MXPA00012809A - Haemophilus parasuis - Google Patents

Abstract

A cellular extract of Haemophilus parasuis that exhibits toxic activity is described. The cellular extract is useful as a vaccine or diagnostic reagent.

Description

VACCINE OF HAEMOPHILUS PARASUIS AND DIAGNOSIS Field of the Invention The invention relates to a preparation of cellular extracts of Haemophilus parasuis, which has toxic activity. The preparation is useful as a vaccine and as a diagnostic agent for Haemophilus parasuis.

Background of the Invention Glásser observed in 1910 a small Gram-negative organism associated with fibrinous serositis and polyarthritis in pigs, which was eventually identified as H. parasuis, and taxonomically distinguished from H. parasuis. It is now accepted that H. parasuis is the infectious agent of porcine polyserositis and arthritis (or Glasser's disease). H. parasuis (Hps) is a small pleomorphic Gram-negative rod, which varies from a coccobacillary form to thin filaments. Growth is supported only in a medium containing nicotinamide adenine dinucleotide (NAD) (heated blood agar, Levinthal's agar), or in blood agar in the vicinity of a stripe of a strain of Staphylococcus (satelitism). A visible growth usually occurs after 36 to 48 hours of incubation. Porcine polyserositis-arthritis is found throughout the world, and was historically considered as a sporadic disease associated with tension in young pigs. More recently, the introduction of the pathogen in large populations of herds free of the specific pathogen (SPF), has demonstrated a devastating effect: The infection can spread as a contagious disease of high morbidity, affecting pigs of all ages s Ln stress factors obvious associates. The disease most frequently afflicts young pigs (2 weeks to 4 months), mainly after the weaning period (5 to 8 weeks). Mortality can reach 50 percent. Serological studies using extracts of autoclaved cells with an agar-gel precipitation assay have shown at least 15 different serotypes that differ in their pathogenicity (Kielstein, J. Clin.Micro.30: 4: 862 (1992)) . Vaccines for H. parasuis are commercially available, which are made from inactivated, or bacterial, bacteria. A drawback of bacterin vaccines is that they elicit antibodies against primarily (lipo) polysaccharides that are only specific for certain serotypes of H. parasuis, and therefore, do not protect against other serotypes of H. parasuis. The degree of protection against infection in the field provided by bacterin vaccines, may also be lower than jj ^^^ jy ^ Aff fMÉl ^^ desired. Even when not currently available, live attenuated H. parasuis vaccines would suffer from the drawbacks normally associated with live vaccines, 5 including the risk of inoculating animals with inadequately attenuated pathogens, and the possibility that attenuated bacteria may be reversed. to a pathogenic state, resulting in disease in the inoculated animal, and the spread of pathogens to other animals.

Accordingly, there is a need for a H. parasuis vaccine that is safe, effective, and that provides heterologous protection (serotype-independent). For this purpose, the present inventors devoted themselves to developing an alternative vaccine. 15 The rapid establishment of the disease, and the severity of internal damage, suggest that the clinical effects may be caused by the expression of one or more Hps toxins. It has been suggested that Hps can induce functional and structural damage to the nasal mucosa, associating initially with the mucosal layer, inducing damage to the underlying mucosa by releasing one or more toxic compounds (Vahle, J.L., Ph.D. thesis, AAI9626072, 960902, Iowa State University (1996)). The chemical nature of any of these toxins, and the way they exercise its effects, it had not been elucidated.

Gram-negative endotoxin has been studied as an possible virulence factor of H. parasuis. Endotoxin is a lipopolysaccharide (LPS) component of the outer membrane of Gram-negative bacteria, and is released with bacterial lysis. The study by Amano et al (J. Vet. Med. Sci. Vol. 59, No. 6, 451-455 (1997)) reported that Gram-negative endotoxin levels were increased in the blood of pigs with acute septicemia caused by . parasuis, and that the H. parasuis antigen was associated with pathological lesions. However, it can not be concluded, based on the findings of Amano et al., That it is endotoxin that is causing the clinical and pathological manifestations of the disease, and the identity of the factors still remains unclear. that cause the pathological effects of infection by y. parasuis It is an object of the present invention to find one or more toxins that contribute to the pathology of H. parasuis infection. It is also an object of the loan me invention to develop vaccines containing one or more toxins. Finally, it is an object of this invention to use this antigenic material as a reagent in a diagnostic assay.

