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Intranasal vaccination of young Holstein calves with Mannheimia haemolytica chimeric protein PlpE–LKT (SAC89) and cholera toxin
Veterinary Immunology and Immunopathology 132 (2009) 232–236
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Intranasal vaccination of young Holstein calves with Mannheimia haemolytica chimeric protein PlpE–LKT (SAC89) and cholera toxin A.W. Confer a,*, S. Ayalew a, D.L. Step b, B. Trojan a, M. Montelongo a a
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 McElroy Hall, Stillwater, OK 74078-2007, United States b Department of Veterinary Clinical Sciences, Center for Veterinary Health Sciences, Boren Veterinary Medical Teaching Hospital, Oklahoma State University, Stillwater, OK 74078-2041, United States
A R T I C L E I N F O
Article history: Received 18 December 2008 Received in revised form 2 March 2009 Accepted 22 April 2009 Keywords: Mannheimia haemolytica Mucosal immunity Chimeric protein Cholera toxin Cattle
1. Introduction A major cause of severe bacterial pneumonia in feedlot and neonatal dairy cattle is Mannheimia haemolytica serotype 1 (S1) (Rice et al., 2007). Current vaccines against M. haemolytica are moderately efficacious against shipping fever pneumonia of beef cattle but generally ineffective against neonatal dairy calf pneumonia (Virtala et al., 1996; Rice et al., 2007). Shewen and Wilkie (1988) demonstrated that immunity against M. haemolytica requires leukotoxin (LKT)neutralizing antibodies as well as antibodies against bacterial cell surface antigens. One of the immunologically important surface antigens is a 45 kDa, surface-exposed, outer membrane lipoprotein, PlpE (Pandher et al., 1999). Cattle vaccinated with commercial M. haemolytica vaccines supplemented with recombinant M. haemolytica S1 PlpE (rPlpE) had significantly greater resistance against experimental challenge with M. haemolytica S1 or S6 than did
* Corresponding author. Tel.: +1 405 744 4542; fax: +1 405 744 5275. E-mail address: [email protected] (A.W. Confer). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.04.018
cattle vaccinated with the commercial vaccine alone (Confer et al., 2003, 2006). The immunodominant epitope region of M. haemolytica S1 PlpE (hereafter referred to as R2) consists of eight imperfect hexapeptide repeats of QAQNAP located near the N-terminal region (Ayalew et al., 2004). The epitope involved in neutralization of LKT is localized to a 32 amino acid region near the C-terminus (Lainson et al., 1996; Rajeev et al., 2001). From these data, we developed several PlpE–LKT chimeric constructs containing the R2 epitope of PlpE and the neutralizing epitope of LKT (NLKT). Vaccination of mice with these chimeric proteins stimulated antiPlpE complement fixing and LKT-neutralizing antibodies (Ayalew et al., 2008). One chimeric protein, designated as SAC89 that contains two copies each of R2 and NLKT, stimulated the best overall responses in mice. When cattle were vaccinated with SAC89 plus a formalin-killed M. haemolytica bacterin, those cattle had significantly enhanced resistance against M. haemolytica challenge compared to bacterin-vaccinated cattle (Confer et al., 2009). Passively acquired anti-M. haemolytica antibodies do not prevent nasal colonization with M. haemolytica or
A.W. Confer et al. / Veterinary Immunology and Immunopathology 132 (2009) 232–236
protect against pneumonia (Prado et al., 2006; Step et al., 2005; Virtala et al., 1996). Parenteral vaccination of young dairy calves with M. haemolytica vaccines does not augment antibody responses or resistance to infection; therefore, improved vaccines are needed to induce solid protective immunity of young dairy calves against natural infection (Confer et al., 2003; Hodgins and Shewen, 1998, 2000; Rice et al., 2007; Shewen et al., 2009). We, therefore, undertook these studies to determine if intranasal vaccination of young dairy calves with the chimeric protein SAC89 that contains two copies each of the immunodominant epitopes of PlpE and LKT (R2-NLKT– R2-NLKT) could stimulate secretory and systemic antibodies against M. haemolytica. Cholera toxin (CT) is a welldocumented adjuvant for stimulating mucosal immunity and was used in this study (Lycke, 2005). Shewen et al. (2009) recently reviewed the challenges of developing mucosal vaccines for cattle. 2. Materials and methods 2.1. Cattle
233
