Poly(methacrylic acid) Hydrogels as Carriers of Bacterial Exotoxins in

Chapter 25

Poly(methacrylic acid) Hydrogels as Carriers of Bacterial Exotoxins in an Oral Vaccine for Cattle 1

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 27, 2016 | http://pubs.acs.org Publication Date: November 30, 1993 | doi: 10.1021/bk-1994-0540.ch025

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T. L. Bowersock, W. S. W. Shalaby , M. Levy , M. L. Samuels , R. Lallone , M. R. White , D. Ryker , and Kinam Park 1

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Schools of Veterinary Medicine, Pharmacy, and Math, Purdue University, West Lafayette, IN 47907 Brookwood Biomedical Institute, Box 26221, Birmingham, AL 35226 4

Poly(methacrylic acid) hydrogels were tested as a delivery system for oral vaccines in cattle. Hydrogels were absorbed with a vaccine composed of culture supernatants of Pasteurella haemolytica, the most common cause of bacterial pneumonia in cattle. Hydrogels absorbed with culture supernatants were administered orally to calves. Calves were then challenged by an intrabronchial instillation of virulent P. haemolytica. For each calf survival time was recorded. All surviving calves were euthanatized 3 days after challenge. A post mortem examination was performed and the lungs of each calf were evaluated for the size and severity of pneumonic lesions. Calves vaccinated orally with culture supernatants had less lung affected by pneumonia, less severe pneumonic lesions, and lived longer than non-vaccinated calves. Results indicate that hydrogels can be used to deliver oral vaccines to calves to enhance resistance to pneumonia caused by P. haemolytica.

Pasteurella haemolytica is the most common bacterium that causes death in cattle affected by pneumonia. Numerous vaccines have been developed to prevent this disease with limited success. The newest vaccines to prevent pneumonic pasteurellosis are composed of culture supernatants(l). Culture supernatants (CS) contain the most immunogenic antigens necessary to stimulate protective immunity in calves, and do not cause disease. However, the efficacy of vaccines containing CS has also been questioned (2). As with other vaccines used to prevent pasteurellosis, this vaccine has been used parenterally in feedlots and sales barns where cattle have already been stressed and exposed to multiple pathogens. Herein lies a major reason for poor success of these vaccines: they are not given prior to the time when calves are stressed and infected. Vaccination prior to exposure is desirable i.e., when calves are still on

0097-6156/94/0540-0288$06.00/0 © 1994 American Chemical Society

Shalaby et al.; Polymers of Biological and Biomedical Significance ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Carriers of Bacterial Exotoxins in Oral Vaccine


the farm of birth on pasture. This requires extensive handling of animals that is logistically impractical. The most efficient way to vaccinate these calves is through the feed or water. The oral administration of vaccines to cattle offers many advantages over parenteral inoculations: reduced handling results in less stress to animals; there is a lower labor cost associated with vaccines administered in the feed because animals do not need to be treated one at a time; easier delivery encourages vaccination at a time when calves are less stressed and more likely to respond to a vaccine, e.g., when on pasture with the dam; and there are less injection site reactions that can result in trimming of expensive cuts of meat at slaughter resulting in less profit to packers and producers. Oral administration can also result in the stimulation of mucosal immunity at a time when calves may have (blocking) maternally derived antibodies. Finally, the size and density of hydrogels used to deliver antigens in this experiment are compatible with those of feed pellets. Hydrogels could easily be administered to calves in the feed without any special handling needed. There is growing interest in the development of vaccines that can be administered orally. Oral vaccination is effective because it results in the stimulation of the gut-associated lymphoid tissue (GALT). Migration of lymphocytes from GALT results in increased immunity at other mucosal sites including the lung (3). Immunity to a variety of infectious agents has been induced at other mucosal surfaces in laboratory animals and man following the oral administration of antigens (4-7). The oral administration of vaccines is much more difficult to achieve in ruminants. Oral vaccination of cattle requires that the antigen be able to withstand the extreme changes in pH and severe action of proteolytic enzymes in the upper gastrointestinal tract (GIT). Cattle have a complex upper GIT composed of 4 stomachs: rumen, reticulum, omasum, and abomasum. The rumen is the first stomach and is the site of cellulolytic microorganisms that initiate digestion of fiber in the ruminant diet. Bypassing the rumen or first stomach is especially demanding since microbial degradation would also be likely to destroy most antigens. Therefore, a carrier is needed that can protect antigens until they reach the lower gastrointestinal tract. Hydrogels are crosslinked polymers that swell in the presence of water. The crosslinking creates a meshwork that can trap drugs, chemicals, or antigens. When the hydrogels are hydrated within the body of an animal, the meshwork expands releasing the trapped material. Hydrogels are currently being investigated as a means of oral delivery for drugs, hormones, and peptides. These materials require a controlled release within the body in order to exert a long lived effect with minimal dosing (812). Hydrogels can also be used to protect drugs or antigens from the proteases present in the stomach and lower intestine (13,14). Recently, we demonstrated that orally administered hydrogels can be used to release drugs slowly over time to dogs. Hydrogels were produced that swelled and were retained in the stomach releasing drug for 54 hours (15). Most studies for the delivery of drugs for biomedical purposes


