Patent Application
20120282273
The swine H1N1 influenza virus is currently causing a world-wide pandemic associated with substantial morbidity and mortalityl1-5. This newly emergent strain is immunologically distinct from other influenza viruses including recent H1N1 strains6 thus leaving a large population of the world highly susceptible to infection by this pandemic virus7. Although there is some B cell cross-reactivity with the seasonal influenza viruses the protective epitopes of the swine H1N1 virus appear to be quite distinct.
Described herein are recombinant antibodies (e.g., human monoclonal antibodies) against the swine H1N1 influenza virus.
Described herein are antibodies derived from plasmablasts isolated from patients during (or shortly after) infection with the novel influenza virus. Among the antibodies described herein is an antibody that binds with particularly high affinity, is highly specific to swine H1N1 virus, and is able to mediate hemagglutination-inhibition at low concentrations. In vivo experiments showed that this antibody is able to fully protect mice challenged with a lethal dose of swine H1N1 virus. The antibody is also able to cure mice in a therapeutic setting when treated as late as up to 60 hours (2.5 days) after infection with swine H1N1 virus. Such antibodies have great potential as a human therapeutic or prophylactic agent against the novel swine H1N1 influenza.
In one aspect, the recombinant antibodies described herein include all or part of the amino acid sequence of SEQ ID NO:1 (light chain) and/or all or part of the amino acid sequence of SEQ ID NO:2 (heavy chain). Within the light chain, the variable domain includes all or part of the sequence of SEQ ID NO:9 and can include one or more of CDR1-light (SEQ ID NO:3), CDR2-light (SEQ ID NO:4) and CDR3-light (SEQ ID NO:5). Within the heavy chain, the variable domain includes all or part of the sequence of SEQ ID NO:10 and can include one or more of CDR1-heavy (SEQ ID NO:6), CDR2-heavy (SEQ ID NO:7) and CDR3-heavy (SEQ ID NO:8).
Described herein is an isolated antibody or an antigen-binding fragment thereof that specifically binds the antigen bound by an H1N1 antibody having a light chain consisting of the amino acid sequence of SEQ ID NO:1 and a heavy chain consisting of the amino acid sequence of SEQ ID NO:2. In various embodiments: the antibody or antigen-binding fragment thereof binds H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd of equal to or less than 10−9, 10−10 or 6×10−11); the antibody or antigen-binding fragment thereof binds recombinat HA from H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd equal to or less than 10−9, 10−10 or 9×10−11); the antibody comprises a light chain variable region comprising the amino acids sequences of SEQ ID NOs: 3, 4, and 5; the antibody comprises a heavy chain variable region comprising the amino acids sequences of SEQ ID NOs: 6, 7, and 8; the antibody is a human antibody; the antibody is an IgG antibody; the antibody is an IgG1 antibody; the antibody is an IgG1, kappa antibody; the antibody is an IgG1, lambda antibody; the antibody is selected from an IgM, IgA, IgD and IgE antibody; the antigen-binding fragment is selected from a Fab, a F(ab′)2 fragment, a Fd fragment, an Fv fragment, and a dAb fragment; the antibody is a scFv.