SUMMARY OF THE INVENTION The present invention provides a novel cellular extract of Haemophilus parasuis, which has a toxic activity. This extract causes pathological lesions 5 characteristics of H. parasuis infection when administered intraperitoneally to pigs. Accordingly, the extract appears to contain at least one toxin, which is referred to herein as HpTx. It is also contemplated that there may be more than one toxin in this extract. Vaccines containing the extract are also provided, which are effective to provide protection against homologous and heterologous aggression. It is contemplated that vaccines containing toxin or isolated toxins such as immunogens will provide this same type of protection. Finally, the extract is useful as a diagnostic tool, and can be used to raise the antibodies that can be used as diagnostic reagents to detect H. parasuis antigen, or it could be used by itself as a diagnostic reagent to detect antibodies to H. parasuis. Cases for diagnostic tests using these materials are also considered part of the present invention. * - ~~ *** ~ * »« ° * > 8 ~ * ** ~~ ** -. - .. - ^ > ^ A ^ ~ gf lg gg ^^ Brief Description of the Figure Figure 1 shows SDS-polyacrylamide gels (SDS-PAGE) of a cell extract prepared as described in Example 1, Preparation C), as well as proteins of external membrane (OMP) purified from serotypes 4 and 5. Figure 2 shows a polyacrylamide gel (Noveks New Page at 4-12 percent) of a cell extract prepared as described in Example 1, Preparation C, on an unfiltered raw material. Figure 3 shows a polyacrylamide gel (Noveks New Page at 4-12 percent) of a cell extract prepared as described in Example 1, Preparation C.

DETAILED DESCRIPTION OF THE INVENTION The cellular extract of the present invention is prepared by essentially lysing the bacterial cells, granulating the remaining cells and large cell debris, and collecting the supernatant. Bacterial cells can be lysed by any conventional method; preferably, high pressure is used, such as passing them through a microfluidizer. Preferably, the bacterial culture is concentrated before use, by centrifugation and resuspension of the cell granule in a smaller volume of the fluid than was originally present.

Any serotype of the H. parasuis strain can be used to prepare the cell extract of the present invention. Serotypes 4 and 5 are preferred; serotype 4 is more preferred. Preferably, the supernatant containing the cell extract is filtered to remove any remaining cells or debris, and this is preferably done using a 0.2 micron filter. The supernatant can be frozen for future use. It is also possible to freeze the cell extract by freezing, and reconstruct the lyophilized material just before use. For parenteral administration, the supernatant material itself can be used as a formulation (ie vaccine, or can be mixed with an appropriate pharmaceutical carrier, as desired. The lyophilized material is reconstituted in a pharmaceutically acceptable vehicle, such as an aqueous medium or a suspension containing water. These vehicles are often mixed with other constituents, for example, in order to increase immunizing activity or shelf life. These constituents may be salts, pH regulators, stabilizers, emulsifiers, and adjuvants to improve. '.a immune response. Examples of adjuvants, s: .n intended to be limited, include oil-in-water and water-in-oil emulsions Vitamin E and other oils, oils ^^^^^^^^^^^^^^^^^^ Ijjl ^ gjjlj ^ j ^^^^^^^^ minerals, metabolizable organic oils, aluminum compounds, muramyl dipeptide, saponins, polyanions, compounds unfriendly, metal salts and block (co) polymers. However, the use of adjuvants 5 is not necessary to provide an immunogenic activity to the compositions of the present invention. The immunogenic preparations of the invention are preferably administered to the pigs parenterally, generally by intramuscular or subcutaneous injection. The The vaccine is most effective if it is administered in a series of at least two separate doses at intervals of two to three weeks, but other immunization schedules can be effective, and can be determined by conventional elements. The amount of cell extract preparation used is the amount that can induce immunity in pigs against aggression by H. parasuis. Immunity is defined as the induction of a significant level of protection in a population of pigs after vaccination, comparing with group not vaccinated. On a per dose basis, the amount of the cell extract in a dose of vaccine can be from LO micrograms of protein A to approximately 10 milligrams of protein per pig. A preferable range is from approximately 100 micrograms to approximately IDO micrograms per pig. An expert in the field can It is easy to determine the appropriate dose, usually according to the age of the pig, The vaccines of the present invention can also be formulated in multivalent vaccines comprising other immunogenic materials. From one or more different pathogens, for example, the vaccine may contain a material from one or more of the following pathogens: pseudorabies virus, transmissible gastroenteritis virus, porcine parvovirus, swine influenza virus, Mycoplasma hyopneu oniae, Escherichia coli, Erysipelothrix rhusiopathiae, Bordetella bronchioseptica, Pasteurel la multocida, and Actinobacillus pleuropneumonia. In a further embodiment of the present invention, the cellular extract of the invention can be used as a diagnostic tool to detect infection by. parasuis For example, the cell extract can be used as an antigen in an immunological assay for H. parasuis antibodies in a test sample (tissue or body fluid, for example blood, plasma, serum, etc.). From Alternatively, the cell extract can be used to raise antibodies (monoclonal or polyclonal) to the antigenic material, and in turn, the antibody or antibodies can be used in an immunological assay for the antigen in the test sample. Any immunological assay format, such as ELISA, Western blo_, sandwich assay which are well known to those skilled in the art. Diagnostic assay kits are also incorporated in the present invention, and may comprise one or both of the cell extract and the antibodies thereto, optionally linked with a solid support, and optionally containing the different reagents (regulators, etc.) used. in the trial. Detectable labels for the antigen and antibody are well known in the art, such as horseradish peroxidase or other brands of enzymes, biotin, fluorochromes, and radiolabels. The compositions and methods of the present invention are illustrated by the following non-limiting examples.