substrate. Both sera and secondary antibodies were diluted in PBS–Tween 20 + 1% bovine serum albumin. Antibody responses were expressed as ng of immunoglobulin binding based on a set of IgG standards on each plate. For determination of anti-M. haemolytica IgA antibodies in nasal secretions, the previous steps were repeated. Because of small nasal secretion samples, only IgA antibodies against rPlpE and LKT were determined. Nasal secretions were diluted 1:2, and antibodies were determined using HRP-conjugated goat anti-bovine IgA (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) secondary antibody at a dilution of 1:400. Antibody responses were expressed as ng of immunoglobulin binding based on a set of IgA standards on each plate. 2.3. Immunogens Recombinant chimeric protein SAC89 has a calculated molecular mass of 46864.5 Da. The design, construction, expression, and purification of SAC89 were recently described (Ayalew et al., 2008). 2.4. Experimental design
Eleven purebred 3–18-day-old female Holstein calves were used. Calves were housed individually in hutches in the Oklahoma State University Dairy Cattle Center. All calves were administered colostrum via an esophageal feeding tube within 12 hours after birth and subsequently fed a commercial milk-replacer formula. This study was approved by the Oklahoma State University Institutional Animal Care and Use Committee (amended protocol #182). 2.2. Enzyme-linked immunosorbent assay (ELISA) Sera were assayed for anti-M haemolytica IgG antibodies using ELISA against partially purified LKT, formalinkilled whole bacterial cells (WC), and recombinant PlpE (rPlpE) as previously described (Confer et al., 1997, 2003, 2006). Ninety-six-well microtiter plates were coated with WC at a concentration equivalent to 108 CFU/well, 50 ng/ well each of LKT or rPlpE diluted in coating buffer. Serum dilutions for the various assays were 1:800 for WC, 1:1600 for LKT, or 1:400 for PlpE, which were in the linear range of dilution curves established in our laboratories (data not shown). Affinity-purified, horseradish peroxidase-conjugated goat anti-bovine IgG [H+L] (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) diluted at 1:400 was used as secondary antibody, and o-phenylenediamine (OPD) tablets (Amresco, Inc., Solon, OH) were used as
The 11 dairy calves were divided among three groups (Table 1), and each calf was vaccinated intranasally with a 2 ml dose vaccine divided equally between the two nasal cavities. Each vaccine contained 20 mg of CT from Vibrio cholera (Sigma, St. Louis, MO). Vaccines were sprayed from a syringe inserted deeply (approximately 5 cm) into the nasal passage on days 0 and 14. Group 1 was vaccinated with CT diluted with PBS. Groups 2 and 3 vaccines were composed of CT plus SAC89 at a concentration of 10 and 100 mg/ml, respectively. Serum samples and nasal secretions were obtained on days 0, 14, and 28 to determine antibody response. To obtain nasal secretions, an absorbent cotton material (Tampax Slender, P>M, Cincinnati, OH) was placed within the right nostril of dairy calves for five minutes. After removal, the absorbent material was flushed with 1 ml of PBS solution and squeezed in a 60 ml syringe. Nasal secretions were collected into microcentrifuge tubes. 2.5. Statistical analysis Antibody responses among the various groups were compared by one-way analysis of variance with Tukey’s HSD post hoc test (Petrie and Watson, 1999). Antibody responses within groups were compared by paired t tests. Differences were considered significant when p < 0.05.
Table 1 Experimental design. Intranasal vaccinationa of 3–18-day-old dairy calves with 10 or 100 mg of M. haemolytica PlpE–LKT chimeric protein (SAC89b) plus 20 mg of cholera toxin (CT). Groups
Antigen (dose per vaccination)
Cholera toxin (CT)
No. of calves
Intranasal vaccination
Sampling: sera
Sampling: nasal secretions
1 2 3
None (PBS) SAC89 (10 mg/ml) SAC89 (100 mg/ml)
CT (20mg per dose) CT (20mg per dose) CT (20mg per dose)
3 4 4
Days 0 and 14 Days 0 and 14 Days 0 and 14
Days 0, 14, and 28 Days 0, 14, and 28 Days 0, 14, and 28
Days 0, 14, and 28 Days 0, 14, and 28 Days 0, 14, & 28
a b
Each vaccine consisted of a 2 ml volume of SAC89 or PBS plus CT and was given as a divided dose with 1 ml deposited in each nasal cavity. SAC89 contains two copies each of the immunodominant surface epitope of outer membrane lipoprotein PlpE (R2) and the neutralizing epitope of LKT.
