WO2012083475A1 - Neutralizing polyclonal antibody against hantavirusandes, production method and pharmaceutical compositions - Google Patents
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/12011—Bunyaviridae
- C12N2760/12111—Hantavirus, e.g. Hantaan virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/175—Bunyaviridae, e.g. California encephalitis virus, Rift valley fever virus, Hantaan virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Definitions
- Viruses of the genus Hantavirus belong to the family Bunyaviridae.
- the genome consists of a single negative strand of RNA that is divided into three segments. Segments S, M and L encode for the nucleocapsid (N protein), two glycoproteins of the Gl and G2 envelope and a viral transcriptase, respectively.
- the genus Hantavirus is the only one of the Bunyaviridae family not transmitted by arthropods. In contrast, the natural Hantavirus reservoir are rodents of the Muridae family.
- the different Hantavirus species have a close association with a specific host species and their circulation depends on the geographical distribution of the different species of the Muridae family.
- ANDV is the only Hantavirus described to date. Its reservoir is the rodent Oligoryzomys longicaudatus (long-tailed mouse) present between the II and XII region. Since 1995, 395 patients with SCPH have been confirmed in Chile, of which 66.5% have been diagnosed during the last four years. While the lethality of the infection in 1997 was 60%, it has fallen to 31% in 2004, possibly due to greater knowledge of both the population and the health staff, which allow a more early and effective intervention. Regionally, the seropositivity rate for ANDV in Chilean populations can reach up to 7.5% (6). However, to date no specific vaccine or treatment has proven effective in humans.
- Cardiopulmonary Syndrome (SCPH) due to ANDV is an acute and endemic acute viral disease in Chile that annually affects 60-70 patients between the III-XII regions of Chile, with a mortality of 37%.
- SCPH Cardiopulmonary Syndrome
- patients who survive the infection maintain immunity from re-infection for life. It has been shown that this immunity is due, at least in part, to the persistence of neutralizing antibodies, which prevent the virus from binding to its receptors on the surface of white cells.
- the natural reservoir of ANDV is the rodent Oligoryzomys longicaudatus, which are infected by horizontal transmission but do not develop the disease, being chronic carriers of the virus.
- the ANDV replicative cycle begins when it enters the airway and binds by some domain of its Gl and G2 proteins to avb3 integrins and other receptors still unknown in the cell membrane of lung endothelial cells, peripheral blood mononuclear cells (PBMC) and dendritic cells of the interstitium.
- PBMC peripheral blood mononuclear cells
- Hantaviruses mostly infect endothelial cells, peripheral blood mononuclear cells (PBMC) and dendritic cells; however, neither Hantavirus infection nor replication is cytopathic for these cells (7-10).
- PBMC peripheral blood mononuclear cells
- the immune response to the virus participates in the pathogenesis of Hantavirus disease. Specifically, the cellular response by T lymphocytes against antigens presented in infected endothelial cells is considered as causing the increased vascular permeability and pulmonary edema of SCPH (1 1-14).
- T lymphocytes against antigens presented in infected endothelial cells is considered as causing the increased vascular permeability and pulmonary edema of SCPH (1 1-14).
- in vivo studies it has been suggested that immunity to virus pathology is directly associated with immunity to Hantavirus (15-18). However, to date it is unknown which epitopes are recognized by these neutralizing antibodies that would mediate permanent immunity. This information is crucial both
- Macaques vaccinated with DNA encoding the M segment developed high antibody titers against G1 / G2.
- SCPH the only existing animal model for SCPH
- US Patent 5614193 (US ARMY) describes vaccines with a peptide selected from SEQ ID NO: 1, 647 amino acids, which codes for Hantavirus Gl protein and which contains, at its 427-441 position, the DMDIWYCNGQRKVI peptide with 80% homology with the immunogenic peptide of the invention.
- peptide 427-441 could have immunogenicity by itself, which describes fundamental differences in its effectiveness.
- Japanese patent JP 8 325 291 (Yoshimatsu et al) discloses a Hantavirus antigenic protein and a monoclonal antibody, where the protein does not contain the peptide of the invention.
- the peptides investigated and used in this patent application for the identification of the relevant epitopes consisted of 15 amino acids each with a linear conformation. Because it was possible that this methodology did not detect all specific antibodies effective in recognizing conformational epitopes present during viral replication, this was resolved using overlapping peptides of 14-21 amino acids in size. In addition, since recombinant proteins also show a linear structure and, in the case of Gl and G2, are extremely unstable, the use of overlapping peptides does not represent disadvantages with respect to other possible approaches.
- the methodology proposed in this application represents an adequate and globally applied technique to detect specific antibodies with or without neutralizing function.
- This methodology allows the detection of G1 / G2 epitopes that are recognized by the plasma antibodies of convalescent patients.
- the present invention contributes to the development of passive and / or active immunotherapy.
- the present application shows the identification of the anti-Gl_2210 antibody of Hantavirus Andes, of high prevalence among patients convalescent to the infection, being detectable with a high sensitivity (100%) by ELISA with linear peptides.
- This antibody can be efficiently purified by affinity chromatography from the patient plasma, while maintaining its functional capacity, being able to recognize in situ the conformational structure of the epitope previously identified by ELISA with linear peptides.
- EXAMPLE 1 Identification and quantification of epitopes of the glycoproproteins Gl and G2 of the Hantavirus Andes (ANDV).
- Group A individuals in whom Hantavirus infection (anti-N IgG is documented
- Group B individuals in whom Hantavirus infection (anti-N IgG positive) is documented with moderate or severe symptoms and who required mechanical ventilation and / or hemodynamic support (VM / SH) or ECMO. In addition, healthy individuals were included.
- Hantavirus infection anti-N IgG positive
- VM / SH hemodynamic support
- ECMO hemodynamic support
- VM / SH mechanical ventilation and / or hemodynamic support or ECMO
- Antibody detection by ELISA Enzyme Immunoemayo: An ELISA based on overlapping peptides was performed encoding the ANDV Gl and G2 proteins and detecting the antibodies present in the patients' plasma (IgG) that specifically bind said peptides. The establishment of this methodology was based on the ELISA protocol by detecting specific IgG antibodies for the recombinant N protein published by the Centers of Disease Control (CDC). Triplicates of 10 mixtures of overlapping and continuous peptides (mix 1 to mix 10: concentration 20 nmol / ml per peptide) were incubated overnight at 4 ° C in a 96-pocket plate. The ANDV recombinant N protein was used as a positive control.
- EXAMPLE 2 Identification of immuno-dominant epitopes of protein N.
- EXAMPLE 3 Identification of immunodominant epitopes of the G2 protein. COMPARATIVE EXAMPLE WITH ART (Tischler et al).
- EXAMPLE 4 Identification of immunodominant epitopes of the Gl protein.
- serial dilutions of anti-Gl_2210 IgG Figure 5 we determine the dilution at which the anti-Gl_2210 antibody signal disappeared. On average, patients with mild SCPH showed an average titer of 1/6933 of anti-Gl_2210 while patients with SCPH severe showed an average grade of 1/3167, without achieving a significant difference analyzed by the student t test.
- EXAMPLE 7 Indirect Immunofluorescence Study (IFI) to study in vitro recognition of the conformational epitope of the anti-Gl_2210 antibody.
- Indirect Immunoflourescence was performed.
- Vero E6 endothelial cells were cultured on 8-well Lab-Tek Chamber Slide plates (Nalge Nunc International) and incubated overnight in a culture chamber at 37 ° C until confluence. They were then infected with ANDV at an approximate concentration of 100 PFU (Plaque Forming Units) / lOOul.
- Figure 2 Peptide and nucleotide sequences of consensus objects of the present invention.
- Figure 3 Hanta virus infection cycle and viral structural composition.
- Figure 4 Anti-Gl_2210 antibody isolation protocol isolated from patients' plasma.
- Figure 5 Working protocol for identification of immunogenic peptides and specific epitopes in patients convalescent to ANDV infection
- Figure 7 Recognition of individual peptides of mix 6 by specific IgGs, determined in 12 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2xSD controls. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10 nmol / ml each) in an ELISA.
- Figure 8 Specific IgGs for individual peptides of mix 32 and 33, determined in 8 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2 standard deviations. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10mg / ml c / u) in an ELISA.
- Figure 9 IgG specific for individual peptides of mix 22, determined in 27 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2xSD control. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10nmol / ml c / u) in an ELISA.
- Figure 10 Specific IgG titration for Gl_2210, determined in 12 patients. The results indicate the absorbance obtained by the different serial serial dilutions of each of the patients. Results represent the average of triplicates in each patient. Plasma of each patient was exposed in different dilutions (1: 50-1: 409600) to 10nmol / ml of Gl_2210 in an ELISA.
- Figure 11 Comparison between the anti-Gl_2210 titer in the plasma prior to chromatography and the eluate titer.
- Figure 12 (63x) The images on the left side correspond to anti-Gl_2210 in dilutions 1: 200, 1: 400 and 1: 800 and the images on the right side correspond to anti-Gl_2210 previously incubated with recombinant N protein (* N). DAPI-stained nuclei (in blue) allow the identification of the N protein, which
- Image 8-b shows, in the same way, the same rabbit plasma but previously incubated for 4 hours at room temperature, with 2 ⁇ g of recombinant N protein. In this, the absence of mareaje is observed, which proves the high effectiveness of this pre-incubation as a method to decrease the binding of antibodies to the viral N protein.
- Plasma rabbit anti-N 13-a Plasma rabbit anti-N 13-b. Plasma rabbit anti-N / * N
- Figure 14 (63x) Images 9-a and 9-b correspond to the negative controls performed with papillomavirus.
- BPV Bovine Papiloma Virus
- HPV Human Papiloma Virus
- Absence of mareaje can be observed, which accounts for a high specificity for the epitope identified in the ANDV Gl protein.