Example 1 - Method of Preparation of Cell Extract Culture conditions H. parasuis serotype 4, strain SW124, was grown in Tryptic Soy Broth containing 0.1 percent NAD and 1 percent yeast extract (H-TSB) with 5 20 percent horse serum in 3,000 milliliters of total volume. The culture was grown at 37 ° C for 5 hours, to approximately 1 x 108 - 1 x 109 CFU / ml.

Preparation A 25 After the previous incubation, 250 were harvested milliliters of the culture by centrifugation at 6,000 rom for 15 minutes to separate the cells. The supernatant was filtered with a 0.2 micron filter (Nalgene). This is a supernatant preparation that would contain any 5 endotoxins and other secreted materials, and is used for comparison purposes.

Preparation B The remaining portion of the culture (2,500 milliliters) is centrifuged at 6,000 rpm for 15 minutes. For this preparation, the cells were concentrated by discarding the supernatant, and returning the suspended granules to a volume of approximately 300 milliliters. Then the concentrated cells were used with one pass through a microfluidizer (Microfluidics, Newton, Mass) at 1120 kg / cm 2. The lysed cells were then centrifuged at 8,000 rpm for 20 minutes to remove cell debris. The supernatant of this test was then collected and filtered with a 0.2 micron filter to remove any remaining cells or large cell debris. This is the preparation of cellular extract that has toxic activity.

Preparation C 25 In a preferred method for preparing the extract Cell, Haemophilus parasuis serotype 4, strain SW124, is grown at 37 ° C in H-TSB for 3 to 5 hours, up to about 30 percent T (1 x 108 - 1 x 109 CFU / ml). The culture is centrifuged at 8,500 xg for 20 minutes. The cell granule is resuspended in sufficient serum equivalent to a concentration of 10 times the cells originally present. The cell concentrate is microfluidized at approximately 1120 kg / cm2. The large cell debris is removed by centrifugation at 15,000 xg 10 for 20 minutes. The supernatant is filtered through a 0.2 micron filter. This filtrate can be used as a fresh preparation, but it can also be frozen or lyophilized for future use. The storage is approximately -70 ° C. Example 2 - Test of Biological Activity in Pigs The preparations of Example 1 were then used to evaluate the biological activity in pigs. Three pigs were inoculated intraperitoneally with 10 milliliters of 7a20 preparation A, 3 with 10 milliliters of preparation B (3 milligrams / milliliter of protein), and two served as controls. The controls were not inoculated. All the animals were kept in the same room and in the same pen during the study. The food was removed 16 to 24 25 hours before the administration of the cell fractions ^ _ ^ _ ^ jft¡-r ^ b¿ ^? &? - * | 3 * ^ ^^^^^^ g ^ j ^^^^ s ^^ jígii ^^^^ i ^^^ ^^ (A and B). Within 24 hours of the inoculation, one of the pigs inoculated with preparation B (No. 1681) had died. The necropsy showed fibrin strands and accumulation of fluid in the peritoneal cavity. The thoracic cavity showed threads (ie fibrin and pleuritis) Several of the remaining pigs (see TABLE 1) showed respiratory signs 24 hours after the inoculation, and then all the remaining pigs were sacrificed and their necropsy was done. show in TABLE 1.