234
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Fig. 1. Serum anti-M. haemolytica IgG antibody responses as measured in ELISAs and reported as ng of IgG binding to the antigens. Dairy calves were intranasally vaccinated twice with PBS, 10 or 100 mg of M. haemolytica chimeric protein SAC89 composed of two copies each of the immunodominant surface epitope of PlpE and the neutralizing epitope of leukotoxin on days 0 and 14. Each vaccine contained 20 mg of cholera toxin. Error bars = standard deviations.
3. Results and discussion On day 0, all calves had low but detectable concentrations of serum antibodies (<0.3 ng of IgG binding) against the three M. haemolytica antigen preparations (Fig. 1). Serum antibody responses following the first vaccination were not detected against any of the antigen preparations. After the second vaccination, calves vaccinated with 10 mg of SAC89 developed a significant antibody response (p < 0.05) to WC but not to rPlpE or to LKT. Volumes of intranasal secretions collected were only sufficient to determine anti-IgA responses to PlpE and to LKT. Vaccination with both doses of SAC89 resulted in significant increases (p < 0.05) in anti-PlpE antibody responses by day 14 (Fig. 2). In the 10-mg dose group, anti-LKT antibody responses were significantly elevated (p < 0.05) on day 14 compared to the PBS- and 100 mgvaccinated groups and on day 28 compared to the PBSvaccinated group. In the 100-mg dose group, anti-PlpE and anti-LKT antibodies were significantly increased (p < 0.05) on day 28 compared to antibody responses of the PBSvaccinated group.
These results demonstrated that intranasal vaccination of dairy calves with M. haemolytica chimeric PlpE–LKT protein with CT stimulated a nasal IgA antibody response to M. haemolytica outer membrane lipoprotein PlpE and to LKT. Serum IgG antibody responses following intranasal vaccination, however, were not as obvious with only the 10 mg-vaccinated group developing a detectable antibody response and only to WC. The reason for the differences between mucosal and systemic antibody responses is not entirely known. Similarly, in a recent study wherein 7week-old dairy calves were intranasally vaccinated with a live aroA-deficient Pasteurella multocida B:2 strain, serum antibodies against that bacterium were undetectable (Hodgson et al., 2005). In the present study, dairy calves had low concentrations of anti-M. haemolytica maternal antibodies that could have reduced the responsiveness to vaccination. Hodgins and Shewen (1998) demonstrated that maternal antibodies partially blocked the serum immune response of young colostrum-fed calves after M. haemolytica vaccination. In addition, Shewen et al. (2009) recently demonstrated development of nasal IgA anti-LKT antibodies in young calves intranasally vaccinated at 4 and 6 weeks of age with immunostimulating complexes
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235
Fig. 2. Nasal anti-M. haemolytica IgA antibody responses as measured in ELISAs and reported as ng of IgA binding to the antigens. Dairy calves were intranasally vaccinated twice with PBS, 10 or 100 mg of M. haemolytica chimeric protein SAC89 composed of two copies each of the immunodominant surface epitope of PlpE and the neutralizing epitope of leukotoxin on days 0 and 14. Each vaccine contained 20 mg of cholera toxin. Error bars = standard deviations.
containing LKT. In that study, however, serum antibody responses were low and delayed compared to the serum antibody responses in subcutaneously vaccinated calves. Chase et al. (2008) reviewed in utero and neonatal immunity in cattle. They pointed out that in neonatal calves many of the components of active immunity are not functional until calves are at least 2–4 weeks of age, and immunologic development continues until puberty. In conclusion, intranasal vaccination of calves with the M. haemolytica PlpE–LKT chimeric protein (SAC89) stimulated nasal antibodies to PlpE and LKT antigens; however, serum antibody responses were limited. Although these data are promising as a potential vaccination approach for young cattle, additional studies are needed to determine dose responses, safety, and efficacy as well as to better understand the immune response of neonatal dairy cattle following intranasal M. haemolytica vaccination. Furthermore, it is unlikely that a CT-adjuvanated vaccine would be licensed for use in cattle; therefore, additional mucosal adjuvants need to be tested. Because intranasal replication of respiratory pathogens is the first phase in development of pneumonia, mucosal vaccination of cattle is a compelling alternative to traditional parenteral vaccination for prevention of respiratory disease especially in neonatal dairy cattle (Frank et al., 2003; Lee et al., 2001, 2008; Shewen et al., 2009). Intranasal immunization of cattle has been done for many years using bovine herpesvirus-1, and other viral vaccines have been pursued for intranasal use (Ellis et al., 2007; Muylkens et al., 2007). Experimentally, intranasal vaccination of weaned beef cattle with a live, LKT-deficient M. haemolytica mutant resulted in reduced nasopharyngeal colonization of feedlot cattle with M. haemolytica compared to nasopharyngeal colonization of non-vaccinated control cattle (Frank et al., 2003). Finally, an alternative mucosal vaccination strategy using plantderived recombinant M. haemolytica antigens in an edible
vaccine is under intense investigation (Lee et al., 2001, 2008; Rice et al., 2005; Shewen et al., 2009).