Also described is an isolated antibody or antigen-binding fragment thereof wherein the antibody comprises: (a) polypeptide comprising the amino acid sequences of one or more of SEQ ID NOs: 3, 4, and 5; and (b) polypeptide comprising the amino acid sequences of one or more of SEQ ID NOs: 6, 7, and 8. In various embodiments: the isolated antibody or antigen-binding fragment thereof comprises: (a) polypeptide comprising the amino acid sequences of two or more of SEQ ID NOs: 3, 4, and 5; and (b) polypeptide comprising the amino acid sequences of two or more of SEQ ID NOs: 6, 7, and 8; the isolated antibody or antigen-binding fragment thereof comprises: (a) polypeptide comprising the amino acid sequences of SEQ ID NOs: 3, 4, and 5; and (b) polypeptide comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8; the isolated antibody or antigen-binding fragment thereof comprises a first polypeptide comprising, in the amino terminal to carboxy terminal direction amino acid sequences of two or more of SEQ ID NOs: 3, 4, and 5, wherein there are 10-20 amino acids between SEQ ID NOs: 3 and 4 and between SEQ ID NOs: 4 and 5; and a second polypeptide comprising, in the amino terminal to carboxy terminal direction amino acid sequences of two or more of SEQ ID NOs: 6, 7, and 8, wherein there are 10-20 amino acids between SEQ ID NOs: 6 and 7 and between SEQ ID NOs: 7 and 8: the antibody or antigen-binding fragment thereof binds H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd of equal to or less than 10−9, 10−10 or 6×10−11); the antibody or antigen-binding fragment thereof binds recombinat HA from H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd equal to or less than 10−9, 10−10 or 9×10−11); the antibody comprises a light chain variable region comprising the amino acids sequences of SEQ ID NOs: 3, 4, and 5; the antibody comprises a heavy chain variable region comprising the amino acids sequences of SEQ ID NOs: 6, 7, and 8; the antibody is a human antibody; the antibody is an IgG antibody; the antibody is an IgG1 antibody; the antibody is an IgG1, kappa antibody; the antibody is an IgG1, lambda antibody; the antibody is selected from an IgM, IgA, IgD and IgE antibody; the antigen-binding fragment is selected from a Fab, a F(ab′)2 fragment, a Fd fragment, an Fv fragment, and a dAb fragment; the antibody is a scFv.
Also described is an isolated antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NOs: 3, 4, and 5 and a heavy chain variable region comprising SEQ ID NOs: 6, 7, and 8. In various embodiments: In various embodiments: the antibody or antigen-binding fragment thereof binds H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd of equal to or less than 10−9, 10−10 or 6×10−11); the antibody or antigen-binding fragment thereof binds recombinat HA from H1N1 (e.g., A/CA/04/2009 H1N1) with a Kd equal to or less than 10−9, 10−10 or 9×10−11); the antibody comprises a light chain variable region comprising the amino acids sequences of SEQ ID NOs: 3, 4, and 5; the antibody comprises a heavy chain variable region comprising the amino acids sequences of SEQ ID NOs: 6, 7, and 8; the antibody is a human antibody; the antibody is an IgG antibody; the antibody is an IgG1 antibody; the antibody is an IgG1, kappa antibody; the antibody is an IgG1, lambda antibody; the antibody is selected from an IgM, IgA, IgD and IgE antibody; the antigen-binding fragment is selected from a Fab, a F(ab′)2 fragment, a Fd fragment, an Fv fragment, and a dAb fragment; the antibody is a scFv.
Also described is a composition comprising an antibody or antigen binding fragment thereof described herein and a pharmaceutically acceptable carrier.
Also described is a method for treating or reducing one or more symptoms of infection with H1N1 in a human subject, the method comprising administering an antibody or antigen binding fragment thereof described herein.
Also described is a method of reducing the risk of becoming infected with H1N1, the method comprising administering an antibody described herein.
Naturally-occurring antibodies are immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies or antigen binding fragments is also according to that of Kabat.
Each CDR can included amino acid residues from a complementarity determining region as defined by Kabat (i.e. about residues 24-34 (CDR-L1), 50-56 (CDR-L2) and 89-97 (CDR-L3) in the light chain variable domain (SEQ ID NO:1) and 31-35 (CDR-H1), 50-65 (CDR-H2) and 95-102 (CDR-H3) in the heavy chain variable domain (SEQ ID NO:2); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a hypervariable loop (i.e. about residues 26-32 (CDR-L1), 50-52 (CDR-L2) and 91-96 (CDR-L3) in the light chain variable domain (SEQ ID NO:1) and 26-32 (CDR-H1), 53-55 (CDR-H2) and 96-101 (CDR-H3) in the heavy chain variable domain (SEQ ID NO:2); Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
Framework regions are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) of SEQ ID NO:1) and the heavy chain FR residues are positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) of SEQ ID NO:2. If the CDRs comprise amino acid residues from hypervariable loops, the light chain FR residues are positioned about at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain (SEQ ID NO:1) and the heavy chain FR residues are positioned about at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain (SEQ ID NO:2). In some instances, when the CDR comprises amino acids from both a CDR as defined by Kabat and those of a hypervariable loop, the FR residues will be adjusted accordingly.