- images 9-c and 9-d 2 plasma samples of patients convalescent to ANDV infection are observed, performed as positive controls. In these, a diffuse pattern of very intense pattern is observed in the perinuclear areas, which would correspond to the extensive screening of all the antibodies developed by these patients.
- Anti-BPV Negative Control
- Anti-HPV Negative Control
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Abstract
The present invention provides for identification of the human anti-G1_2210 polyclonal antibody, which is specific for the glycoprotein G1 of Hantavirus Andes (ANDV), which is highly prevalent amongst patients convalescing from infection, and which can be detected with a high degree of sensitivity (100%) by means of ELISA with linear peptides. This antibody may be efficiently purified by means of affinity chromatography from patients' plasma, retaining the functional capability thereof, being capable of recognizing in situ the epitope conformational structure identified previously by means of ELISA with linear peptides. The anti-G1_2210 antibody may be used as neutralizing agent in vivo and in vitro against infection with ANDV or other Hantaviruses in the form of passive or active immunotherapy. Also described are the immunogenic peptide, compositions, vaccines and kits, and the use thereof against Hantavirus infections or for screening biological samples with Hantavirus.
Description
ANTICUERPO POLICLONAL NEUTRALIZANTE CONTRA HANTAVIRUS ANDES, PROCEDIMIENTO DE PRODUCCIÓN Y COMPOSICIONES FARMACÉUTICAS. MEMORIA DESCRIPTIVA NEUTRALIZING POLYCLONAL ANTIBODY AGAINST HANTAVIRUS ANDES, PRODUCTION PROCEDURE AND PHARMACEUTICAL COMPOSITIONS. DESCRIPTIVE MEMORY
Los virus del género Hantavirus pertenecen a la familia Bunyaviridae. El genoma está constituido por una sola hebra negativa de RNA que se divide en tres segmentos. Los segmentos S, M y L codifican para la nucleocápside (proteína N), dos glicoproteínas de la envoltura Gl y G2 y una transcriptasa viral, respectivamente. El género Hantavirus es el único de la familia Bunyaviridae no transmitido por artrópodos. Por el contrario, el reservorio natural de Hantavirus son roedores de la familia Muridae. Las diferentes especies de Hantavirus tienen una asociación estrecha con una determinada especie hospedera y su circulación depende de la distribución geográfica de las diferentes especies de la familia Muridae. Estos roedores, una vez infectados por transmisión horizontal entre ejemplares maduros sexualmente, portarían el virus en forma crónica, sin desarrollar enfermedad. La transmisión del virus al humano se produce principalmente por la inhalación de aerosoles de orina, saliva u otras secreciones de los roedores. Se han descrito dos principales síndromes clínicos producidos por Hantavirus (1): los Hantavirus circulantes en el viejo mundo, conocidos desde la década de 1940, causan una fiebre hemorrágica con síndrome renal (FHSR), de severidad variable según la especie, con una letalidad entre menos del 1 al 15%; en cambio, los Hantavirus del nuevo mundo, descritos recién el año 1993 producen un Síndrome Cardiopulmonar (SCPH), cuya severidad también es variable, pero que puede alcanzar tasas de letalidad de hasta un 60%. Viruses of the genus Hantavirus belong to the family Bunyaviridae. The genome consists of a single negative strand of RNA that is divided into three segments. Segments S, M and L encode for the nucleocapsid (N protein), two glycoproteins of the Gl and G2 envelope and a viral transcriptase, respectively. The genus Hantavirus is the only one of the Bunyaviridae family not transmitted by arthropods. In contrast, the natural Hantavirus reservoir are rodents of the Muridae family. The different Hantavirus species have a close association with a specific host species and their circulation depends on the geographical distribution of the different species of the Muridae family. These rodents, once infected by horizontal transmission between sexually mature specimens, would carry the virus in chronic form, without developing disease. The transmission of the virus to the human is mainly caused by the inhalation of urine, saliva or other rodent secretions. Two main clinical syndromes produced by Hantavirus (1) have been described: the circulating Hantaviruses in the old world, known since the 1940s, cause a hemorrhagic fever with renal syndrome (FHSR), of varying severity depending on the species, with lethality between less than 1 to 15%; on the other hand, the Hantaviruses of the new world, described just in 1993, produce a Cardiopulmonary Syndrome (SCPH), whose severity is also variable, but can reach lethality rates of up to 60%.
Alrededor de 200.000 personas se infectan anualmente con Hantavirus, la mayoría de ellas en Asia y Europa (2, 3). Sin embargo, el número de casos descritos en América va en aumento en la medida en que se ha ido conociendo la enfermedad y actualmente ha sido descrita en casi todos los países americanos. Las especies con mayor distribución en América del Norte y en el cono
sur americano (Chile y Argentina) son Sin Nombre Virus (SNV) y Andes Virus (ANDV) y se caracterizan por una alta letalidad. Clínicamente, el SCPH se desarrolla 7-39 días después la exposición al virus con síntomas prodrómicos de fiebre, cefalea, decaimiento y molestias gastrointestinales. Después de algunos días aparece tos seca y disnea que puede llevar en pocas horas a una insuficiencia respiratoria grave con infiltrados pulmonares rápidamente progresivos, plaquetopenia y falla cardíaca que requieren ventilación mecánica y soporte hemodinámico (1, 4). Muchos de los pacientes que fallecen lo hacen dentro de las primeras 24-48 horas de ingresar al hospital. Around 200,000 people are infected annually with Hantavirus, most of them in Asia and Europe (2, 3). However, the number of cases described in America is increasing as the disease has been known and has now been described in almost all American countries. The most widely distributed species in North America and in the cone South American (Chile and Argentina) are Sin Nombre Virus (SNV) and Andes Virus (ANDV) and are characterized by high lethality. Clinically, SCPH develops 7-39 days after exposure to the virus with prodromal symptoms of fever, headache, decay and gastrointestinal discomfort. After a few days, dry cough and dyspnea appear, which can lead to severe respiratory failure in a few hours with rapidly progressive pulmonary infiltrates, platelet and heart failure requiring mechanical ventilation and hemodynamic support (1, 4). Many of the patients who die do so within the first 24-48 hours of entering the hospital.
En Chile, ANDV es el único Hantavirus descrito hasta la fecha. Su reservorio es el roedor Oligoryzomys longicaudatus (ratón de cola larga) presente entre la II y la XII región. Desde 1995 se han confirmado 395 pacientes con SCPH en Chile, de los cuales se han diagnosticado 66,5% durante los últimos cuatro años. Mientras la letalidad de la infección en 1997 era de 60%, ésta ha descendido a un 31% el año 2004, posiblemente debido a un mayor conocimiento tanto de la población como del personal de salud, que permiten una intervención más precoz y efectiva. Regionalmente la tasa de seropositividad para ANDV en populaciones chilenas puede alcanzar hasta un 7,5% (6). Sin embargo, hasta la fecha ninguna vacuna o tratamiento específico ha comprobado ser efectivo en humanos. In Chile, ANDV is the only Hantavirus described to date. Its reservoir is the rodent Oligoryzomys longicaudatus (long-tailed mouse) present between the II and XII region. Since 1995, 395 patients with SCPH have been confirmed in Chile, of which 66.5% have been diagnosed during the last four years. While the lethality of the infection in 1997 was 60%, it has fallen to 31% in 2004, possibly due to greater knowledge of both the population and the health staff, which allow a more early and effective intervention. Regionally, the seropositivity rate for ANDV in Chilean populations can reach up to 7.5% (6). However, to date no specific vaccine or treatment has proven effective in humans.
Patogenia y respuesta inmune Pathogenesis and immune response
El Síndrome Cardiopulmonar (SCPH) por ANDV es una enfermedad viral aguda emergente y endémica en Chile que afecta anualmente 60-70 pacientes entre las III-XII regiones de Chile, con una mortalidad de 37%. Sin embargo, los pacientes que sobreviven la infección mantienen inmunidad frente a una re-infección de por vida. Se ha mostrado que aquella inmunidad se debe, por lo menos en parte, a la persistencia de anticuerpos neutralizantes, que impiden la unión del virus a sus receptores en la superficie de las células blancos.
El reservorio natural del ANDV son los roedores Oligoryzomys longicaudatus, los cuales se infectan por transmisión horizontal pero no desarrollan la enfermedad, siendo portadores crónicos del virus. Su transmisión se produce por la inhalación de aerosoles de orina, excrementos y saliva, produciendo el SCPH cuya base patogénica estaría basada en la respuesta inmune montada en respuesta al virus y no un efecto citopático de este. El ciclo replicativo de ANDV comienza cuando ingresa por la vía aérea y se une por algún dominio de sus proteínas Gl y G2 a integrinas avb3 y otros receptores aún desconocidos en la membrana celular de células endoteliales del pulmón, células mononucleares de sangre periférica (PBMC) y células dendríticas del intersticio. Cardiopulmonary Syndrome (SCPH) due to ANDV is an acute and endemic acute viral disease in Chile that annually affects 60-70 patients between the III-XII regions of Chile, with a mortality of 37%. However, patients who survive the infection maintain immunity from re-infection for life. It has been shown that this immunity is due, at least in part, to the persistence of neutralizing antibodies, which prevent the virus from binding to its receptors on the surface of white cells. The natural reservoir of ANDV is the rodent Oligoryzomys longicaudatus, which are infected by horizontal transmission but do not develop the disease, being chronic carriers of the virus. Its transmission is produced by the inhalation of urine, excrement and saliva aerosols, producing SCPH whose pathogenic base would be based on the immune response mounted in response to the virus and not a cytopathic effect of it. The ANDV replicative cycle begins when it enters the airway and binds by some domain of its Gl and G2 proteins to avb3 integrins and other receptors still unknown in the cell membrane of lung endothelial cells, peripheral blood mononuclear cells (PBMC) and dendritic cells of the interstitium.