TABLE 1 u ___ ^ ag * ^ j ^ ¡^ ¡g ^ Pig lung material 1677 was cultured on a Haemophilus (HTM) and blood agar (BA) assay medium. No growth was seen after 24 hours at 37 ° C over any means, demonstrating in this way that clinical signs were not due to bacterial infection.

Example 3 - Vero Cell toxicity For the determination of the cytotoxicity of the In 10 preparations, Vero cells were cultured in a minimal Eagles Non-Essential Amino Acid Medium (EMNE) (Gibco) with 2 percent bovine calf serum to a confluence of approximately 80 to 100 percent on a plate of u ^ 96 wells. The cell monolayers were washed 2 to 3 times with a basal EMNE medium, and then Preparation C was added in double dilutions through the plate. Cells were graded for cytotoxicity, ie cell rounding, enlargement, lysis, and substrate separation, at regular intervals 24 hours after inoculation. At 48-72 hours, the culture showed cytotoxicity. Accordingly, the cell extract of the present invention causes cytotoxicity in Vero cells.

Example 4 - Efficacy of the Mouse Vaccine To evaluate the potential of the cell extract vaccine, mice were vaccinated intraperitoneally with 0 2 milliliters of cell extract (3 milligrams / milliliter of protein) prepared as in Example 1 (serotype 4), Preparation C, mixed at 1: 1 with adjuvant Diluvac Forte (Intervet, BV), or serum (no adjuvant) .The mice were vaccinated at 0 and at 14 days, and were attacked with H. parasuis serotype 4 (homologous aggression). , or H. parasuis serotype 5 (heterologous aggression) 10 days after the second vaccination The results are shown in TABLE 2.

TABLE 2 As seen in Table 2, vaccination with the cell extract of the present invention provided good protection in mice, even in the absence of an added adjuvant. In addition, the cell extract of the present invention, made of serotype 4, provided protection against the heterologous aggression of serotype 5, as well as against homologous aggression by serotype 4.

Example 5 - Efficacy in pigs To evaluate the vaccine potential of the cell extract in pigs a total of 4 pigs were vaccinated intramuscularly with 2.5 milliliters of the cell extract, prepared as in Example 1 (Preparation C) from serotype 4, mixed to 1: 1 with adjuvant Diluvac Forte ™ in 3 milligrams of protein per dose. The pigs were vaccinated at 3 and 6 weeks, and were attacked at 8 weeks with H. parasuis serotype 4 (homologous aggression), or H. parasu is serotype 5 (heterologous aggression). The controls (14 pigs in each control group) for the study were unvaccinated animals, and whole cell vaccine groups. The whole cell vaccine contained whole cells from H. parasuis serotypes 4 and 5 mixed with Diluvac adjuvant Forte MR. Each test group was divided in half, with 7th half assaulted with serotype 4, and the other half assaulted with serotype 5. All animals were sacrificed at 9 weeks, and were scored according to the following method. . 20 A necropsy score is given to each pig, based on the rating system shown below. A final Necropsy Score is calculated by adding the qualified total points. The Group Necropsy Ratings are calculated by taking the sum of the Necropsy Qualifications of individual pigs, and dividing by the number of pigs in the group. All pigs that die before slaughter and have typical signs of H. parasuis infection at necropsy receive a 25-point necropsy score. 1. Peritoneal cavity 0 = normal 1 = fibrin strands present 2 = accumulation of fluid 2 = accumulation of fibrin mark 5 = adhesion of membranes and / or organs to one another or to the body wall. 2. Thoracic cavity 0 = normal pleura and pericardium 2 = accumulation of fluid 2 = accumulation of fibrin mark 2 = pleuritis 3 = pericarditis The necropsy scores are out of a possible 19 (maximum possible points of the pigs that survive). The results are shown in TABLE 3.