An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.
The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F(ab′)2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.).
Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
The antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
An antigen binding portion of an antibody specifically binds to an antigen (e.g., H1N1). It has been shown that the antigen-binding function of an antibody can be performed by portions of a full-length antibody, all of which are encompassed by the general term antibody, including: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544 546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883). Single chain Fv and other forms of single chain antibodies, such as diabodies are also encompassed by the general term antibody. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak et al. (1994) Structure 2:1121).
An antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047). Antibody portions, such as Fab and F(ab′)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.
Human antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
Recombinant antibodies are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (Taylor et al. (1992) Nucl. Acids Res. 20:6287) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences or variants thereof to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences or variants thereof. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that may not naturally exist within the human antibody germline repertoire in vivo.
The studies described below analyzed the B cell responses in patients infected with swine H1N1 virus. As part of these studies we generated a panel of virus specific human monoclonal antibodies. These antibodies were isolated from plasmablasts that were activated by infection providing a means to directly evaluate the breadth and repertoire of the antibody response elicited by swine H1N1 virus. Interestingly, a majority of these antibodies also reacted with seasonal influenza viruses. In fact, several of the antibodies bound with higher affinity to past influenza strains than to the current swine H1N1 virus. These findings suggest that the swine H1N1 virus predominantly activated memory B cells previously generated against cross-reactive but non-protective epitopes present in annual influenza strains. Of the influenza specific antibodies generated five bound to recombinant hemagglutinin (HA) protein and of these only one antibody showed hemagglutination-inhibition (HAI) activity against the swine H1N1 influenza virus. In contrast to most of the other antibodies generated, this neutralizing antibody was highly specific for the swine H1N1 virus and did not cross-react with the other H1N1 influenza viruses, confirming that the critical HA active-site epitopes in this new virus are quite unique. In vivo experiments showed that this antibody was able to protect mice challenged with a lethal dose of mouse-adapted swine H1N1 influenza virus. Moreover, it was effective therapeutically even when administered 60 hours after infection and could thus potentially be developed as a therapeutic agent against the swine H1N1 influenza virus pandemic.
The novel 2009 pandemic swine H1N1 influenza virus is characterized by a unique genetic make-up1,2,8 that results in little or no pre-existing serum antibody mediated protection against infection7,9. It is currently unclear what effect this has on the repertoire of responding B cells in infected patients and whether infection with this novel virus leads to activation of cross-reactive memory B cells or if the response is dominated by newly induced naive B cells. To analyze the repertoire of the responding B cells after infection and to generate monoclonal antibodies (mAbs) against the swine H1N1 influenza strain, we examined the B cell responses in five patients infected with swine H1N1 virus. The clinical details about these patients are given in the supplemental methods section. Blood samples were taken 1-2 weeks after onset of clinical symptoms and were used to isolate infection-induced plasmablasts (CD19+, CD20−, CD3−, CD38high and CD27high cells) by flow cytometry based cell sorting (
In order to determine how specific the antibody response was to the swine H1N1 virus strain, the 53 monoclonal antibodies were screened by ELISA for reactivity to various influenza antigens (
It is worth noting that the sole HAI+mAb, EM4C04 (
As indicated in
The studies show that the antibody responses induced in patients infected with the novel swine H1N1 influenza appear to be dominated by a recall response of non-protective memory B cells that are cross-reactive to annual influenza strains. Of the 25 virus-specific monoclonal antibodies generated herein only one displayed HAI activity against the swine H1N1 virus. This low frequency of cells producing protective antibodies after infection differs significantly as compared to previous work on seasonal influenza vaccines12, where 40% of the virus specific antibodies bound with high affinity to HA and half of those antibodies had HAI activity against the influenza vaccine viral strains. As the novel swine H1N1 vaccine is now becoming widely available15-18, it will be of interest to compare the vaccine induced antibody responses to the responses induced by infection as described herein. Finally, the in vivo protection experiments presented here demonstrate that the human monoclonal antibody EM4C04 has impressive prophylactic and therapeutic activity in mice and shows potential for development as a therapeutic agent against the pandemic swine H1N1 influenza virus in humans.