Los Hantavirus infectan mayormente a las células endoteliales, células mononucleares de sangre periférica (PBMC) y células dendríticas; sin embargo, ni la infección ni la replicación de Hantavirus son citopáticos para estas células (7-10). Hay evidencias de que la respuesta inmune al virus participa en la patogenia de la enfermedad por Hantavirus. Específicamente la respuesta celular por linfocitos T contra antígenos presentados en células endoteliales infectadas, está considerada como causante del aumento de la permeabilidad vascular y el edema pulmonar del SCPH (1 1-14). Por otra parte, en estudios in vivo se ha sugerido que la inmunidad a la patología virus está directamente asociada a la inmunidad a Hantavirus (15-18). Sin embargo, a la fecha se desconocen cuáles son los epítopes reconocidos por estos anticuerpos neutralizantes que mediarían una inmunidad permanente. Esta información es crucial tanto para una posible terapia por inmunidad pasiva, como para el desarrollo de una vacuna preventiva que induzca una inmunidad humoral activa. Hantaviruses mostly infect endothelial cells, peripheral blood mononuclear cells (PBMC) and dendritic cells; however, neither Hantavirus infection nor replication is cytopathic for these cells (7-10). There is evidence that the immune response to the virus participates in the pathogenesis of Hantavirus disease. Specifically, the cellular response by T lymphocytes against antigens presented in infected endothelial cells is considered as causing the increased vascular permeability and pulmonary edema of SCPH (1 1-14). On the other hand, in vivo studies it has been suggested that immunity to virus pathology is directly associated with immunity to Hantavirus (15-18). However, to date it is unknown which epitopes are recognized by these neutralizing antibodies that would mediate permanent immunity. This information is crucial both for possible passive immunity therapy, and for the development of a preventive vaccine that induces active humoral immunity.
Antecedentes Bibliográficos Bibliographic Background
Varios estudios con diferentes especies de Hantavirus han sugerido la existencia de anticuerpos neutralizantes en el plasma de pacientes (16, 19, 20). Dichas evidencias sugieren que tanto la erradicación del virus como la inmunidad frente a una re-infección, son mediadas por
anticuerpos neutralizantes. El efecto protector de los anticuerpos neutralizantes también ha sido sugerido en pacientes con SCPH por SNV: existe una correlación entre el nivel de anticuerpos neutralizantes al momento del ingreso al hospital y la gravedad de la enfermedad (18). Además de los estudios in Vitro en que el efecto neutralizante se pudo asignar a los anticuerpos contra Gl y/o G2 mientras que los anticuerpos contra la proteína N no eran neutralizantes (17, 21-25), recientemente se publicó un estudio que comprobó in vivo la capacidad neutralizante de anticuerpos contra G1/G2. Macacos vacunados con ADN codificando para el segmento M desarrollaron altos títulos de anticuerpos contra G1/G2. Al transfundir el plasma de estos macacos a hámster sirios (el único modelo animal existente para SCPH (26), los anticuerpos protegieron de la muerte al 100% de los hamsters infectados previamente (4-5 días antes) con ANDV (27). Several studies with different Hantavirus species have suggested the existence of neutralizing antibodies in the plasma of patients (16, 19, 20). Such evidence suggests that both the eradication of the virus and immunity against re-infection are mediated by neutralizing antibodies The protective effect of neutralizing antibodies has also been suggested in patients with SCPH due to SNV: there is a correlation between the level of neutralizing antibodies at the time of hospital admission and the severity of the disease (18). In addition to the in vitro studies in which the neutralizing effect could be assigned to antibodies against Gl and / or G2 while antibodies against N protein were not neutralizing (17, 21-25), a study was recently published that found in I live the neutralizing ability of antibodies against G1 / G2. Macaques vaccinated with DNA encoding the M segment developed high antibody titers against G1 / G2. By transfusing the plasma of these macaques to Syrian hamsters (the only existing animal model for SCPH (26), the antibodies protected 100% of hamsters previously infected (4-5 days earlier) with ANDV from death) (27).
Se ha demostrado en pacientes con infección previa por SNV, que anticuerpos específicos y neutralizantes se encuentran en el plasma en títulos entre 1 :100 y 1 :3200 hasta más de 2000 días (5 años) después la aparición de síntomas (29). Importantemente, no había una relación significativa entre los títulos encontrados y la gravedad de la enfermedad previa. Segundo, péptidos traslapados han sido exitosamente utilizados en determinar los epítopes de linfocitos B en estudios de Hepatitis E (HEV) (19), VIH (30) y Coronavirus causante del SARS (Severe acute respiratory syndrome) (31 , 32) entre otros. It has been shown in patients with prior SNV infection, that specific and neutralizing antibodies are found in the plasma in titres between 1: 100 and 1: 3200 up to more than 2000 days (5 years) after the onset of symptoms (29). Importantly, there was no significant relationship between the titles found and the severity of the previous illness. Second, overlapping peptides have been successfully used to determine B-cell epitopes in studies of Hepatitis E (HEV) (19), HIV (30) and Coronavirus causing SARS (Severe acute respiratory syndrome) (31, 32) among others.
La literatura de patentes también aborda el problema técnico planteado en la presente solicitud. La patente norteamericana US 5614193 (US ARMY), describe vacunas con un péptido seleccionado de la SEQ ID NO:l, de 647 aminoácidos, que codifica para la proteína Gl de Hantavirus y que contiene, en su posición 427-441 el péptido DMDIWYCNGQRKVI con un 80% de homología con el péptido inmunogénico de la invención. No obstante, en este documento no hay ninguna indicación de que el péptido 427-441 pudiese tener inmunogenicidad por sí mismo, lo que describe diferencias fundamentales en su efectividad. Por otro lado, la patente japonesa JP 8 325 291 (Yoshimatsu et al), divulga una proteína antigénica de Hantavirus
y un anticuerpo monoclonal, donde la proteína no contiene el péptido de la invención. La publicación internacional de patente WO 2006/088478 (Novartis Vaccines and Diagnostics INC.) protege métodos y reactivos para el diagnóstico de infecciones por Hantavirus, que emplea combinaciones de antí genos recombinantes N o Gl, los que no incluyen el péptido antigénico de la invención, ni su secuencia codificante. Otras patentes tales como WO 2004/058 808 (US ARMY), WO 0238174 (Humboldt Uni Zu Berlín Univers) y WO 9727 302 (HEIL JEDANG CORP) describen vacunas peptídicas, anticuerpos y vacunas de DNA que no comprenden ni el péptido inmunogénico de la presente invención, ni su secuencia codificante. De acuerdo a esto, resulta evidente que la literatura tanto científica como de patentes no describe la esencia de la presente invención y permite establecer que ella representa un aporte en el campo técnico. The patent literature also addresses the technical problem raised in this application. US Patent 5614193 (US ARMY) describes vaccines with a peptide selected from SEQ ID NO: 1, 647 amino acids, which codes for Hantavirus Gl protein and which contains, at its 427-441 position, the DMDIWYCNGQRKVI peptide with 80% homology with the immunogenic peptide of the invention. However, there is no indication in this document that peptide 427-441 could have immunogenicity by itself, which describes fundamental differences in its effectiveness. On the other hand, Japanese patent JP 8 325 291 (Yoshimatsu et al) discloses a Hantavirus antigenic protein and a monoclonal antibody, where the protein does not contain the peptide of the invention. International Patent Publication WO 2006/088478 (Novartis Vaccines and Diagnostics INC.) Protects methods and reagents for the diagnosis of Hantavirus infections, which employs combinations of recombinant antigens N or Gl, which do not include the antigenic peptide of the invention. , nor its coding sequence. Other patents such as WO 2004/058 808 (US ARMY), WO 0238174 (Humboldt Uni Zu Berlin Univers) and WO 9727 302 (HEIL JEDANG CORP) describe peptide vaccines, antibodies and DNA vaccines that do not comprise the immunogenic peptide of the present invention, nor its coding sequence. Accordingly, it is evident that both scientific and patent literature does not describe the essence of the present invention and allows it to be established that it represents a contribution in the technical field.
