibody response. A “an effective amount” of an influenza immunogenic composition/vaccine is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polypeptide (e.g., length, three-dimensional structure, and/or amino acid composition), other components of the composition/vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of an influenza immunogenic composition/vaccine provides an induced or boosted immune response as a function of antigen production in the cells of the subject.
In some embodiments, an immunogenic composition further comprises a carrier selected from biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. In some embodiments, an immunogenic composition further comprises an excipient and/or adjuvant.
The cell cultures described herein, in some embodiments, include a human small airway-on-chip device. The device, in some embodiments, comprises a polymer chip comprising a membrane that separates (a) an air channel; (b) a microvascular channel; and (c) a membrane, wherein the membrane comprises an epithelium layer exposed to the air channel and an endothelium layer exposed to the microvascular channel. In some embodiments, the air channel is above and/or parallel to the microvascular channel.
In some embodiments, the polymer chip comprises poly(dimethylsiloxane) (PDMS). Other polymers may be used.
In some embodiments, the air channel has a height of 0.5 mm to 2 mm (e.g., 0.5 mm, 1.0 mm, 1.5 mm, or 2 mm). In some embodiments, the air channel has a width of 0.5 mm to 2 mm (e.g., 0.5 mm, 1.0 mm, 1.5 mm, or 2 mm). In some embodiments, the air channel has a diameter of 0.5 mm to 2 mm (e.g., 0.5 mm, 1.0 mm, 1.5 mm, or 2 mm).
In some embodiments, the microvascular channel has a height of 0.1 mm to 2 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1 mm, 1.5 mm, or 2 mm). In some embodiments, the microvascular channel has a width of 0.1 mm to 2 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1 mm, 1.5 mm, or 2 mm). In some embodiments, the microvascular channel has a diameter of 0.1 mm to 2 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 1 mm, 1.5 mm, or 2 mm).
In some embodiments, the membrane is a porous membrane. In some embodiments, the porous membrane comprises 0.2 μm to 10 μm pores (e.g., 0.2 am, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 m.
In some embodiments, membrane has a thickness of 5 am to 15 am (e.g., 5 am, 6 am, 7 m, 8 μm, 9 μm, or 10 μm.
In some embodiments, the membrane is a polyester membrane. Other membrane materials may be used. In some embodiments, the membrane is coated with collagen, for example, type IV collagen. As discussed below, the epithelium layer of the membrane, in some embodiments, comprises primary human lung airway epithelial cells (hLAECs). In some embodiments, the endothelium layer of the membrane comprises primary human lung microvascular endothelial cells (hLMVECs). In some embodiments, the epithelium and/or endothelium layer(s) comprises lung airway epithelial cells and/or lung microvascular endothelial cells that are generated from induced pluripotent stem cells (iPSCs).
The device (e.g., microfluidic device), in some embodiments, has at least one channel (e.g., microchannel) comprising human airway cells, a port at both ends of each microchannel, and one or more pumps for moving a fluid across the at least one microchannel. A microchannel is a channel with a diameter that is less than or equal to 1 millimeter (mm). The ports are sites for the introduction of agents, factors, or cells into the device and for the removal of fluid from the device.
A device of the present disclosure may comprise more than one microchannel. In some embodiments, the device comprises at least two channels (e.g., microchannels). The channels may be configured to mimic a human airway, in which there is an upper microchannel and a lower microchannel separated by a membrane. The membrane may be porous to allow passage of liquids, cells, agents, and/or factors between the upper and the lower channels. In some embodiments, the membrane is coated with extracellular matrix (ECM) proteins to facilitate culture of airway cells. In some embodiments, the ECM proteins are type I collagen, type II collagen, type III collagen, and/or type IV collagen.
Influenza virus primarily infects cells of the airway (e.g., airway epithelium, lung epithelium, airway endothelium, lung endothelium, alveoli). Cells are cultured in the device to mimic the airway of a subject. Airway cells are cells found in the airway of mammals (e.g., humans). The airway refers to the respiratory system, which comprises cells of the pharynx, trachea, and lung (e.g., bronchus, bronchioles, and alveoli). Non-limiting examples of airway cells include epithelial cells, endothelial cells, blood cells, immune cells, cartilaginous cells, and alveoli. The airway comprises epithelial cell and endothelial cell layers, in some embodiments. The epithelial cells are the primary site of influenza infection. Infected epithelial cells signal to endothelial cells to initiate immune cell recruitment. In some embodiments, the cells are epithelial cells (e.g., airway epithelium, lung epithelium). In some embodiments, the cells are endothelial cells (e.g., airway endothelium, lung endothelium). In some embodiments, the cells are epithelial cells and endothelial cells.
Infecting airway cells with an influenza virus refers to contacting airway cells with an influenza virus under conditions that allow infection (e.g., 37° C., 5% CO2). Infection of airway cells may be confirmed by any method known or developed in the art. Non-limiting methods of confirming influenza virus infection include microscopy to detect the presence of viral particles in the cytoplasm of cells, identification of virial particles budding and being secreted from infected cells, and quantitative PCR (qPCR) using primers that hybridize to influenza virus genes, but not airway cell genes.
Different types of microfluidic chips made of poly(dimethylsiloxane) (PDMS) containing an upper channel (1 mm high×1 mm wide, similar to the radius of human bronchiole) and a parallel lower microvascular channel (0.2 mm high×1 mm wide) separated by a thin (10 μm), porous, polyester membrane coated on both sides with type IV collaged to construct the human small airway structures (
Therefore, different from the previous models, the human lung airway-on-a-chip could effectively recapitulate the structures and functions of in vivo healthy lung bronchioles and sustain them for more than two months in vitro.