Patients were recruited with IRB approval and had ongoing or recent verified swine H1N1 infections. HAI titers, inhibiting antibody concentrations, and viral neutralization were determined by standard procedures as previously described12,19. The ASCs were identified herein as CD3−/CD20−/low/CD19+/CD27hi/CD38hi cells as previously described11,12. The single cell RT-PCR methods and the procedures for production of recombinant mAbs were as previously describee10-12. Monoclonal antibodies were screened against fresh influenza virions grown in chicken eggs. ELISA was performed on starting concentrations of 10 ug/ml of virus or rHA and on 1:20 dilution of the vaccines and antibody affinities (Kd) were calculated by nonlinear regression analysis as previously described12. For immunoprecipitation, 1 μg each of recombinant HA protein and antibody were incubated at 4° C. overnight in 100 μl NP40 Buffer prior to precipitation with Protein G-Sepharose. The samples were denatured for 5 min at 95° C. in Laemmli gel sample buffer followed by centrifugation to remove the Protein GSepharose and analysis on 12% Tris-Glycine polyacrylamide gels. Precipitated protein bands were identified by staining with Sypro-orange and Fluorescence imaging. For the challenge experiments, female Balb/c mice (8 weeks old) were challenged intra-nasally with 3×xLD50 of a highly pathogenic, mouse-adapted swine H1N1 influenza virus (A/California/04/09) that was passaged in mice for five generations. Mice were treated intraperitoneally with 200 ug (10 mg/kg of body weight) of the specific mAb EM4C04 at all time points. All mice were monitored daily for morbidity and body weight changes.
All studies were approved by the Emory University, University of Chicago and Columbia University institutional review boards (Emory IRB#22371 and 555-2000, U of C IRB# 16851E, CU IRB#AAAE1819). Patient 1 (EM) is a 30-year old healthy woman who developed fever, cough and progressive dyspnea over 8 days prior to hospital admission. She was diagnosed with acute respiratory syndrome (ARDS), which required mechanical ventilation. Her nasopharyngeal swab on admission was positive for influenza by RTPCR. She continued shedding virus (hospital day 13) despite treatment with oseltamivir, but had cleared the virus by day 15 with continued treatment. Her course was further complicated by bacterial pneumonia, pulmonary embolism, and a requirement for prolonged oscillatory ventilator support and tracheostomy. She gradually recovered and was discharged to home two months after becoming ill. Blood samples for PBMC preparation were collected 19 days and 29 days after the onset of symptoms. Patient 2 (SF) is a 37-year old man with a history of hypertension and interstitial lung disease of unknown etiology who was hospitalized with symptoms of fever, cough, shortness of breath, nausea and vomiting for 3 days. He was diagnosed with pneumonia, acute sinusitis and acute renal failure. His nasopharyngeal swab on admission was positive for influenza virus by culture and was confirmed as the swine H1N1 influenza virus by RTPCR. He was initially treated with oseltamivir for 5 days but was continuing to shed influenza virus and was discharged with a course of zanamivir. He was hospitalized for a total of 8 days and recovered. PBMCs were collected 18 days after the onset of symptoms. Patient 3 is a 25 year old male who developed cough and fever to 103° F. The diagnosis of 2009 H1N1 influenza was confirmed by RT-PCR. He was treated with oseltamivir and his symptoms lasted for 4 days. He recovered completely and blood samples were collected 9 days after the onset of symptoms. Patient 4 is a previously healthy, 40-year old man who developed symptoms consistent with mild upper respiratory tract illness, including cough, rhinorrhea, and fever. MassTag PCR analysis of a nasopharyngeal swab specimen obtained 6 days after symptom onset identified H1N1 influenza virus; the presence of swine H1N1 influenza virus was subsequently confirmed by RT-PCR. Blood samples for PBMC isolation were obtained 13 days after the onset of symptoms. Patient 5 is a 52 year old female whose diagnosis of 2009 H1N1 influenza A was confirmed by RT-PCR. Her symptoms included fever, cough, pharyngitis, myalgias, nausea, headache, and gastrointestinal symptoms. She was treated with oseltamivir and her symptoms resolved after 6 days and she recovered completely. Blood samples were collected 10 days after the onset of symptoms.