DESCRIPCION DETALLADA DE LA INVENCION DETAILED DESCRIPTION OF THE INVENTION
A pesar de varios intentos de desarrollar vacunas preventivas para diferentes Hantavirus, la eficacia de aquellas queda por comprobar. Además, a pesar de que Ribavirina ha sido sugerido como tratamiento antiviral específico, estudios recientes no pudieron demostrar efectos significativos (33). Hasta la fecha no existe una vacuna preventiva ni un tratamiento farmacéutico específico contra la infección por ANDV. Una posible terapia específica que se pretende evaluar, es la transfusión de plasma que contenga altos títulos de anticuerpos neutralizantes in Vitro, o sea, el traspaso de inmunidad pasiva. Este tratamiento ya se aplica con éxito en la Fiebre Hemorrágica Argentina (FHA) por virus Junín (34-36). Basado en la experiencia con estos pacientes se estima que 600mL de plasma de un paciente con un título de 1 :800 de anticuerpos neutralizantes sería suficiente de tratar dos pacientes de 80kg cada uno. Sin embargo, al desconocer el(los) epítope(s) de los anticuerpos neutralizantes, la determinación cualitativa y cuantitativa de anticuerpos neutralizantes requiere experimentos extensos in Vitro utilizando cultivos virales que requieren un nivel de bioseguridad. A pesar que un donante con
título alto puede donar plasma varios veces por año, dichos factores limitantes hacen poco probable identificar suficientes donantes para la necesidad de Chile con actualmente 57 casos (año 2004) de SCPH. El encuentro de un donante con título alto de anticuerpos neutralizantes, implica un "screening"" de numerosos pacientes y costosos análisis. El desconocimiento de la especificidad de los anticuerpos neutralizantes tampoco permite la purificación de éstos a títulos altos desde plasma donado, o la síntesis de anticuerpos policlonales por roedores vacunados. Así, la inmunoterapia pasiva requiere la transfusión de plasma completo incluyendo anticuerpos neutralizantes y no neutralizantes y encerrando el riesgo potencial del contagio de enfermedades infecciosas. Despite several attempts to develop preventive vaccines for different Hantaviruses, the effectiveness of those remains to be proven. In addition, although Ribavirin has been suggested as a specific antiviral treatment, recent studies failed to demonstrate significant effects (33). To date there is no preventive vaccine or a specific pharmaceutical treatment against ANDV infection. A possible specific therapy that is intended to be evaluated is plasma transfusion that contains high titers of neutralizing antibodies in vitro, that is, passive immunity transfer. This treatment is already successfully applied in the Argentine Hemorrhagic Fever (FHA) due to Junín virus (34-36). Based on experience with these patients, it is estimated that 600mL of a patient's plasma with a 1: 800 titer of neutralizing antibodies would be sufficient to treat two patients of 80kg each. However, upon ignoring the epitope (s) of neutralizing antibodies, the qualitative and quantitative determination of neutralizing antibodies requires extensive in vitro experiments using viral cultures that require a level of biosecurity. Although a donor with High titre can donate plasma several times a year, such limiting factors make it unlikely to identify enough donors for Chile's need with currently 57 cases (2004) of SCPH. The encounter of a donor with a high titer of neutralizing antibodies implies a "screening" of numerous patients and costly analyzes.The lack of specificity of the neutralizing antibodies does not allow their purification at high titers from donated plasma, or the synthesis of polyclonal antibodies by vaccinated rodents Thus, passive immunotherapy requires the transfusion of complete plasma including neutralizing and non-neutralizing antibodies and enclosing the potential risk of infectious diseases.
El estado del arte actual permite plantear que posiblemente en humanos, al igual que en hamsters, la inmunidad a ANDV sea mediada por anticuerpos específicos para las glicoproteínas Gl y G2. Al respecto, hasta la fecha se desconocen los epítopes blancos dentro G1/G2 de los anticuerpos neutralizantes en humanos. The current state of the art makes it possible to state that possibly in humans, as in hamsters, immunity to ANDV is mediated by antibodies specific for Gl and G2 glycoproteins. In this regard, white epitopes within G1 / G2 of neutralizing antibodies in humans are unknown.
De acuerdo a estos antecedentes, nos propusimos detectar anticuerpos específicos para las glicoproteínas Gl y G2 de ANDV y determinar los epítopes reconocidos por dichos anticuerpos. Para ello se utilizó plasma de pacientes que han presentado una infección previa por ANDV y se incubaron con péptidos traslapados de 15 aminoácidos cada uno, que incluían la secuencia completa de las glicoproteínas Gl y G2 del ANDV chileno (R123, NC_003467) (28). According to these antecedents, we set out to detect antibodies specific for the ANDV Gl and G2 glycoproteins and determine the epitopes recognized by said antibodies. For this, plasma was used from patients who had a previous ANDV infection and were incubated with overlapping peptides of 15 amino acids each, which included the complete sequence of the glycoproteins Gl and G2 of the Chilean ANDV (R123, NC_003467) (28).
La detección de los anticuerpos contra Gl y G2 de ANDV como la identificación de sus epítopes principales, señalada en la presente invención, permite los siguientes avances: The detection of antibodies against Gl and G2 of ANDV as the identification of its main epitopes, indicated in the present invention, allows the following advances:
• La determinación de la función neutralizante in vitro (por Ensayos de Reducción de Placas) e in vivo (por ej. hámster sirio infectado con ANDV) de anticuerpos monoclonales y específicos para los epítopes identificados en este estudio. • The determination of neutralizing function in vitro (by Plaque Reduction Assays) and in vivo (eg, Syrian hamster infected with ANDV) of monoclonal antibodies specific for epitopes identified in this study.
• La rápida y fácil detección de los anticuerpos neutralizantes, una vez, la función neutralizante esté asignada a uno o varios epítope(s).
• La purificación y concentración de los anticuerpos neutralizantes desde el plasma de individuos con infección pasada para su uso como inmunoterapia pasiva. • The quick and easy detection of neutralizing antibodies, once, the neutralizing function is assigned to one or several epitopes (s). • Purification and concentration of neutralizing antibodies from the plasma of individuals with past infection for use as passive immunotherapy.
• La síntesis de anticuerpos policlonales por roedores vacunados o monoclonales por hibridomas in Vitro. • The synthesis of polyclonal antibodies by vaccinated or monoclonal rodents by in vitro hybridomas.
• El desarrollo de una vacuna preventiva que induzca inmunidad humoral activa y esterilizante en humanos. • The development of a preventive vaccine that induces active and sterilizing humoral immunity in humans.
Los péptidos investigados y utilizados en esta solicitud de patente para la identificación de los epítopes relevantes consistieron de 15 aminoácidos cada uno con una conformación lineal. Debido a que era posible que esta metodología no detectara todos los anticuerpos específicos efectivos en reconocer epítopes conformacionales presentes durante la replicación viral, ello se resolvió utilizando péptidos traslapados de 14-21 aminoácidos de tamaño. Además, dado que proteínas recombinantes también muestran una estructura lineal y, en el caso de Gl y G2, son extremamente inestables, la utilización de péptidos traslapados no representa desventajas respecto de otros acercamientos posibles. The peptides investigated and used in this patent application for the identification of the relevant epitopes consisted of 15 amino acids each with a linear conformation. Because it was possible that this methodology did not detect all specific antibodies effective in recognizing conformational epitopes present during viral replication, this was resolved using overlapping peptides of 14-21 amino acids in size. In addition, since recombinant proteins also show a linear structure and, in the case of Gl and G2, are extremely unstable, the use of overlapping peptides does not represent disadvantages with respect to other possible approaches.
En resumen, la metodología propuesta en la presente solicitud representa una técnica adecuada y globalmente aplicada para detectar anticuerpos específicos con o sin función neutralizantes. Esta metodología permite detectar los epítopes de G1/G2 que son reconocidos por los anticuerpos del plasma de pacientes convalecientes. Así la presente invención contribuye al desarrollo de una inmunoterapia pasiva y/o activa. In summary, the methodology proposed in this application represents an adequate and globally applied technique to detect specific antibodies with or without neutralizing function. This methodology allows the detection of G1 / G2 epitopes that are recognized by the plasma antibodies of convalescent patients. Thus the present invention contributes to the development of passive and / or active immunotherapy.
En conjunto este conocimiento es de gran importancia para el desarrollo de una inmunoterapia pasiva en otros países con SCPH, especialmente por ANDV, como Argentina. No podemos excluir la posibilidad que anticuerpos neutralizantes y específicos para ANDV muestren una reacción cruzada y sean capaces de neutralizar otros Hantavirus. Sin embargo, mientras la secuencia de la proteína N está altamente conservada entre los diferentes Hantavirus, aquellas de las glicoproteinas son más variables.
Basado en el protocolo original del "Center for Disease Control and Prevention (CDC)" iniciamos el establecimiento de un ELISA con los péptidos traslapados para poder detectar anticuerpos específicos para aquellos péptidos. Dado que los péptidos usados (un total de 310 péptidos) tenían características de hidrofobicidad y carga eléctrica muy diversas, era necesario modificar el protocolo original de la CDC de tal manera que se pegaran cantidades suficientes de los péptidos para luego unirse a los anticuerpos específicos. Por lo tanto incubamos placas de 96 pocilios con mezclas de 6-11 péptidos traslapados (concentración final por péptido: 10nmol/ml) y comparamos diferentes condiciones de temperatura (4°C vs. 37°C, overnight y de tampón solvente bicarbonato, pH 9,6 o PBS. Se observó que las placas preparadas en condiciones de PBS a 37°C tuvieron el mejor rendimiento en términos de sensibilidad y especificidad. Utilizando la técnica por péptidos previamente establecida, evaluamos la presencia de inmunoglobulinas contra las proteínas N, Gl y G2 en el plasma de pacientes (figuras 6a-c). Los resultados obtenidos mostraron que las mezclas de la proteína N fueron más reconocidos (promedio de 17% positivos) que las de la proteína Gl y G2 (8% y 10%, respectivamente). Dentro de las mezclas más reconocidas destacaron la mezcla 6 de la proteína N (11/27 pacientes positivos, figura 6a), la mezcla 6 de la proteína Gl (6/27, figura 6b) y las mezclas 30 y 34 de la proteína G2 (5/27 c/u, figura 6c). Together this knowledge is of great importance for the development of passive immunotherapy in other countries with SCPH, especially by ANDV, such as Argentina. We cannot exclude the possibility that neutralizing and ANDV-specific antibodies show a cross reaction and are capable of neutralizing other Hantaviruses. However, while the N protein sequence is highly conserved among the different Hantaviruses, those of the glycoproteins are more variable. Based on the original protocol of the "Center for Disease Control and Prevention (CDC)" we began the establishment of an ELISA with overlapping peptides in order to detect specific antibodies to those peptides. Since the peptides used (a total of 310 peptides) had very different hydrophobicity and electrical charge characteristics, it was necessary to modify the original CDC protocol so that sufficient amounts of the peptides were attached to then bind to specific antibodies. Therefore we incubated 96-well plates with mixtures of 6-11 overlapping peptides (final concentration per peptide: 10nmol / ml) and compared different temperature conditions (4 ° C vs. 37 ° C, overnight and bicarbonate solvent buffer, pH 9.6 or PBS Plates prepared under PBS conditions at 37 ° C were observed to have the best performance in terms of sensitivity and specificity.Using the previously established peptide technique, we evaluated the presence of immunoglobulins against N, Gl proteins and G2 in the plasma of patients (Figures 6a-c). The results obtained showed that mixtures of the N protein were more recognized (average of 17% positive) than those of the Gl and G2 protein (8% and 10%, respectively) Within the most recognized mixtures, the mixture 6 of the N protein (11/27 positive patients, figure 6a), the mixture 6 of the Gl protein (6/27, figure 6b) and the mixtures 30 and 34 of G2 protein (5/27 each, figure 6c).