Acknowledgements This work was funded in part by grants #2002-02232 and #2006-01684 from the USDA CSREES National Research Initiative Competitive Grants Program, grants #AR031-14 and #ARO61-202 from the Oklahoma Center for Advancement of Science and Technology, support from Native Americans in Biological Sciences Program Grant Number 5 R25 GM069516-03, National Institutes of Health, National Institute of General Medical Sciences, and a grant from the Noble Foundation of Ardmore, Oklahoma. The authors thank OSU Dairy Cattle Center staff, Josh Wray and Jason Thorne for technical assistance. References Ayalew, S., Confer, A.W., Garrels, K.D., Ingram, K., Montelongo, M., 2008. Mannheimia haemolytica chimeric protein vaccine composed of the major surface-exposed epitope of outer membrane lipoprotein PlpE and the neutralizing epitope of leukotoxin. Vaccine 26, 4955–4961. Ayalew, S., Confer, A.W., Blackwood, E.R., 2004. Characterization of immunodominant and potentially protective epitopes of Mannheimia haemolytica serotype 1 outer membrane lipoprotein PlpE. Infect. Immun. 72, 7265–7274. Chase, C.C., Hurley, D.J., Reber, A.J., 2008. Neonatal immune development in the calf and its impact on vaccine response. Vet. Clin. N. Am. Food Anim. Pract. 24, 87–104. Confer, A.W., Ayalew, S., Montelongo, M., Step, D.L., Wray, J.H., Hansen, R.D., Panciera, R.J., 2009. Immunity of cattle following vaccination with a Mannheimia haemolytica chimeric PlpE–LKT (SAC89) protein. Vaccine 27, 1771–1776. Confer, A.W., Ayalew, S., Panciera, R.J., Montelongo, M., Whitworth, L.C., Hammer, J.D., 2003. Immunogenicity of recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE and augmentation of a commercial vaccine. Vaccine 21, 2821–2829. Confer, A.W., Ayalew, S., Panciera, R.J., Montelongo, M., Wray, J.H., 2006. Recombinant Mannheimia haemolytica serotype 1 outer membrane
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protein PlpE enhances commercial M. haemolytica vaccine-induced resistance against serotype 6 challenge. Vaccine 24, 2248–2255. Confer, A.W., Clinkenbeard, K.D., Gatewood, D.M., Driskel, B.A., Montelongo, M., 1997. Serum antibody responses of cattle vaccinated with partially purified native Pasteurella haemolytica leukotoxin. Vaccine 15, 1423–1429. Ellis, J., Gow, S., West, K., Waldner, C., Rhode, C., Mutwiri, G., Rosenberg, H., 2007. Response of calves to challenge exposure with virulent bovine respiratory syncytial virus following intranasal administration of vaccines formulated for parenteral administration. J. Am. Vet. Med. Assoc. 230, 233–243. Frank, G.H., Briggs, R.E., Duff, G.C., Hurd, H.S., 2003. Effect of intranasal exposure to leukotoxin-deficient Mannheimia haemolytica at the time of arrival at the feedyard on subsequent isolation of M. haemolytica from nasal secretions of calves. Am. J. Vet. Res. 64, 580–585. Hodgins, D.C., Shewen, P.E., 1998. Serologic responses of young colostrum fed dairy calves to antigens of Pasteurella haemolytica A1. Vaccine 20, 2018–2025. Hodgins, D.C., Shewen, P.E., 2000. Vaccination of neonatal colostrumdeprived calves against Pasteurella haemolytica A1. Can. J. Vet. Res. 64, 3–8. Hodgson, J.C., Finucane, A., Dagleish, M.P., Ataei, S., Parton, R., Coote, J.G., 2005. Efficacy of vaccination of calves against hemorrhagic septicemia with a live aroA derivative of Pasteurella multocida B:2 by two different routes of administration. Infect. Immun. 73, 1475–1481. Lainson, F.A., Murray, J., Davies, R.C., Donachie, W., 1996. Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin. Microbiology 142, 2499–2507. Lee, R.W., Cornelisse, M., Ziauddin, A., Slack, P.J., Hodgins, D.C., Strommer, J.N., Shewen, P.E., Lo, R.Y., 2008. Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis. J. Biotechnol. 135, 224–231. Lee, R.W., Strommer, J., Hodgins, D., Shewen, P.E., Niu, Y., Lo, R.Y., 2001. Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1 leukotoxin 50 fusion protein. Infect. Immun. 69, 5786–5793.