TABLE 3 Vaccination Average score of necropsy aggression with serotype 5 serotype 5 Cell extract, serotype 4 3.00 5.50 Whole cells 4 and 5 6.43 4.57 Not vaccinated 15.57 9.40 Table 3 shows that vaccination with the cell extract preparation provides good protection against homologous and heterologous aggression in pigs. Example 6 - Protein Characteristics of Cellular Extract A. SDS-PAGE Gels The cell extract preparations (Preparation C, Example 1) were tested on SDS-acrylamide gels (SDS-10 PAGE), together with purified outer membrane proteins. (OMP) of serotypes 4 and 5. The protein content was determined by the BCA method (Pierce). The results are shown in Figure 1. The loaded materials are as follows: track 2 - cell extract, 7.25 micrograms; lane 3 - molecular weight standards; lane 4 - cellular extract, 7.25 micrograms; track 5 - 45 Kd marker; ^ «? ^ Track 6 - Rainbow Markers; lane 7 - molecular weight standards; lane 8 - OMP, serotype 4, 8 micrograms; track 9 - rainbow markers; 5 track 10 - 45 Kd marker; lane 11 - molecular weight standards; lane 12 - OMP, serotype 5, 7.6 micrograms; and track 13 - rainbow markers. (Rainbow markers are color markers 10 previously stained where the bands of different sizes are different in color). Polyacrylamide gradient gels (Novex Nupage at 4 12 percent) were also tested, and are shown in Figures 2 and 3. The clues on the gel shown in Figure 15 2 are as follows: Track 1 - Cellular extract from supernatant not filtered. Track 2 - same as track 1, except for the addition (ie SDS + Beta-Mercaptoethanol at room temperature) Track 3 - same as track 1, except for the addition of SDS at room temperature. track 1, but after 2 hours, treatment at 60 ° C. Runway 5 - after 2 hours treatment at 70 ° C. Runway 6 - after treatment for 2 hours at 90 ° C. Runway 7 - after 2 hours, treatment at 100 ° C.

Track 8 - after treatment for 2 hours or 35 ° C. Track 9 - same as track 8, except that in addition it takes treatment for 2 hours at 60 ° C. Track 10 - same as track 1, but with addition of Beta-mercaptoethanol at room temperature. Lane 11 - then treatment for 5 minutes at 100 ° C and addition of beta-mercaptoethanol. Track 12 - wide range markers (Bio-Rad). The tracks illustrated in Figure 3 are as follows: Lane 1 - filtered supernatant (cell extract). Track 2 - same as track 1, except for the addition (ie SDS + Beta-Mercaptoethanol at room temperature) Track 3 - same as track 1, except for the addition of SDS at room temperature Track 4 - same as track 1, but after two hours treatment at 60 ° C. Runway 5 - after 2 hours, treatment at 70 ° C. Runway 6 - after treatment for 2 hours at 90 ° C. Runway 7 - after 2 hours treatment at 100 ° C. Track 8 - after treatment for 2 hours at 35 ° C. Runway 9 - same as runway 8, except that in addition it is treated for 2 hours at 60 ° C. Runway 10 - same as runway 1, but with addition of Beta-Mercaptoethanol at room temperature, Track 11 - after treatment for 5 minutes at 100 ° C, and addition of beta-mercaptoethanol. Track 12 - wide range markers (Bio-Rad).