Por lo tanto, a través de las investigaciones y antecedentes mostrados anteriormente en la memoria descriptiva, hemos descubierto de manera sorprendente que los péptidos traslapados permiten aislar anticuerpos policlonales específicos contra Gl y G2 y por lo tanto, la presente solicitud plantea como problema técnico la provisión de anticuerpos, composiciones, productos, y procedimientos de obtención de éstos con fines terapéuticos. Therefore, through the investigations and background shown above in the specification, we have surprisingly discovered that overlapping peptides allow the isolation of specific polyclonal antibodies against Gl and G2 and therefore, the present application raises the provision as a technical problem. of antibodies, compositions, products, and methods of obtaining them for therapeutic purposes.
La presente solicitud muestra entonces la identificación del anticuerpo anti-Gl_2210 de Hantavirus Andes, de alta prevalencia entre pacientes convalecientes a la infección, siendo detectable con una alta sensibilidad (100%) mediante ELISA con péptidos lineales. Este anticuerpo puede ser purificado eficientemente mediante cromatografía de afinidad desde el
plasma de pacientes, conservando su capacidad funcional, siendo capaz de reconocer in situ la estructura conformacional del epítope identificado previamente mediante ELISA con péptidos lineales. Lo anterior junto con que la secuencia blanco de anti-Gl_2210 se encuentra en la parte extramembranea de Gl, permite plantear la posibilidad de que el anticuerpo anti-Gl_2210 posee una función neutralizante in vitro y posiblemente in vivo frente a la infección por ANDV. The present application then shows the identification of the anti-Gl_2210 antibody of Hantavirus Andes, of high prevalence among patients convalescent to the infection, being detectable with a high sensitivity (100%) by ELISA with linear peptides. This antibody can be efficiently purified by affinity chromatography from the patient plasma, while maintaining its functional capacity, being able to recognize in situ the conformational structure of the epitope previously identified by ELISA with linear peptides. The foregoing, together with the fact that the white sequence of anti-Gl_2210 is found in the extramembrane part of Gl, allows us to raise the possibility that the anti-Gl_2210 antibody has a neutralizing function in vitro and possibly in vivo against ANDV infection.
A continuación se describen los experimentos que ejemplifican el procedimiento y los hallazgos de la presente solicitud de patente. Estos ejemplos son preferidos pero no excluyentes de otros aspectos protegibles de la invención: EJEMPLOS The experiments that exemplify the procedure and the findings of the present patent application are described below. These examples are preferred but not exclusive of other protectable aspects of the invention: EXAMPLES
EJEMPLO 1. Identifícación y cuantífícación de epítopes de las glicopro teínas Gl y G2 del Hantavirus Andes (ANDV). EXAMPLE 1. Identification and quantification of epitopes of the glycoproproteins Gl and G2 of the Hantavirus Andes (ANDV).
Pacientes investigados: Todos los pacientes fueron reclutados en Chile. Para el efecto de correlacionar la calidad (especificidad) de los anticuerpos específicos con el cuadro clínico se clasificaron los pacientes en los siguientes grupos Investigated patients: All patients were recruited in Chile. In order to correlate the quality (specificity) of the specific antibodies with the clinical picture, patients were classified in the following groups
Grupo A, individuos en que se documenta infección por Hantavirus (anti-N IgG Group A, individuals in whom Hantavirus infection (anti-N IgG is documented
positivos), que no presentaron síntomas o síntomas leves y no requirieron ventilación mecánica ni soporte hemodinámico (VM/SH). positive), which presented no mild symptoms or symptoms and did not require mechanical ventilation or hemodynamic support (VM / SH).
Grupo B, individuos en que se documenta infección por Hantavirus (anti-N IgG positivos) con síntomas moderados o graves y que requirieron ventilación mecánica y/o soporte hemodinámico (VM/SH) o ECMO. Además se incluyeron individuos sanos. Group B, individuals in whom Hantavirus infection (anti-N IgG positive) is documented with moderate or severe symptoms and who required mechanical ventilation and / or hemodynamic support (VM / SH) or ECMO. In addition, healthy individuals were included.
(Grupo C) sin infección por Hantavirus (anti-N IgM +IgG negativos) como población control para excluir errores sistémicos. (Group C) without Hantavirus infection (anti-N IgM + IgG negative) as a control population to exclude systemic errors.
Se incluyeron pacientes mayores de 18 años con anticuerpos positivos contra la proteína N de Hantavirus, con consentimiento informado o individuos negativos para anticuerpos contra la
proteína N como individuos de control, los cuales se encuentran aprobados por los Comités de Ética de la Clínica Alemana (ambos consentimientos, porque las muestras de controles se obtienen solo en la Clínica Alemana/UDD) y la Universidad de la Frontera (solo consentimiento para pacientes. Patients older than 18 years with positive antibodies against Hantavirus N protein, with informed consent or negative individuals for antibodies against Protein N as control individuals, which are approved by the Ethics Committees of the German Clinic (both consents, because control samples are obtained only at the German Clinic / UDD) and the Universidad de la Frontera (consent only to patients
La descripción de los grupos de estudio se establece en la siguiente tabla: The description of the study groups is established in the following table:
VM/SH: ventilación mecánica y/o soporte hemodinámico o ECMO VM / SH: mechanical ventilation and / or hemodynamic support or ECMO
Muestras: La participación en este estudio requirió la donación de 10ml de sangre que se obtuvo por una punción de una vena periférica. De la muestra de sangre se obtuvieron aproximadamente 2ml de plasma por cada 10ml de sangre después de su centrifugación. Samples: Participation in this study required the donation of 10ml of blood that was obtained by a puncture of a peripheral vein. Approximately 2ml of plasma was obtained from the blood sample for every 10ml of blood after centrifugation.
Detección de anticuerpos por ELISA (Inmunoemayo enzimático): Se realizó un ELISA basado en péptidos traslapados codificando las proteínas Gl y G2 de ANDV y detectando los anticuerpos presentes en el plasma de los pacientes (IgG) que se unen específicamente a dichos péptidos. El establecimiento de esta metodología se basó en el protocolo de ELISA detectando anticuerpos IgG específicos para la proteína N recombinante publicado por Centres of Disease Control (CDC). Se incubaron triplicados de 10 mezclas de péptidos traslapados y continuados (mix 1 a mix 10: concentración 20 nmol/ml por péptido) por una noche a 4°C en una placa de 96 bolsillos. Como control positivo se usó la proteína N recombinante de ANDV. Bolsillos sin proteína y sin péptido servirán como controles negativos. Luego se lavará y se incubará la placa con plasma diluido del paciente como se indica en el protocolo. Después de la fase de incubación y del lavado se incubó la placa con anticuerpos contra IgG humano conjugada a HRPO permitiendo hacer evidente macroscópicamente la unión (entre IgG y anti-IgG) al agregar un
sustrato de ABTS. Luego se determinó la absorbancia en un lector de ELISA a 405nm con 595 nm como referencia. Este primer paso constituyó una fase de ""screening"" lo cual permitió la detección de anticuerpos contra G1/G2 de ANDV de manera altamente eficiente. En caso de una reacción positiva para una de las mezclas en el paso de ""screening"" se repitió el experimento con el plasma del mismo paciente utilizando los péptidos individuales de aquella mezcla. Así determinamos las secuencias específicas que eran más comúnmente reconocidas por los anticuerpos presentes en el plasma de los pacientes convalecientes. Antibody detection by ELISA (Enzyme Immunoemayo): An ELISA based on overlapping peptides was performed encoding the ANDV Gl and G2 proteins and detecting the antibodies present in the patients' plasma (IgG) that specifically bind said peptides. The establishment of this methodology was based on the ELISA protocol by detecting specific IgG antibodies for the recombinant N protein published by the Centers of Disease Control (CDC). Triplicates of 10 mixtures of overlapping and continuous peptides (mix 1 to mix 10: concentration 20 nmol / ml per peptide) were incubated overnight at 4 ° C in a 96-pocket plate. The ANDV recombinant N protein was used as a positive control. Pockets without protein and without peptide will serve as negative controls. The plate will then be washed and incubated with diluted plasma of the patient as indicated in the protocol. After the incubation and washing phase, the plate was incubated with antibodies against human IgG conjugated to HRPO allowing the binding (between IgG and anti-IgG) to be made macroscopically apparent by adding a ABTS substrate. The absorbance was then determined in an ELISA reader at 405 nm with 595 nm as a reference. This first step constituted a phase of "screening" which allowed the detection of antibodies against G1 / G2 of ANDV in a highly efficient way. In case of a positive reaction for one of the mixtures in the "screening" step, the experiment with the same patient's plasma was repeated using the individual peptides of that mixture. Thus we determined the specific sequences that were most commonly recognized by the antibodies present in the plasma of convalescent patients.
EJEMPLO 2: Identificación de epítopes inmuno-dominantes de la proteína N. EXAMPLE 2: Identification of immuno-dominant epitopes of protein N.