Lycke, N., 2005. Targeted vaccine adjuvants based on modified cholera toxin. Curr. Mol. Med. 5, 591–597. Muylkens, B., Thiry, J., Kirten, P., Schynts, F., Thiry, E., 2007. Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. Vet. Res. 38, 181–209. Pandher, K., Murphy, G.L., Confer, A.W., 1999. Identification of immunogenic, surface-exposed outer membrane proteins of Pasteurella haemolytica serotype 1. Vet. Microbiol. 65, 215–226. Petrie, A., Watson, P., 1999. Statistics for Veterinary and Animal Science. Blackwell Press, London, pp. 81–8, 92–6. Prado, M.E., Confer, A.W., Prado, T.M., 2006. Maternally and naturally acquired antibodies to Mannheimia haemolytica and Pasteurella multocida in beef calves. Vet. Immunol. Immunopathol. 111, 301– 307. Rajeev, S., Kania, S.A., Nair, R.V., McPherson, J.T., Moore, R.N., Bemis, D.A., 2001. Bordetella bronchiseptica fimbrial protein-enhanced immunogenicity of a Mannheimia haemolytica leukotoxin fragment. Vaccine 19, 4842–4850. Rice, J., Ainley, W.M., Shewen, P., 2005. Plant-made vaccines: biotechnology and immunology in animal health. Anim. Health Res. Rev. 6, 199– 209. Rice, J.A., Carrasco-Medina, L., Hodgins, D.C., Shewen, P.E., 2007. Mannheimia haemolytica and bovine respiratory disease. Anim. Health Res. Rev. 8, 117–128. Shewen, P.E., Carrasco-Medina, L., McBey, B.A., Hodgins, D.C., 2009. Challenges in mucosal vaccination of cattle. Vet. Immunol. Immunopathol. 128, 192–198. Shewen, P.E., Wilkie, B.N., 1988. Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica. Can. J. Vet. Res. 52, 30–36. Step, D.L., Confer, A.W., Kirkpatrick, J.G., Richards, J.B., Fulton, R.W., 2005. Respiratory tract infections in dairy calves from birth to breeding age: detection by laboratory isolations and seroconversions. Bov. Pract. 39, 44–53. Virtala, A.M., Mechor, G.D., Grohn, Y.T., Erb, H.N., Dubovi, E.J., 1996. Epidemiologic and pathologic characteristics of respiratory tract disease in dairy heifers during the first three months of life. J. Am. Vet. Med. Assoc. 208, 2035–2042.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Short communication
Intranasal vaccination of young Holstein calves with Mannheimia haemolytica chimeric protein PlpE–LKT (SAC89) and cholera toxin A.W. Confer a,*, S. Ayalew a, D.L. Step b, B. Trojan a, M. Montelongo a a
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 McElroy Hall, Stillwater, OK 74078-2007, United States b Department of Veterinary Clinical Sciences, Center for Veterinary Health Sciences, Boren Veterinary Medical Teaching Hospital, Oklahoma State University, Stillwater, OK 74078-2041, United States
A R T I C L E I N F O
Article history: Received 18 December 2008 Received in revised form 2 March 2009 Accepted 22 April 2009 Keywords: Mannheimia haemolytica Mucosal immunity Chimeric protein Cholera toxin Cattle
1. Introduction A major cause of severe bacterial pneumonia in feedlot and neonatal dairy cattle is Mannheimia haemolytica serotype 1 (S1) (Rice et al., 2007). Current vaccines against M. haemolytica are moderately efficacious against shipping fever pneumonia of beef cattle but generally ineffective against neonatal dairy calf pneumonia (Virtala et al., 1996; Rice et al., 2007). Shewen and Wilkie (1988) demonstrated that immunity against M. haemolytica requires leukotoxin (LKT)neutralizing antibodies as well as antibodies against bacterial cell surface antigens. One of the immunologically important surface antigens is a 45 kDa, surface-exposed, outer membrane lipoprotein, PlpE (Pandher et al., 1999). Cattle vaccinated with commercial M. haemolytica vaccines supplemented with recombinant M. haemolytica S1 PlpE (rPlpE) had significantly greater resistance against experimental challenge with M. haemolytica S1 or S6 than did