All work with samples from infected patients was performed in a designated BSL2+ facility at Emory University. Peripheral blood mononuclear cells (PBMC) were isolated using Vacutainer tubes (Becton Dickinson, BD), washed, and resuspended in PBS with 2% FCS for immediate use or frozen for subsequent analysis. Plasma samples were saved in −80C.
The Swine H1N1 influenza virus (A/California/04/2009) was kindly provided by Dr. Richard J Webby at St. Jude Childrens Hospital. Influenza virus stocks used for the assays were freshly grown in eggs, prepared and purified as described19 and the hemagglutination activity (HA) was determined using turkey red blood cells (Lampire Biological Laboratories, Pipersville, Pa.) as previously described12,19 or purchased as inactivated preparations (ProSpec-Tany TechnoGene Ltd., Rehovot, Israel) and included: A/California/04/2009 (H1N1), A/FM/1/47 (H1N1), A/PR8/34 (H1N1), A/New Jersey/76 (H1N1), A/New Caledonia/20/9 (H1N1), A/Solomon Island/3/2006, A/Wisconsin/67/2005 (H3N2), and B/Malaysia/2506/2004. Vaccines tested included the 2006/7 vaccine from Chiron Vaccines Limited (Liverpool, UK) and the 2008/9 formulation from Sanofi Pasteur Inc. (Swiftwater, Pa.). Recombinant HA proteins were provided by the influenza reagent resource (IRR; influenza reagent resource.org) of the CDC (rHA from A/California/04/2009 (H1N1) (#FR-180), A/Brisbane/10/2007 (H1N1) (#FR-61), A/Brisbane/59/2007 (H3N2) (#FR-65)) or by Biodefense & Emerging Infections research repository (BEI; www.beiresources.org) (rHA from A/Indonesia/05/2005).
Analytical flow cytometry analysis was performed on whole blood following lysis of erythrocytes and fixing in 2% PFA. All live cell sorting was performed on purifiedPBMCs in the BSL-3 facility at the Emory Vaccine Center. All antibodies for bothanalytical and cell sorting cytometry were purchased from Pharmingen, except anti-CD27 that was purchased from ebiosciences. Anti-CD3-PECy7 or PerCP, anti-CD20-PECy7 or PerCP, anti-CD38-PE, anti-CD27-APC and anti-CD19-FITC. ASCs were gated and isolated as CD19+CD3−CD20lowCD27highCD38high cells. Flow cytometry data was analyzed using FlowJo software.
Identification of antibody variable region genes were done essentially as previously described10,11. Briefly, single ASCs were sorted into 96-well PCR plates containing RNase inhibitor (Promega). VH and Vic genes from each cell were amplified by RT-PCR and nested PCR reactions using cocktails of primers specific for both IgG and IgA as previously describee10,11 and then sequenced. To generate recombinant antibodies, restriction sites were incorporated by PCR with primers to the particular variable and junctional genes. VH or Vκ genes amplified from each single cell were cloned into IgG1 or Igκ expression vectors as previously describee10,11. Heavy/light chain plasmids were co-transfected into the 293A cell line for expression and antibodies purified with protein A sepharose.