Expusimos plasma de 12 pacientes a los péptidos individuales del mix 6. En la mayoría de las muestras se encontró respuesta contra el péptido N_603 (241-255aa) y N_604 (246-260aa) con un 83% (10/12) y un 75% (9/12), respectivamente (figura 2). El control sano no mostró IgG específicos para los péptidos N_601-606. Considerando que los péptidos utilizados están traslapados por 10 aminoácidos, este resultado permite concluir que el epítope inmunodominante se encuentra en la región traslapada. Cabe destacar que esta región no ha sido identificada previamente. El experimento permite concluir que mediante este método se detectan epítopes de linfocitos B hasta ahora desconocidos. We exposed plasma from 12 patients to the individual peptides of mix 6. In most of the samples a response was found against peptide N_603 (241-255aa) and N_604 (246-260aa) with 83% (10/12) and 75 % (9/12), respectively (figure 2). The healthy control showed no specific IgG for peptides N_601-606. Considering that the peptides used are overlapped by 10 amino acids, this result allows us to conclude that the immunodominant epitope is in the overlapping region. It should be noted that this region has not been previously identified. The experiment allows to conclude that by this method epitopes of B lymphocytes hitherto unknown are detected.
EJEMPLO 3: Identificación de epítopes inmunodominantes de la proteína G2. EJEMPLO COMPARATIVO CON EL ARTE (Tischler et al). EXAMPLE 3: Identification of immunodominant epitopes of the G2 protein. COMPARATIVE EXAMPLE WITH ART (Tischler et al).
Expusimos plasma de los 4 pacientes previamente identificados como positivos y de 4 pacientes previamente identificados como negativos. Además expusimos los plasma de estos 8 pacientes a péptidos de la mezcla que contiene los péptidos G2_3201 , G2_3208 y G2_3209 (figura 3) que cubren las regiones aminoacídicas 918-930 y 955-970 de la proteína G2, previamente publicadas. Los resultados de este experimento indican que G2_3306 fue el péptido más reconocido por IgG de 3/8 (37,5%) de los pacientes mientras G2_3201, G2_3208 y G2_3209 no
superaban ser reconocidos por más que un paciente (12,5%). A pesar del número pequeño de pacientes investigados, estos resultados sugieren que la detección de IgGs utilizando mezclas de péptidos es un método de alta especificidad (100%) pero de baja sensibilidad (50%). We exposed plasma from the 4 patients previously identified as positive and from 4 patients previously identified as negative. We also exposed the plasma of these 8 patients to peptides of the mixture containing peptides G2_3201, G2_3208 and G2_3209 (Figure 3) covering the amino acid regions 918-930 and 955-970 of the G2 protein, previously published. The results of this experiment indicate that G2_3306 was the most recognized IgG peptide of 3/8 (37.5%) of the patients while G2_3201, G2_3208 and G2_3209 were not exceeded being recognized by more than one patient (12.5%). Despite the small number of patients investigated, these results suggest that the detection of IgGs using peptide mixtures is a method of high specificity (100%) but of low sensitivity (50%).
Las dos regiones inmunodominantes anteriormente descritas en la literatura [Tischler et al. (G2_3201, G2 3208 y G2_3209)], fueron solamente reconocidas por un paciente, respectivamente, lo que denota que las regiones G_3306 son mejores epítopes inmunoidominantes. The two immunodominant regions previously described in the literature [Tischler et al. (G2_3201, G2 3208 and G2_3209)], were only recognized by one patient, respectively, which indicates that the G_3306 regions are better immunoidominant epitopes.
EJEMPLO 4. Identificación de epítopes inmunodominantes de la proteína Gl. EXAMPLE 4. Identification of immunodominant epitopes of the Gl protein.
Con el objetivo de averiguar el(los) péptido(s) que contienen(n) el(los) epítope(s) inmunodominantes de Gl, expusimos los plasmas de los 6 pacientes positivos a los péptidos individuales. Todos los pacientes resultaron positivos para el péptido Gl_2210. Sorprendentemente también los pacientes (n=21) previamente negativos mostraron IgGs específicos para el péptido Gl_2210 mientras los controles sanos (n=5) resultaron negativos. Dado que la In order to find out the peptide (s) containing the immunodominant epitope (s) of Gl, we exposed the plasmas of the 6 patients positive to the individual peptides. All patients were positive for the Gl_2210 peptide. Surprisingly, also the previously negative patients (n = 21) showed specific IgGs for the Gl_2210 peptide while the healthy controls (n = 5) were negative. Given that the
seroprevalencia de anticuerpos anti-Gl_2210 es de 100% (figura 4) en pacientes convalecientes este anticuerpo constituye un excelente candidato para representar un anticuerpo neutralizante. seroprevalence of anti-Gl_2210 antibodies is 100% (figure 4) in convalescent patients this antibody constitutes an excellent candidate to represent a neutralizing antibody.
EJEMPLO 5. Estudio de correlación de anti-Gl_2210 con la evolución clínica de SCPH. Dado que el objetivo general de nuestra propuesta era identificar epítopes que podrían ser blancos de anticuerpos neutralizantes (= epítopes neutralizantes), y dado que Gl_2210 fue reconocido en 100% de los pacientes convalecientes, nuestros datos sugieren que el epítope G1 2210 es altamente inmunogénico y potencialmente representa un epítope neutralizante. Realizando diluciones seriadas de IgG anti-Gl_2210 (figura 5) determinamos la dilución en la que desaparecía la señal de los anticuerpos anti-Gl_2210. En promedio, pacientes con SCPH leve mostraron ün título promedio de 1/6933 de anti- Gl_2210 mientras pacientes con SCPH
grave mostraron un título promedio de 1/3167, sin lograr una diferencia significativa analizada mediante el test t de student. EXAMPLE 5. Correlation study of anti-Gl_2210 with the clinical evolution of SCPH. Since the general objective of our proposal was to identify epitopes that could be targets of neutralizing antibodies (= neutralizing epitopes), and since Gl_2210 was recognized in 100% of convalescent patients, our data suggest that epitope G1 2210 is highly immunogenic and It potentially represents a neutralizing epitope. By making serial dilutions of anti-Gl_2210 IgG (Figure 5) we determine the dilution at which the anti-Gl_2210 antibody signal disappeared. On average, patients with mild SCPH showed an average titer of 1/6933 of anti-Gl_2210 while patients with SCPH severe showed an average grade of 1/3167, without achieving a significant difference analyzed by the student t test.
EJEMPLO 6. Aislamiento de anti-Gl_2210 por cromatografía de afinidad. EXAMPLE 6. Isolation of anti-Gl_2210 by affinity chromatography.
Dado que anti-Gl_2210 se detectó utilizando péptidos lineales, era posible que la estructura terciana de los péptidos no refleje la estructura conformacional de la secuencia viral correspondiente in vivo. Por lo tanto una alta tasa de reconocimiento de un anticuerpo in Vitro no implica necesariamente una funcionalidad de este in vivo. De acuerdo a esto, a partir de 6 pacientes que habían presentado altos títulos de este anticuerpo en la titulación previa, se purificó 56C^g del anticuerpo anti-Gl_2210 mediante cromatografía de afinidad y luego se probó mediante Inmunofluorescencia Indirecta (IFI) su capacidad de reconocer la estructura conformacional in Vitro sobre un co-cultivo de células endoteliales infectadas por ANDV. La fracción purificada de anti-Gl_2210 mostró un título 8 veces superior a lo del plasma correspondiente (figura 6). En cambio la respuesta contra proteína N disminuyó significativamente, sugiriendo una alta especificidad de la cromatografía realizada. Experimentos de Western Blot realizados posteriormente revelaron que el aislamiento del anticuerpo realmente no había sido completamente específico, y se encontraba mezclado con anticuerpos anti- proteína N. Esto se explicaría tanto por la alta antigenicidad de esa proteína, como por la alta concentración que tiene en el plasma de los pacientes con respecto al anticuerpo anti-Gl_2210, siendo estos dos factores lo que habrían permitido su unión no específica al péptido en la columna de cromatografía. Since anti-Gl_2210 was detected using linear peptides, it was possible that the tertiary structure of the peptides does not reflect the conformational structure of the corresponding viral sequence in vivo. Therefore, a high recognition rate of an in vitro antibody does not necessarily imply its functionality in vivo. Accordingly, from 6 patients who had presented high titers of this antibody in the previous titration, 56C ^ g of the anti-Gl_2210 antibody was purified by affinity chromatography and then its Indirect Immunofluorescence (IFI) capacity was tested. recognize the conformational structure in vitro on a co-culture of endothelial cells infected by ANDV. The purified fraction of anti-Gl_2210 showed a titer 8 times higher than that of the corresponding plasma (Figure 6). On the other hand, the response against protein N decreased significantly, suggesting a high specificity of the chromatography performed. Western Blot experiments carried out later revealed that the isolation of the antibody had not really been completely specific, and was mixed with N-protein antibodies. This would be explained both by the high antigenicity of that protein, and by the high concentration it has in the plasma of the patients with respect to the anti-Gl_2210 antibody, these two factors being what would have allowed their non-specific binding to the peptide in the chromatography column.
EJEMPLO 7. Estudio de Inmunofluorescencia Indirecta (IFI) para estudiar reconocimiento in Vitro del epítope conformacional del anticuerpo anti-Gl_2210. EXAMPLE 7. Indirect Immunofluorescence Study (IFI) to study in vitro recognition of the conformational epitope of the anti-Gl_2210 antibody.