* Corresponding author. Tel.: +1 405 744 4542; fax: +1 405 744 5275. E-mail address: [email protected] (A.W. Confer). 0165-2427/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2009.04.018
cattle vaccinated with the commercial vaccine alone (Confer et al., 2003, 2006). The immunodominant epitope region of M. haemolytica S1 PlpE (hereafter referred to as R2) consists of eight imperfect hexapeptide repeats of QAQNAP located near the N-terminal region (Ayalew et al., 2004). The epitope involved in neutralization of LKT is localized to a 32 amino acid region near the C-terminus (Lainson et al., 1996; Rajeev et al., 2001). From these data, we developed several PlpE–LKT chimeric constructs containing the R2 epitope of PlpE and the neutralizing epitope of LKT (NLKT). Vaccination of mice with these chimeric proteins stimulated antiPlpE complement fixing and LKT-neutralizing antibodies (Ayalew et al., 2008). One chimeric protein, designated as SAC89 that contains two copies each of R2 and NLKT, stimulated the best overall responses in mice. When cattle were vaccinated with SAC89 plus a formalin-killed M. haemolytica bacterin, those cattle had significantly enhanced resistance against M. haemolytica challenge compared to bacterin-vaccinated cattle (Confer et al., 2009). Passively acquired anti-M. haemolytica antibodies do not prevent nasal colonization with M. haemolytica or
A.W. Confer et al. / Veterinary Immunology and Immunopathology 132 (2009) 232–236
protect against pneumonia (Prado et al., 2006; Step et al., 2005; Virtala et al., 1996). Parenteral vaccination of young dairy calves with M. haemolytica vaccines does not augment antibody responses or resistance to infection; therefore, improved vaccines are needed to induce solid protective immunity of young dairy calves against natural infection (Confer et al., 2003; Hodgins and Shewen, 1998, 2000; Rice et al., 2007; Shewen et al., 2009). We, therefore, undertook these studies to determine if intranasal vaccination of young dairy calves with the chimeric protein SAC89 that contains two copies each of the immunodominant epitopes of PlpE and LKT (R2-NLKT– R2-NLKT) could stimulate secretory and systemic antibodies against M. haemolytica. Cholera toxin (CT) is a welldocumented adjuvant for stimulating mucosal immunity and was used in this study (Lycke, 2005). Shewen et al. (2009) recently reviewed the challenges of developing mucosal vaccines for cattle. 2. Materials and methods 2.1. Cattle
233
substrate. Both sera and secondary antibodies were diluted in PBS–Tween 20 + 1% bovine serum albumin. Antibody responses were expressed as ng of immunoglobulin binding based on a set of IgG standards on each plate. For determination of anti-M. haemolytica IgA antibodies in nasal secretions, the previous steps were repeated. Because of small nasal secretion samples, only IgA antibodies against rPlpE and LKT were determined. Nasal secretions were diluted 1:2, and antibodies were determined using HRP-conjugated goat anti-bovine IgA (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) secondary antibody at a dilution of 1:400. Antibody responses were expressed as ng of immunoglobulin binding based on a set of IgA standards on each plate. 2.3. Immunogens Recombinant chimeric protein SAC89 has a calculated molecular mass of 46864.5 Da. The design, construction, expression, and purification of SAC89 were recently described (Ayalew et al., 2008). 2.4. Experimental design