To develop the human small airway-on-a-chip as an influenza virus infection model, the epithelium was inoculated with influenza virus via the air channel, mimicking the infection in vivo (
Immunofluorescence confocal microscopic analysis showed that the influenza virus infection damaged the junctions and tissue integrity of epithelium and endothelium (
Rapid and direct assessment of the replication capacity of an influenza virus in the upper and conducting airways of humans can provide an important parameter used to assess the zoonotic and pandemic threat posed by emerging influenza viruses. To verify the ability of human small airway chip to assess the viral replication competence, the replication kinetics of influenza A/WSN/33 (H1N1) virus was compared and a human influenza virus strain, e.g., A/Hong Kong/8/68/(H3N2), on chips constructed with human small airway cells from healthy individuals or people with COPD. It was found that the viral titers of both H1N1 and H3N2 viruses increased gradually after inoculation (
Cellular tropism could strongly influence influenza severity and pathogenicity [Am J Pathol. 2010 April; 176(4):1614-8]. To show the small airway chip can be used to explore the tropism of influenza viruses, the cellular tropism of three influenza viruses, e.g., H1N1, H3N2, and H5N1 was tested (data not shown). They exhibited different cellular tropism: all three influenza viruses infected goblet cells; a high number of ciliated cells were infected by H1N1 and H3N2 viruses, with none infected by H5N1 virus; a small portion of club cells were infected by all three influenza viruses; and basal cells were infected by H5N1 but not H1N1 or H3N2 (data not shown). Thus, the model can be used to explore the viral tropism of different influenza strains in human and provide information for the prediction of influenza severity and the study of viral pathogenicity.
Collectively, the influenza infection model in the human small airway chip provided results that were consistent with the those observed in clinical studies. Thus, this method can be exploited as an alternative physiologically relevant experimental model for broadening virology research in human physiological environment. In particular, this could include investigation of virus infectivity, replication competence, virulence, and tissue tropism in humans in vitro that could be used to assess the pandemic threat of the emerging influenza viruses, which is a major goal of the World Health Organization (WHO).
The human small airway-on-a-chip influenza infection model was used to identify a subset of influenza variants that could potentially emerge as a result of evolution during spread from human to human. Knowing these variants would allow one to develop vaccines that can be manufactured in advance and administered to populations as soon as a given variant is identified in the population.
The clinically approved anti-influenza drugs amantadine and oseltamivir were used to identify drug-resistant influenza strains using the human small airway-on-a-chip model. Amantadine targets the M2 protein of influenza viruses, which is an ion channel allowing protons to move through the viral envelope to uncoat viral RNA and thus, it blocks the release of viral RNA into the cytoplasm. Oseltamivir (TAMIFLU®) targets the neuraminidase (NA) protein of influenza virus, inhibiting its enzymatic activity and causing the tethered progeny virus to be unable to escape from the host cell.
1 μM of amantadine inhibited ˜90% amantadine-sensitive influenza A/WSN/33 strain (H1N1) (
The results show that the inhibition rate of 1 μM of amantadine on influenza virus is ˜90% (
The propensity of oseltamivir to induce viral resistance was also explored (
The ability of the human small airway-on-a-chip model to mimic the influenza virus evolution through gene recombination that causes antigen drift and shift of influenza virus sequences, which is often responsible for the reduction of the efficacy of influenza vaccines in clinical populations was studied. In this proof-of-principle study, the human airway chips were co-infected by the two virus strains, e.g., influenza A/WSN/33 (H1N1) virus (MOI=0.01) and influenza A/Hong Kong/8/68 (H3N2) virus (MOI=0.01), and cultured for 48 h. The progeny virus strains were isolated through plaque purification, and their genomes were sequenced. The sample preparation procedure for sequencing is as follows: The isolated drug-resistant virus strains were cultured in MDCK.2 cells, total RNA was isolated from cells using TRIzol. Then the first strand of cDNA was synthesized using AMV reverse transcriptase (Promega, Madison, Wis., USA) with a random primer and an oligo (dT) primer, according to manufacturer's specifications.
Ten reassortant virus strain variants were detected in the progeny viruses isolated from human airway co-infected by H1N1 and H3N2 viruses (
These results suggest that the influenza human small airway-on-a-chip can be used to mimic the gene recombination of influenza viruses and predict potentially novel emerging reassortants that might cause pandemics. Hundreds of influenza viruses have been identified have been identified in the past. Gene recombination and reassortant between these hundreds of influenza viruses can be explored extensively in the human airway chip so that we can predict the potential emerging reassortants that have increased virulence, and hence may cause influenza pandemics. Therefore, the model can provide substantial information for influenza vaccine design.
PCR was carried out using the Phusion Hot Start Flex 2× Master Mix (New England BioLab, USA) with 30 μl of a reaction mixture containing primers specific for different influenza A/WSN/33 (H1N1) gene segments. The PCR conditions were 1 cycle at 98° C. for 2 min, followed by 30 cycles at 98° C. for 15 sec, 55° C. for 30 sec, 72° C. (30 sec/kb), and finally 1 cycle at 72° C. for 5 min. The resulting PCR products were gene sequenced. The viral genome sequencing could be also done through next generation sequencing service provided by many sequencing companies.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
The terms “about” and “substantially” preceding a numerical value mean±10% of the recited numerical value.
Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/850,113, filed May 20, 2019, which is incorporated by reference herein in its entirety.
This invention was made with government support under HL141797 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/033590 | 5/19/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62850113 | May 2019 | US |