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POLYMERS OF BIOLOGICAL AND BIOMEDICAL SIGNIFICANCE

use hydrogels that are biocompatible which are broken down within the body by enzymes or pH. Our approach in the present experiment was to use non-absorbable hydrogels made of poly(methacrylic acid) (PMA). This polymer was selected for several reasons: it can absorb and release large proteins efficiently; it does not swell too quickly thereby retaining the density needed to bypass the rumen; it is chemically inert and does not stimulate an immune response; and it is resilient and therefore capable of controlled release of drug in the presence of greatfrictionalactivity in the upper GIT of ruminants. This is necessary because if the hydrogel broke down too soon, it would release antigen before leaving the rumen resulting in the proteolytic loss of the antigen. Being non- degradable the PMA hydrogels pass unabsorbed from the body in the feces without causing adverse reactions in the host animal. We have recently demonstrated that orally administered PMA hydrogels can bypass the rumen to deliver a model antigen to the lower GIT of sheep for 96 hours (16). In the present study we hypothesized that hydrogels could be absorbed with culture supernatants of the bacterium P. haemolytica. administered as an oral vaccine, and stimulate protective immunity to pneumonic pasteurellosis in calves.

Materials and Methods Preparation of hydrogels - Poly(methacrylic acid) hydrogels were prepared by polymerizing 40% (w/v) methacrylic acid (Aldrich Chemical Co.) in the presence of 0.8% Ν, N - methylenebisacrylamide (Bio-Rad Laboratories), a crosslinking agent. Ammonium persulfate (Polysciences, Inc.) and sodium bisulfite (J.T. Baker Chemical Co.) were used as the initiator and co- initiator, respectively. The solutions were degassed by exposure to a vacuum followed by purging with nitrogen. Polymerization was carried out at 60° C for 18 hours under nitrogen. The gels were removed from a mold, cut into 5 mm diameter χ 3 mm length discs, and washed exhaustively over a 3 day period in distilled deionized water. The gels were then dried at 37 C for at least one week. 1

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Preparation gf culture supernatants - E. haemolytica biotype A l was grown as previously described (17). Briefly, colonies from a blood agar plate were used to inoculate tryptic soy broth. This culture was incubated at 37 C for 4.5 hours. This broth was used to inoculatefreshtryptic soy broth which was incubated for 1 hour at 37° C with 5% CO2 bubbled through it. The broth was stirred as rapidly as possible without foaming. After 1 hour of incubation bacteria were removed from the culture by ultrafiltration (Pellicon, Millipore, Inc.). Culture supernatantsfreeof bacteria were lyophilized and stored dessicated at -20° C until loaded into hydrogels. 0

Loading hvdrogels with culture supernatants - Culture supernatants (CS) of E. haemolytica contain many antigens, including a proteinaceous exotoxin (leukotoxin) 102 kd in size. This is the primary component of the CS and an essential antigen necessary in a vaccine. Therefore, it was necessary to determine whether CS