Utilizando el anticuerpo anti- Gl_2210 aislado, y con el objetivo de comprobar la capacidad de este anticuerpo por reconocer la estructura conformacional in vitro de su epítope en la Gl viral,
se realizó Inmunoflourescencia Indirecta. Para ello se cultivaron células endoteliales Vero E6, sobre placas Lab-Tek Chamber Slide (Nalge Nunc International) de 8 pocilios y se incubaron por una noche en cámara de cultivo a 37°C hasta confluencia. Luego se infectaron con ANDV a una concentración aproximada de 100 PFU (Plaque Forming Units) / lOOul. Teniendo las placas con células endoteliales infectadas por ANDV, se probó el reconocimiento con lOOul del anticuerpo anti- G1 2210 purificado en diluciones 1 :200, 1 :400 y 1 : 800. Como controles se incluyeron muestras de plasma de pacientes convalecientes a la infección por ANDV, proteína N recombinante de ANDV, virus Papiloma Bovino (BPV), Virus Papiloma Humano (HPV) y plasma de conejo inmunizado con proteína N (Rabbit anti- N serum). Para el mareaje de los IgG anti-Gl_2210 se utilizó anticuerpo anti- IgG humana conjugado con FITC (Anti-human IgG FITC) de Boehringer Mannheim. Los núcleos fueron marcados con 4,6-Diamidino-2-Fenilindol diclorhidrato (DAPI) de Merck S.A. Tanto las diluciones del anti-Gl_2210 como todos los controles se realizaron con pocilios en duplicado. La mezcla del anti-Gl_2210 con anticuerpos anti-N anteriormente descrita representó un problema para nuestro objetivo de estudiar la capacidad de anti-Gl_2210 de reconocer específicamente el epítope conformacional, debido a que no era posible distinguir si la marca era el anticuerpo estudiado anti- Gl_2210 u otros anticuerpos anti-N contaminantes a la fracción de anti-Gl_2210. Para resolver lo anterior, previo a la IFI se realizó con una alícuota del anticuerpo una pre-incubación de 4 horas a temperatura ambiente con proteína N recombinante. De esta forma se produjo la unión de los anticuerpos anti-N contaminantes y posteriormente en el ensayo de IFI se suprimió significativamente la unión a la proteína N viral. Using the isolated anti-Gl_2210 antibody, and in order to check the ability of this antibody to recognize the in vitro conformational structure of its epitope in the viral Gl, Indirect Immunoflourescence was performed. For this, Vero E6 endothelial cells were cultured on 8-well Lab-Tek Chamber Slide plates (Nalge Nunc International) and incubated overnight in a culture chamber at 37 ° C until confluence. They were then infected with ANDV at an approximate concentration of 100 PFU (Plaque Forming Units) / lOOul. Having the plates with endothelial cells infected with ANDV, recognition with lOOul of the purified anti-G1 2210 antibody was tested in dilutions 1: 200, 1: 400 and 1: 800. As controls were included plasma samples of patients convalescent to infection by ANDV, recombinant N protein of ANDV, Bovine Papilloma virus (BPV), Human Papilloma Virus (HPV) and rabbit plasma immunized with N protein (Rabbit anti-N serum). For the mapping of anti-Gl_2210 IgG, anti-human IgG antibody conjugated to FITC (Boehringer Mannheim FITC) was used. The nuclei were labeled with 4,6-Diamidino-2-Phenylindole dihydrochloride (DAPI) from Merck SA Both dilutions of anti-Gl_2210 and all controls were performed with duplicate wells. The mixture of anti-Gl_2210 with anti-N antibodies described above represented a problem for our purpose of studying the ability of anti-Gl_2210 to specifically recognize the conformational epitope, because it was not possible to distinguish whether the brand was the antibody studied anti- Gl_2210 or other anti-N antibodies contaminating the fraction of anti-Gl_2210. To solve the above, prior to IFI, a 4-hour pre-incubation at room temperature with recombinant N protein was performed with an aliquot of the antibody. In this way the binding of the contaminating anti-N antibodies occurred and subsequently in the IFI assay the binding to the viral N protein was significantly suppressed.
DESCRIPCIÓN DE LOS DIBUJOS: DESCRIPTION OF THE DRAWINGS:
Figura 1: Casos de SCPH Confirmados y Letalidad en Chile hasta 31 Octubre, 2006 (Fuente: Departamento Epidemiología Ministerio de Salud de Chile). Figure 1: Cases of SCPH Confirmed and Lethality in Chile until October 31, 2006 (Source: Department of Epidemiology Ministry of Health of Chile).
Figura 2: Secuencias peptídicas y nucleotídicas de consenso objetos de la presente invención.
Figura 3: Ciclo de infección de virus Hanta y composición estructural viral. Figure 2: Peptide and nucleotide sequences of consensus objects of the present invention. Figure 3: Hanta virus infection cycle and viral structural composition.
Figura 4: Protocolo de aislamiento de anticuerpo anti-Gl_2210 aislado desde el plasma de pacientes. Figure 4: Anti-Gl_2210 antibody isolation protocol isolated from patients' plasma.
Figura 5: Protocolo de trabajo para identificación de péptidos inmunogénicos y los epítopes específicos en pacientes convalecientes a la infección por ANDV Figure 5: Working protocol for identification of immunogenic peptides and specific epitopes in patients convalescent to ANDV infection
Figura 6: Reconocimiento de mezclas de péptidos traslapados. Figure 6: Recognition of overlapping peptide mixtures.
a: reconocimiento de mezclas de péptidos traslapados representando la proteína N de ANDV.a: recognition of overlapping peptide mixtures representing ANDV N protein.
Corte de significancia para cada mezcla = promedio + 2 x SD controles sanos. Significance cut for each mix = average + 2 x SD healthy controls.
b: reconocimiento de mezclas de péptidos traslapados representando la proteína Gl de ANDV. Corte de significancia para cada mezcla = promedio + 2 x SD controles sanos. b: recognition of mixtures of overlapping peptides representing the ANDV Gl protein. Significance cut for each mix = average + 2 x SD healthy controls.
c: reconocimiento de mezclas de péptidos traslapados representando la proteína G2 de ANDV.c: recognition of mixtures of overlapping peptides representing the G2 protein of ANDV.
Corte de significancia para cada mezcla = promedio + 2 x SD controles sanos. Significance cut for each mix = average + 2 x SD healthy controls.
Figura 7: Reconocimiento de péptidos individuales del mix 6 por IgG específicos para, determinado en 12 pacientes. Los resultados indican el porcentaje de pacientes cuyas muestras (aplicadas en triplicados) mostraban igual o más absorbancia que el promedio de los controles sanos + 2xSD controles. Plasma de cada paciente fue expuesto en un dilución 1 :50 a cada uno de los péptidos indicados (10 nmol/ml c/u) en un ELISA. Figure 7: Recognition of individual peptides of mix 6 by specific IgGs, determined in 12 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2xSD controls. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10 nmol / ml each) in an ELISA.
Figura 8: IgG específicos para péptidos individuales de los mix 32 y 33, determinado en 8 pacientes. Los resultados indican el porcentaje de pacientes cuyas muestras (aplicadas en triplicados) mostraban igual o más absorbancia que el promedio de los controles sanos + 2 desviaciones estándar. Plasma de cada paciente fue expuesto en un dilución 1 :50 a cada uno de los péptidos indicados (10mg/ml c/u) en un ELISA. Figure 8: Specific IgGs for individual peptides of mix 32 and 33, determined in 8 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2 standard deviations. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10mg / ml c / u) in an ELISA.
Figura 9: IgG específicos para péptidos individuales del mix 22, determinado en 27 pacientes. Los resultados indican el porcentaje de pacientes cuyas muestras (aplicadas en triplicados) mostraban igual o más absorbancia que el promedio de los controles sanos + 2xSD control.
Plasma de cada paciente fue expuesto en un dilución 1 :50 a cada uno de los péptidos indicados (10nmol/ml c/u) en un ELISA. Figure 9: IgG specific for individual peptides of mix 22, determined in 27 patients. The results indicate the percentage of patients whose samples (applied in triplicates) showed equal or more absorbance than the average of the healthy controls + 2xSD control. Each patient's plasma was exposed in a 1: 50 dilution to each of the indicated peptides (10nmol / ml c / u) in an ELISA.
Figura 10: Titulación de IgG específicos para Gl_2210, determinado en 12 pacientes. Los resultados indican la absorbancia obtenida por las diferentes diluciones seriadas de plasma de cada uno de los pacientes. Resultados representan el promedio de triplicados en cada paciente. Plasma de cada paciente fue expuesto en diferentes diluciones (1 :50 - 1 :409600) a 10nmol/ml de Gl_2210 en un ELISA. Figure 10: Specific IgG titration for Gl_2210, determined in 12 patients. The results indicate the absorbance obtained by the different serial serial dilutions of each of the patients. Results represent the average of triplicates in each patient. Plasma of each patient was exposed in different dilutions (1: 50-1: 409600) to 10nmol / ml of Gl_2210 in an ELISA.
Figura 11: Comparación entre el título de anti-Gl_2210 en el plasma previo a la cromatografía y el título del eluído. Figure 11: Comparison between the anti-Gl_2210 titer in the plasma prior to chromatography and the eluate titer.