B. Sizing Determinations: To help determine the size of any toxin in the cell extract, the cell extract was filtered through Amicon 100 Kd and 300 Kd filters. The filtrates and retentates were applied to Vero cells and pig alveolar macrophages in double dilutions, in order to look for cytotoxicity. In addition, 17 milliliters of cell extract was ultracentrifuged at 28,000 g for 5 hours, and applied to Vero cells and to alveolar macrophages of pigs in double dilutions. None of the cell extract preparations had any effect on the macrophages. In Vero cells, the retentates of 100 Kd and 300 Kd showed cytotoxicity, as well as the supernatant. These results suggest that either the entity with the toxic activity in the cell extract is greater than 300 Kd, or the toxic entity forms aggregates that are greater than 300 Kd under non-reducing conditions.

C. Effect of Digestion with Proteinase K on 7a Toxicity of Cellular Extract The cell extract was digested with proteinase K in calcium chloride. The toxicity of the M ** m í í m »* á ** ,, .. ^. _ * > .... '_ _ * -_- * _ ----.-__ ^ ¿j ^ ¡^ ^ Digested cellular material and controls of CaCl2 in pigs. Qualifications of the Pigs From the above results, it can be concluded that the toxic material in the extract is proteinaceous, because the digested cell extract loses its toxicity.

Cytotoxicity in Vero Cells The immobilized beads with proteinase K (Merck Cat. 10 No. 1.1293) were washed according to the instructions. 500 milliliters of cell extract was added to 250 milliliters of beads in a tube with 1 milliliter instantaneous cap. The tube was gently shaken for 2 minutes at room temperature, and then centrifuged at 10,000 xg. for 30 seconds. The supernatant was pipetted, and centrifuged again for 15 minutes at 20,000 xg to granulate any remaining beads.

These samples treated with PK were added to Vero cells at the time they were coated. The untreated cell extract was used as a positive control. The toxic cellular extract was added at dilutions of 1: 2, twice as high as 5: 256. Plates were incubated at 37 ° C in C02 at 5 ° C for 7 days. The wells were checked to determine cytotoxicity. The untreated cell extract (positive control) was cytotoxic up to 1:16, and the cell extract treated with PK was not cytotoxic in any dilution. These 10 results are additional evidence that the toxic entity is proteinaceous.

Claims (16)

1. A cell-free composition comprising cytotoxic material from Haemophilus parasuis.

2. A cellular extract of Haemophilus parasuis, which is immunogenic and has toxic activity.

3. A composition comprising the extract of claim 2, and a pharmaceutically acceptable excipient.

4. A vaccine for the protection of H. parasuis infection, which comprises the cell extract of claim 2.

5. The vaccine of claim 4, which further comprises a pharmaceutically acceptable excipient.

6. The vaccine of claim 4, which further comprises an adjuvant. The vaccine of claim 4, wherein the cell extract is from H. parasuis, serotype 4. 8. A method for the preparation of a cellular extract of Haemophilus parasuis, which comprises: a. centrifuge a culture of Haemophilus parasuis; b. remove the supernatant from step a), re-suspend the granulated cells, and lyse the resuspended cells; c. centrifuge to remove any cells f || - ... - .. ^^ as ^ -, .. to the rest and cellular waste; d. obtain the supernatant of step c), thereby obtaining the cell extract. 9. The method of claim 8, wherein "parasuis is serotype 4. 10. A method for immunizing animals against parasuis, which comprises administering the vaccine of claim 4. to the animal. Claim 10, wherein the vaccine is administered parenterally 12. A method for diagnosing H. parasuis infection, which comprises incubating the cellular extract of claim 2 with a test sample in a manner that allows the formation of a Antigen-antibody complex; 15 detect the complex, whereby the presence of a complex indicates the past or present infection by H. parasuis. The method of claim 12, wherein the complex is detected with a labeled antibody. 14. A method for diagnosing H. parasuis infection, which comprises incubating antibodies that have been raised against the cell extract of claim 2, with a test sample in a manner that allows the formation of a antigen-antibody; and detect 25 the complex, whereby, the presence of a complex indicates infection by H. parasuis. 15. The method of claim 14, wherein the complex is detected with a labeled antibody. 16. A test kit comprising the cell extract of claim 2. J ^^ '- ** ^ **. ** ^ ** ^. * .. *. . . z. . ,, ^^ .. ^ &