25. BOWERSOCK ET AL.



could be loaded into and releasedfromPMA hydrogels. The CS were resuspended to a 22% (w/v) solution in distilled deionized water. Poly(methacrylic acid) hydrogels were placed in the CS and allowed to fully absorb for 48 hours at 37° C. Loaded hydrogels (containing 635 ug of total protein of CS per gel) were then dried to a hard glassy consistency by placing them in a 37°C incubator for 48 hours. Antigen release studies in vitro - To test for release of the CS antigens, 3 loaded hydrogel discs were placed into individual test tubes containing phosphate buffered saline and allowed to hydrate for 48 hours. Eluent was removed daily from each hydrogel for 3 days after the hydrogels were fully hydrated. Fresh saline was placed on the gels after removing the eluent. The eluents were then tested for the presence of leukotoxin, the primary protein antigen present in CS by an enzyme-linked immunosorbent assay (ELISA). The ELISA was performed by placing 50 ul of each daily sample of eluent into a well on a 96 well polystyrene plate (Immulon 2, Dynatech Laboratories) and allowing the material to bind overnight at 4° C. Three wells were tested for each eluent sample. A polyvalent rabbit antibody made in our laboratory to the 102 kd leukotoxin of R haemolvtica was used to detect the presence of leukotoxin in eluents by incubation at 37° C for 3 hours. A secondary anti-rabbit IgG (Bethyl Laboratories, Inc., Bethyl, TX) conjugated to horseradish peroxidase was placed on the plate and incubated at 37° C for 2 hours. The substrate orthophenyldiamine (Sigma Chemical Company) was added to each well and the plate incubated at room temperature for 30 min. The reaction was stopped by adding sulfuric acid to each well and the plate was read at 490 nm using an EIA spectrophotometer (Molecular Devices Inc.). Calf vaccine trial - Twelve 4 month old Holstein-Friesian calves, purchased from a local dairy, were divided into 2 groups. These two groups were further divided into 2 groups for 2 separate trials which consisted of 3 experimental and 3 control calves. Experimental calves (CS vaccinates) were given 300 CS- loaded hydrogels per day for 5 days placed in two 15 ml gelatin boluses. Control calves (nonvaccinates) were given 300 plain hydrogels. The hydrogels within the gelatin boluses were administered to the calves by use of a (pill) balling gun. Three weeks after the first day of vaccination each calf was challenged with an intrabronchial inoculation of 25 ml of 10 -10 CFU/ml of virulent R. haemolvtica. Calves were monitored for clinical signs of disease and euthanatized if in respiratory distress. Calves which survived for seventy-two hours were euthanatized and a post mortem examination performed. 9

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At the post mortem examination, lungs were scored for the percentage of pneumonic lesions, and scored for the severity of gross and histopathological lesions. Lesions were scored on a scale of 0-3 with 0 equal to no lesion noted and 3 equal to a severe lesion. Parameters scored for severity included edema, consolidation, fibrin, cellular infiltration, purulent material, and hemorrhage for both gross and histopathological lesions. The maximum score possible for each calf for gross lesions was 24 and for histopathology was 42. The scoring and determination of areas of


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pneumonia were performed by the pathologist who had no knowledge of which group a given calf belonged. The data for each calf was ranked (by trial) by survival time and analyzed in conjunction with the percentage pneumonic lung, gross and histopathological lesion score using the Wilcoxon rank sum statistical test.


Results and Discussion Antigen release studies - The mean absorbance values for the eluents as tested by ELISA indicated the presence of leukotoxin in the hydrogels for at least 72 hours after elution (Table 1). These results demonstrated that the hydrogels could be loaded with CS including the 102 kd protein antigen and that the leukotoxin was not irreversibly trapped within, but was able to diffuse out of the hydrogels. The fact that the polyclonal antibodies recognized the leukotoxin is important because it indicates that the chemicals and treatments used to load the gels did not adversely affect the epitopes recognized by the polyclonal antibodies. Thus, the leukotoxin retained the epitopes necessary to interact with the immune system. It is likely that other smaller (outer membrane protein) antigens were also present in the CS. We assumed that if the largest antigen of interest could be absorbed into the hydrogels, that smaller antigens would also be absorbed. Calf challenge studies - Results for the survival time post- challenge in hours, per cent pneumonic lung, gross and histopathological lesion scores for each calf are shown in Table 2. Scores for calves in thefirsttrial were not as sharply defined by group as scores in the second trial due to a difference in the dose of challenge bacteria used in each trial. In trial 1 the challenge dose contained 10 CFU/ml of bacteria whereas in trial 2 the challenge contained 10 CFU/ml. The greater challenge dose overwhelmed most of the calves in trial 1 within a few hours. However, it is interesting to note that the severity of the lesions was still greater for the control calves. 10

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In trial 2, when ten-fold fewer bacteria were used in the challenge, all the control calves died and all the CS vaccinated calves survived to the end of the trial. When evaluated in combination with survival time, there was a significantly lower percentage of pneumonic lung, a lower gross lesion score, and a lower histopathological lesion score for CS vaccinated calves compared to sham vaccinates. Passage of hydrogels through the gastrointestinal tract Feces were examined during the time calves were being vaccinated and for 5 days after vaccination for the presence of hydrogels. Onlyfivehydrogels, mildly hydrated to non-hydrated, were found in feces. This supports the results of studies performed in sheep in which no intact hydrogels were found in the intestinal contents or the feces over a period of 7 days following the oral administration of hydrogels (16). The hydrogels administered to the sheep were the same size, density, and material as used in the present experiment. Although the presence of leukotoxin was not determined in the hydrogels recoveredfromthe calves, the low number found reflects the high percentage of retention in the reticulum. The reason hydrogels were found in the feces


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Table 1. Absorbance values of ELISA indicating the presence of leukotoxin in eluentsfromhydrogels

Sample Tested

Absorbance Reading

Culture supernatants used to load hydrogels Tryptic soy broth alone Eluent 1 Eluent 2 Eluent 3