Eleven purebred 3–18-day-old female Holstein calves were used. Calves were housed individually in hutches in the Oklahoma State University Dairy Cattle Center. All calves were administered colostrum via an esophageal feeding tube within 12 hours after birth and subsequently fed a commercial milk-replacer formula. This study was approved by the Oklahoma State University Institutional Animal Care and Use Committee (amended protocol #182). 2.2. Enzyme-linked immunosorbent assay (ELISA) Sera were assayed for anti-M haemolytica IgG antibodies using ELISA against partially purified LKT, formalinkilled whole bacterial cells (WC), and recombinant PlpE (rPlpE) as previously described (Confer et al., 1997, 2003, 2006). Ninety-six-well microtiter plates were coated with WC at a concentration equivalent to 108 CFU/well, 50 ng/ well each of LKT or rPlpE diluted in coating buffer. Serum dilutions for the various assays were 1:800 for WC, 1:1600 for LKT, or 1:400 for PlpE, which were in the linear range of dilution curves established in our laboratories (data not shown). Affinity-purified, horseradish peroxidase-conjugated goat anti-bovine IgG [H+L] (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) diluted at 1:400 was used as secondary antibody, and o-phenylenediamine (OPD) tablets (Amresco, Inc., Solon, OH) were used as
The 11 dairy calves were divided among three groups (Table 1), and each calf was vaccinated intranasally with a 2 ml dose vaccine divided equally between the two nasal cavities. Each vaccine contained 20 mg of CT from Vibrio cholera (Sigma, St. Louis, MO). Vaccines were sprayed from a syringe inserted deeply (approximately 5 cm) into the nasal passage on days 0 and 14. Group 1 was vaccinated with CT diluted with PBS. Groups 2 and 3 vaccines were composed of CT plus SAC89 at a concentration of 10 and 100 mg/ml, respectively. Serum samples and nasal secretions were obtained on days 0, 14, and 28 to determine antibody response. To obtain nasal secretions, an absorbent cotton material (Tampax Slender, P>M, Cincinnati, OH) was placed within the right nostril of dairy calves for five minutes. After removal, the absorbent material was flushed with 1 ml of PBS solution and squeezed in a 60 ml syringe. Nasal secretions were collected into microcentrifuge tubes. 2.5. Statistical analysis Antibody responses among the various groups were compared by one-way analysis of variance with Tukey’s HSD post hoc test (Petrie and Watson, 1999). Antibody responses within groups were compared by paired t tests. Differences were considered significant when p < 0.05.
Table 1 Experimental design. Intranasal vaccinationa of 3–18-day-old dairy calves with 10 or 100 mg of M. haemolytica PlpE–LKT chimeric protein (SAC89b) plus 20 mg of cholera toxin (CT). Groups
Antigen (dose per vaccination)
Cholera toxin (CT)
No. of calves
Intranasal vaccination
Sampling: sera
Sampling: nasal secretions
1 2 3
None (PBS) SAC89 (10 mg/ml) SAC89 (100 mg/ml)
CT (20mg per dose) CT (20mg per dose) CT (20mg per dose)
3 4 4
Days 0 and 14 Days 0 and 14 Days 0 and 14
Days 0, 14, and 28 Days 0, 14, and 28 Days 0, 14, and 28
Days 0, 14, and 28 Days 0, 14, and 28 Days 0, 14, & 28
a b
Each vaccine consisted of a 2 ml volume of SAC89 or PBS plus CT and was given as a divided dose with 1 ml deposited in each nasal cavity. SAC89 contains two copies each of the immunodominant surface epitope of outer membrane lipoprotein PlpE (R2) and the neutralizing epitope of LKT.
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Fig. 1. Serum anti-M. haemolytica IgG antibody responses as measured in ELISAs and reported as ng of IgG binding to the antigens. Dairy calves were intranasally vaccinated twice with PBS, 10 or 100 mg of M. haemolytica chimeric protein SAC89 composed of two copies each of the immunodominant surface epitope of PlpE and the neutralizing epitope of leukotoxin on days 0 and 14. Each vaccine contained 20 mg of cholera toxin. Error bars = standard deviations.