Figura 12 (63x) Las imágenes del lado izquierdo corresponden a anti- Gl_2210 en diluciones 1 :200, 1 :400 y 1 :800 y las imágenes del lado derecho corresponden a anti- Gl_2210 previamente incubado con proteína N recombinante (*N). Los núcleos teñidos con DAPI (en azul) permiten identificar el mareaje de la proteína N, que Figure 12 (63x) The images on the left side correspond to anti-Gl_2210 in dilutions 1: 200, 1: 400 and 1: 800 and the images on the right side correspond to anti-Gl_2210 previously incubated with recombinant N protein (* N). DAPI-stained nuclei (in blue) allow the identification of the N protein, which
característicamente se observa con un patrón intenso más cercana al núcleo. En las imágenes del lado derecho se puede apreciar una significativa disminución del mareaje, lo que representa el mareaje únicamente de los anti-Gl_2210, con una baja o nula presencia de anticuerpos anti-N. Figura 13: (63x) Con el objetivo de determinar la efectividad del método establecido de pre- incubación del anti- Gl_2210 con proteína N recombinante para disminuir el mareaje de los anticuerpos anti-N contaminates, se realizó experimentos en paralelo con plasma de conejo inmunizado con proteína N recombinante, es decir plasma de conejo con una alta concentración de anticuerpos anti-N. La imagen 8-a corresponde al plasma de conejo intacto, y se observa una alta intensidad de mareaje. En la imagen 8-b se observa en tanto, el mismo plasma de conejo pero previamente incubado por 4hrs a temperatura ambiente, con 2 μg de proteína N recombinante. En esta se observa ausencia de mareaje lo que comprueba la alta efectividad de esta pre-incubación como método para disminuir la unión de los anticuerpos a la proteína N viral. Lo anterior comprueba que las imágenes 7-b, 7-d y 7-f corresponden efectivamente al
mareaje de anticuerpos anti-Gl_2210 reconociendo con alta especificidad y sensibilidad (diluciones 1 :200 hasta 1 : 800) al epítope conformacional en la proteína viral in vitro. characteristically it is observed with an intense pattern closer to the nucleus. In the images on the right side a significant decrease in the marking can be seen, which represents the marking of only the anti-Gl_2210, with a low or no presence of anti-N antibodies. Figure 13: (63x) In order to determine the effectiveness of the established method of pre-incubation of the anti-Gl_2210 with recombinant N protein to decrease the anti-N contaminant antibody tide, parallel experiments with immunized rabbit plasma were performed with recombinant N protein, ie rabbit plasma with a high concentration of anti-N antibodies. The 8-a image corresponds to the intact rabbit plasma, and a high intensity of marking is observed. Image 8-b shows, in the same way, the same rabbit plasma but previously incubated for 4 hours at room temperature, with 2 μg of recombinant N protein. In this, the absence of mareaje is observed, which proves the high effectiveness of this pre-incubation as a method to decrease the binding of antibodies to the viral N protein. The above verifies that the images 7-b, 7-d and 7-f correspond effectively to the anti-Gl_2210 antibody mapping recognizing with high specificity and sensitivity (dilutions 1: 200 to 1: 800) to the conformational epitope in the viral protein in vitro.
13- a. Plasma conejo anti- N 13-b. Plasma conejo anti- N/ *N 13- a. Plasma rabbit anti-N 13-b. Plasma rabbit anti-N / * N
Figura 14: (63x) Imágenes 9-a y 9-b corresponden a los controles negativos realizados con papiloma virus. (BPV: Bovine Papiloma Virus, HPV: Human Papiloma Virus). Se puede observar ausencia de mareaje, lo que da cuenta de una alta especificidad para el epítope identificado en la proteína Gl de ANDV. En las imágenes 9-c y 9-d se observan 2 muestras de plasma de pacientes convalecientes a la infección por ANDV, realizadas como controles positivos. En estas se observa un mareaje de patrón difuso muy intenso en las zonas perinucleares, lo correspondería al mareaje extenso de todos los anticuerpos desarrollados por estos pacientes. Figure 14: (63x) Images 9-a and 9-b correspond to the negative controls performed with papillomavirus. (BPV: Bovine Papiloma Virus, HPV: Human Papiloma Virus). Absence of mareaje can be observed, which accounts for a high specificity for the epitope identified in the ANDV Gl protein. In images 9-c and 9-d, 2 plasma samples of patients convalescent to ANDV infection are observed, performed as positive controls. In these, a diffuse pattern of very intense pattern is observed in the perinuclear areas, which would correspond to the extensive screening of all the antibodies developed by these patients.
14- a. Anti- BPV (Control Negativo) 14-b. Anti- HPV (Control Negativo) 14 to. Anti-BPV (Negative Control) 14-b. Anti-HPV (Negative Control)
14-c. Plasma ·# 1 (Control Positivo) 14-d. Plasma ·# 2 (Control Positivo)
14-c. Plasma · # 1 (Positive Control) 14-d. Plasma · # 2 (Positive Control)
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Claims
1. Un anticuerpo policlonal humano CARACTERIZADO porque une la proteína Gl de Bunyaviridae, reconociendo una secuencia aminoacídica que posee más de un 80% de homología con respecto a DQDVWYCNGQKKVI (Gl_2210). 1. A human polyclonal antibody CHARACTERIZED because it binds the Gl protein of Bunyaviridae, recognizing an amino acid sequence that has more than 80% homology with respect to DQDVWYCNGQKKVI (Gl_2210).
2. Un péptido inmunogénico CARACTERIZADO porque posee más de un 80% de homología con la secuencia DQDVWYCNGQKKVI (Gl_2210). 2. A CHARACTERIZED immunogenic peptide because it has more than 80% homology with the sequence DQDVWYCNGQKKVI (Gl_2210).
3. Vacuna contra el Hantavirus CARACTERIZADA porque posee el péptido inmunogénico de la reivindicación 2 y excipientes farmacéuticamente aceptables. 3. Vaccine against the CHARACTERIZED Hantavirus because it possesses the immunogenic peptide of claim 2 and pharmaceutically acceptable excipients.
4. Vacuna de acuerdo a la reivindicación 3 CARACTERIZADA porque sirve para prevenir la infección por Hantavirus andes (ANDV). 4. Vaccine according to claim 3 CHARACTERIZED because it serves to prevent infection by Hantavirus andes (ANDV).
5. Vacuna de acuerdo a la reivindicación 3 CARACTERIZADA porque sirve para prevenir la infección por Hantavirus Sin Nombre Virus (SNV). 5. Vaccine according to claim 3 CHARACTERIZED because it serves to prevent infection by Hantavirus Sin Nombre Virus (SNV).
6. Secuencia nucleotídica de ADN CARACTERIZADA porque codifica para la cadena liviana y/o pesada del anticuerpo de la reivindicación 1. 6. Nucleotide sequence of CHARACTERIZED DNA because it codes for the light and / or heavy chain of the antibody of claim 1.
7. Una composición farmacéutica CARACTERIZADA porque comprende un anticuerpo policlonal humano de la reivindicación 1 y excipientes farmacéuticamente aceptables. 7. A pharmaceutical composition CHARACTERIZED in that it comprises a human polyclonal antibody of claim 1 and pharmaceutically acceptable excipients.
8. Uso de un anticuerpo de la reivindicación 1 CARACTERIZADO porque sirve para preparar medicamentos para neutralizar Hantavirus. 8. Use of an antibody of claim 1 CHARACTERIZED because it serves to prepare medicaments to neutralize Hantavirus.
9. Uso de un anticuerpo según la reivindicación 8 CARACTERIZADO porque sirve para preparar medicamentos para neutralizar el Hantavirus andes (ANDV). 9. Use of an antibody according to claim 8 CHARACTERIZED because it serves to prepare medicaments to neutralize the Hantavirus andes (ANDV).
10. Uso de un anticuerpo según la reivindicación 8 CARACTERIZADO porque sirve para preparar medicamentos para neutralizar Hantavirus Sin Nombre Virus (SNV). 10. Use of an antibody according to claim 8 CHARACTERIZED because it serves to prepare medicaments to neutralize Hantavirus Sin Nombre Virus (SNV).
1 1. Uso de un péptido de la reivindicación 2 CARACTERIZADO porque sirve para diseñar un método de inmunoterapia pasiva o activa. 1 1. Use of a peptide of claim 2 CHARACTERIZED because it serves to design a passive or active immunotherapy method.
12. Uso de un péptido de la reivindicación 2 CARACTERIZADO porque sirve para diseñar un método de inmunoterapia pasiva o activa contra Hantavirus andes (ANDV). 12. Use of a peptide of claim 2 CHARACTERIZED because it serves to design a passive or active immunotherapy method against Hantavirus andes (ANDV).
13. Uso de un péptido de la reivindicación 2 CARACTERIZADO porque sirve para diseñar un método de inmunoterapia pasiva o activa contra Hantavirus Sin Nombre Virus (SNV). 13. Use of a peptide of claim 2 CHARACTERIZED because it serves to design a passive or active immunotherapy method against Hantavirus Sin Nombre Virus (SNV).
14. Un método de screening de muestras biológicas CARACTERIZADO porque se utiliza como primer anticuerpo el anticuerpo anti-Gl_2210 de la reivindicación 1 y como segundo anticuerpo un anticuerpo marcado, con fluorescencia, fosforescencia, luminiscencia, radioactividad o una actividad enzimática, donde la reacción puede ser leída en un instrumento adecuado. 14. A biological sample screening method CHARACTERIZED in that the anti-Gl_2210 antibody of claim 1 is used as the first antibody and as a second antibody a labeled antibody, with fluorescence, phosphorescence, luminescence, radioactivity or an enzymatic activity, where the reaction can Be read in a suitable instrument.
15. Un kit CARACTERIZADO porque posee un anticuerpo anti Gl_2210, un segundo anticuerpo marcado de acuerdo a la reivindicación 14 y se utiliza para la detección in vitro de muestras positivas para Hantavirus. 15. A CHARACTERIZED kit because it has an anti Gl_2210 antibody, a second labeled antibody according to claim 14 and is used for in vitro detection of Hantavirus positive samples.
16. Uso de un kit de acuerdo a la reivindicación 15 CARACTERIZADO porque se utiliza para la detección in vitro de muestras positivas para Hantavirus andes. 16. Use of a kit according to claim 15 CHARACTERIZED because it is used for in vitro detection of samples positive for Hantavirus andes.
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| WO2015130722A1 (en) * | 2014-02-25 | 2015-09-03 | Albert Einstein College Of Medicine Of Yeshiva University | Methods and assays for treating hantavirus infections |
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| WO2006088478A2 (en) * | 2004-06-18 | 2006-08-24 | Novartis Vaccines And Diagnostics Inc. | Methods and reagents for diagnosing hantavirus infection |
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| US20040053216A1 (en) * | 1999-01-29 | 2004-03-18 | Hooper Jay W. | DNA vaccines against hantavirus infections |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015130722A1 (en) * | 2014-02-25 | 2015-09-03 | Albert Einstein College Of Medicine Of Yeshiva University | Methods and assays for treating hantavirus infections |
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