Whole virus, recombinant HA or vaccine-specific ELISA was performed on starting concentrations of 10 ug/ml of virus or rHA and on 1:20 dilution of the vaccine as previously described12. The hemagglutination inhibition (HAI) titers were determined as previously described11,19. Affinity estimates were calculated by nonlinear regression analysis of curves from 8 dilutions of antibody (10 to 0.125 μg/ml) using GraphPad Prism.
For immunoprecipitation, 100 μl NP40 Buffer (20 mM Tris-HCl PH8.0, 137 mM NaCl, 10% Glycerol, 1% NP-40, 2 mM EDTA) containing complete Protease Inhibitors (Roche) was mixed with 1 μg of recombinant HA protein and incubated on ice for 30 min. One microgram of monoclonal antibody was then added. The antibody and HA mixture was incubated at 4° C. overnight with constant agitation. On the next day, Protein G-Sepharose (GE Healthcare) was prepared in NP40 buffer at a volume of 10 μl/sample. Protein GSepharose was incubated with the antibody and HA mixture at 4C for 4 hrs with constant agitation. The protein G-Sepharose was centrifuged for 3 min at 3000 rpm and the pellet was washed with 400 μl of NP40 buffer for 3 times. Finally the pellet was resuspended into 25 μl of Laemmli gel sample buffer (Bio-Rad). The samples were then boiled for 5 min at 95C. The protein G was pelleted and 15 μl of supernatant was loaded onto 12%Tris-Glycine polyacrylamide gels. The gels were run in 1×TGS at 70V for 30 min, followed by 120V till the frontline ran out of the gel. The gels were stained with 1× Sypro-orange (Invitrogen) in 7.5% acetic acid for 1 hr, and then gels were destained with 7.5% acetic acid for 3 min. Gels were finally scanned in a Typhoon 9410 Fluorescence imaging system (GE Healthcare).
In vivo Protection Experiments
Female Balb/c mice 6-8 weeks old were used for the challenge studies. Mice were inoculated intra-nasally with 3xLD50 of a highly pathogenic, mouse-adapted swine H1N1 influenza virus (A/California/04/09) that was passaged in mice five generations. The LD50 was determined by the method of Reed and Muench. The experiments were conducted in accordance with ethical procedures and policies approved by the Emory University's Institutional Animal Care and Use Committee. In order to determine the prophylactic efficacy of the mAb, mice were treated intraperitoneally with 200 μg (10 mg/kg of body weight) of the specific mAb EM4C04. Twelve hours later mice were challenged with 3xLD50 of the mouse adapted H1N1 virus. All mice were monitored daily for any signs of morbidity and mortality. Body weight changes were registered daily for a period of 14 days. All mice that lost more that 25% of their initial body weight were sacrificed according to the IACUC guideless. In order to determine the therapeutic efficacy of the EM4C04 mAb, mice were challenged with 3xLD50 of the mouse-adapted swine H1N1 virus. At various times post infection (12, 24, 36, 48, 60 hours) mice were treated intraperitoneally with 200 μg (10 mg/kg of body weight) of the specific mAb EM4C04. All mice were monitored daily and the body weight changes were registered daily as described above.
Data was collected and graphed using MS Excel and Graphpad Prism software. Efficacy of the therapeutic and challenge experiments was evaluated by ANOVA using Graphpad Prism software.
Described below are the sequences of the EM4C04 heavy chain and light chain
The CDR described herein can be grafted into the following vectors encoding human IgG and kappa chains, as well as others: Fully human IgG (GenBank® Accession No: FJ475055) and Fully human kappa (GenBank® Accession No: FJ475056).