.421 .207 .391 .351 .468

of the calf and not the sheep is most likely attributed to the fact that the calves received a total of 10 times the number of hydrogels the sheep received. It was far more desirable to have the hydrogels retained in the reticulum over time and for the CS to be released as the gels swelled than to have the hydrogels pass through the gut intact with very little chance of beeorning hydrated and releasing the CS. Figure 1 shows an example of one of the hydrogels recovered from the feces and a hydrogel that had not been administered. The recovered hydrogel was swollen, eroded at the edges, and discolored presumably due to the absorbance of bile or pigments from ingesta. At post mortem the entire gastrointestinal tract (rumen to anus) was examined and no intact or recognizable parts of hydrogels were found. This suggests that most hydrogels were probably eroded in the reticulum due to the extremely coarse consistency of the ingesta. The CS were released by diffusion out of the hydrogels as indicated by in vitro studies shown in Table 1, and as indicated previously by chromium release studies performed in sheep (16). It is possible that the hydrogels began to erodefromthe time they entered the reticulum and this further enhanced the diffusion of CS from the hydrogels. This hypothesis cannot be proven until further tests are performed. Most importantly, CS were releasedfromthe orally administered hydrogels which resulted in the protection of calves challenged by P. haemolytica. Conclusions The oral administration of vaccines to prevent respiratory disease has been investigated extensively in laboratory animals primarily for the prevention of influenza in humans (18-19). The oral administration of antigens to prevent respiratory disease in mminants has been relatively unexplored. In this study, poly(methacrylic acid) hydrogels were absorbed with CS of R haemolytica containing a mixture of bacterial antigens including the proteinaceous exotoxin 102 kd in size. Calves vaccinated orally with these hydrogels orally lived longer and had less pulmonary lesions than control calves when challenged with viable P. haemolvtica. The results of this study


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44.2 28.8 31.3

9.5 8.5 10.5

Percentage Gross score Dneumonic lung 0.4 6 2.1 6 25.8 12

1.2 0.9 1.6

72** 72** 72**

Histopathological Survival score time (hours) 3.5 3.5 5.8 3.5 15.8 23*

CS Vaccinates

* Calf euthanatized due to severe pneumonia ** Calf euthanatized to end study Survival plus Percentage Pneumonic Lung P=.040 Survival plus Gross Lesion Score P=.035 Survival plus Histopathology Lesion Score P=.045

•7082 •7084 •7087

£MJ

•7074 •7077 •7078

Ca!f#

Thai/

•7081 •7083 •7085

•7075 •7076 •7080

Caif#

100 100 100

11 10 10

Percentage Gross score Dneumonic lung 53.7 15 61.4 13.5 24.2 17

3.5 6.8 2.5

12 12 20

Histopathological Survival score time (hours) 6.2 3.5 7.7 3.5 4.4 72**

Sham vaccinates

Table 2. Gross and histopathological lesion scores, percentage pneumonic lung, and survival time following challenge in CS vaccinated and sham vaccinated calves



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Carriers of Bacterial Exotoxins in Oral Vaccine 295

Figure 1. Hydrogel (on right) was recovered from the feces of a CS-vaccinated calf within 5 days after oral administration. Compare the swelling and surface erosion of this hydrogel to that of a hydrogel (on the left) that had not been administered to a calf. The hydrogel on the left was amber in color. The hydrogel on therightwas dark green in color presumably due to absorption of bile and pigments from the ingesta in the calf. are consistent with those of previous studies in which intraduodenal administration of CS of P. haemolytica resulted in enhanced pulmonary antibodies in calves as well as decreased pulmonary lesions in calves challenged by R haemolytica (17). The decreased lesions in vaccinated calves in the present study show that antigen released by hydrogels was as effective as the direct intraduodenal inoculation of antigen in stimulating protective immunity in the lungs of calves. Oral administration of antigens induces antigen specific lymphocytes that migrate to other mucosal sites and produce antibodies. Recent studies suggest that cellular immunity may also be enhanced by the oral administration of antigens (20). Further studies are needed to determine the immune mechanisms responsible for protection in these calves. This study showed that hydrogels can deliver antigens orally to ruminants resulting in enhanced immunity at distant mucosal sites. This was demonstrated by reduced pulmonary lesions in calves challenged by a viruluent respiratory bacterium. Studies are underway to determine what other antigens can be loaded into hydrogels and retain their immunogenicity when released into the lower GIT of cattle. Hydrogels provide a practical, safe, economical way to deliver oral vaccines to a large number of animals to prevent diseases which begin at a mucosal surface.


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Acknowledgments This work was supported by Animal Health and Disease Research Funds at the School of Veterinary Medicine, Purdue University, West Lafayette, IN.


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Poly(methacrylic acid) Hydrogels as Carriers of Bacterial Exotoxins in

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