3. Results and discussion On day 0, all calves had low but detectable concentrations of serum antibodies (<0.3 ng of IgG binding) against the three M. haemolytica antigen preparations (Fig. 1). Serum antibody responses following the first vaccination were not detected against any of the antigen preparations. After the second vaccination, calves vaccinated with 10 mg of SAC89 developed a significant antibody response (p < 0.05) to WC but not to rPlpE or to LKT. Volumes of intranasal secretions collected were only sufficient to determine anti-IgA responses to PlpE and to LKT. Vaccination with both doses of SAC89 resulted in significant increases (p < 0.05) in anti-PlpE antibody responses by day 14 (Fig. 2). In the 10-mg dose group, anti-LKT antibody responses were significantly elevated (p < 0.05) on day 14 compared to the PBS- and 100 mgvaccinated groups and on day 28 compared to the PBSvaccinated group. In the 100-mg dose group, anti-PlpE and anti-LKT antibodies were significantly increased (p < 0.05) on day 28 compared to antibody responses of the PBSvaccinated group.
These results demonstrated that intranasal vaccination of dairy calves with M. haemolytica chimeric PlpE–LKT protein with CT stimulated a nasal IgA antibody response to M. haemolytica outer membrane lipoprotein PlpE and to LKT. Serum IgG antibody responses following intranasal vaccination, however, were not as obvious with only the 10 mg-vaccinated group developing a detectable antibody response and only to WC. The reason for the differences between mucosal and systemic antibody responses is not entirely known. Similarly, in a recent study wherein 7week-old dairy calves were intranasally vaccinated with a live aroA-deficient Pasteurella multocida B:2 strain, serum antibodies against that bacterium were undetectable (Hodgson et al., 2005). In the present study, dairy calves had low concentrations of anti-M. haemolytica maternal antibodies that could have reduced the responsiveness to vaccination. Hodgins and Shewen (1998) demonstrated that maternal antibodies partially blocked the serum immune response of young colostrum-fed calves after M. haemolytica vaccination. In addition, Shewen et al. (2009) recently demonstrated development of nasal IgA anti-LKT antibodies in young calves intranasally vaccinated at 4 and 6 weeks of age with immunostimulating complexes
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Fig. 2. Nasal anti-M. haemolytica IgA antibody responses as measured in ELISAs and reported as ng of IgA binding to the antigens. Dairy calves were intranasally vaccinated twice with PBS, 10 or 100 mg of M. haemolytica chimeric protein SAC89 composed of two copies each of the immunodominant surface epitope of PlpE and the neutralizing epitope of leukotoxin on days 0 and 14. Each vaccine contained 20 mg of cholera toxin. Error bars = standard deviations.
containing LKT. In that study, however, serum antibody responses were low and delayed compared to the serum antibody responses in subcutaneously vaccinated calves. Chase et al. (2008) reviewed in utero and neonatal immunity in cattle. They pointed out that in neonatal calves many of the components of active immunity are not functional until calves are at least 2–4 weeks of age, and immunologic development continues until puberty. In conclusion, intranasal vaccination of calves with the M. haemolytica PlpE–LKT chimeric protein (SAC89) stimulated nasal antibodies to PlpE and LKT antigens; however, serum antibody responses were limited. Although these data are promising as a potential vaccination approach for young cattle, additional studies are needed to determine dose responses, safety, and efficacy as well as to better understand the immune response of neonatal dairy cattle following intranasal M. haemolytica vaccination. Furthermore, it is unlikely that a CT-adjuvanated vaccine would be licensed for use in cattle; therefore, additional mucosal adjuvants need to be tested. Because intranasal replication of respiratory pathogens is the first phase in development of pneumonia, mucosal vaccination of cattle is a compelling alternative to traditional parenteral vaccination for prevention of respiratory disease especially in neonatal dairy cattle (Frank et al., 2003; Lee et al., 2001, 2008; Shewen et al., 2009). Intranasal immunization of cattle has been done for many years using bovine herpesvirus-1, and other viral vaccines have been pursued for intranasal use (Ellis et al., 2007; Muylkens et al., 2007). Experimentally, intranasal vaccination of weaned beef cattle with a live, LKT-deficient M. haemolytica mutant resulted in reduced nasopharyngeal colonization of feedlot cattle with M. haemolytica compared to nasopharyngeal colonization of non-vaccinated control cattle (Frank et al., 2003). Finally, an alternative mucosal vaccination strategy using plantderived recombinant M. haemolytica antigens in an edible
vaccine is under intense investigation (Lee et al., 2001, 2008; Rice et al., 2005; Shewen et al., 2009).
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