Antibodies described herein can be used in any method that antibodies produced by other means cane be used. Thus, they can be used in passive therapy and diagnosis. Passive antibody immunization can provide a state of immediate immunity that can last for weeks and possibly months. Some human IgG isotypes have serum half-lives in excess of 30 days, which would confer long-lived protection to passively immunized persons. Where active vaccines are available, they may be administered together with antibodies to both immediate and long-lasting protection. In addition, the antibodies can be administered in conjunction with one or more therapeutic drugs for treatment or prevention of infection or for treatment of infection. Administration of antibodies produced as described herein will follow the general protocols for passive immunization. Antibodies for administration be prepare in a formulation suitable for administration to a host. Aqueous compositions comprise an effective amount of an antibody dispersed in a pharmaceutically acceptable carrier and/or aqueous medium. The phrases “pharmaceutically and/or pharmacologically acceptable” refer to compositions that do not produce an adverse, allergic and/or other untoward reaction when administered to an animal, and specifically to humans, as appropriate.
As used herein, “pharmaceutically acceptable carrier” includes any solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents and the like. The use of such media or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For administration to humans, preparations should meet sterility, pyrogenicity, general safety and/or purity standards as required by FDA Office of Biologics standards.
Antibodies will generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, or even intraperitoneal routes. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation or in such amount as is therapeutically effective. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
1. Dawood, F. S., et al. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 360, 2605-2615 (2009).
2. Garten, R. J., et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325, 197-201 (2009).
3. Webby, R. J. & Webster, R. G. Are we ready for pandemic influenza? Science 302, 1519-1522 (2003).
4. Yen, H. L. & Webster, R. G. Pandemic influenza as a current threat. Curr Top Microbiol Immunol 333, 3-24 (2009).
5. Palese, P. Influenza: old and new threats. Nat Med 10, S82-87 (2004).
6. Steel, J., et al. Transmission of pandemic H1N1 influenza virus and impact of prior exposure to seasonal strains or interferon treatment. J Virol (2009).
7. Hancock, K., et al. Cross-Reactive Antibody Responses to the 2009 Pandemic H1N1 Influenza Virus. N Engl J Med (2009).
8. Brockwell-Staats, C., Webster, R. G. & Webby, R. J. Diversity of Influenza Viruses in Swine and the Emergence of a Novel Human Pandemic Influenza A (H1N1). Influenza Other Respi Viruses 3, 207-213 (2009).
9. Ahmed, R., Oldstone, M. B. & Palese, P. Protective immunity and susceptibility to infectious diseases: lessons from the 1918 influenza pandemic. Nat Immunol 8, 1188-1193 (2007).
10. Wardemann, H. et al. Predominant autoantibody production by early human B cell precursors. Science 301, 1374-1377 (2003).
11. Smith, K., et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4, 372-384 (2009).
12. Wrammert, J., et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667-671 (2008).
13. Itoh Y., et al., In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature 460, 1021-1025 (2009).
14. Doherty, P. C., Turner, S. J., Webby, R. G. & Thomas, P. G. Influenza and the challenge for immunology. Nat Immunol 7, 449-455 (2006).
15. Clark, T. W., et al. Trial of Influenza A (H1N1) 2009 Monovalent MF59-Adjuvanted Vaccine—Preliminary Report. N Engl J Med (2009).
16. Greenberg, M.E., et al. Response after One Dose of a Monovalent Influenza A (H1N1) 2009 Vaccine—Preliminary Report. N Engl J Med (2009).
17 Rappuoli, R., et al. Public health. Rethinking influenza. Science 326, 50 (2009).
18. Horimoto, T. & Kawaoka, Y. Designing vaccines for pandemic influenza. Curr Top Microbiol Immunol 333, 165-176 (2009).
19. Compans, R. W. Hemagglutination-inhibition: rapid assay for neuraminic acid-containing viruses. J. Virol 14, 1307-1309 (1974).
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/52274 | 10/12/2010 | WO | 00 | 6/26/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61250479 | Oct 2009 | US | |
| 61260650 | Nov 2009 | US |
