CpG motif-based adjuvant enhances immunogenicity of a recombinant LHRH vaccine and noninvasive monitoring of adrenal and gonadal function in the jaguar (Panthera onca)

Valeria Amorim Conforti

doi:10.7273/000005783

A recombinant ovalbumin-luteinizing hormone-releasing hormone (ova-LHRH) antigen has been developed for immunocontraception. In the first study, a novel immunostimulant for ova-LHRH immunization, CpG Oligodeoxynucleotide (ODN) 2006, was compared against Mycobacterium butyricum in female rats. Also, the immunogenicity of ova-LHRH after lyophilization and exposure to organic solvents was assessed. Rats received either ova-LHRH solubilized in urea; lyophilized ova-LHRH; lyophilized ova-LHRH exposed to methylene chloride; or lyophilized ova-LHRH exposed to ethyl acetate. After lyophilization, there was a decrease in immunogenicity of ova-LHRH. Exposure to ethyl acetate further decreased immunogenicity of ova-LHRH. CpG ODN 2006 was a more effective immunostimulant than M. butyricum for LHRH immunization. In the second study, ova-LHRH fusion protein was used for immunosterilization of heifers. Two adjuvants were compared for the dose of 3.4 mg ovaLHRH: Freund's complete adjuvant (FCA) and a novel CpG motif-based oligodeoxynucleotide (CpG ODN 2006). Additionally, increasing doses of ova-LHRH in CpG ODN in a water-in-oil (w-o) emulsion were tested. Seven treatment groups (n = 8 heifers per group) were used in this study: 1) untreated control; 2) 1.5 mg ova-LHRH with CpG ODN in w-o; 3) 2.3 mg ova-LHRH with CpG ODN in w-o; 4) 3.4 mg ova-LHRH with CpG ODN in w-o; 5) 5.1 mg ova-LHRH with CpG ODN in w-o; 6) 7.6 mg ova-LHRH with CpG ODN in w-o; and 7) 3.4 mg ova-LHRH in FCA. Animals received two immunizations at weeks 0 and 14. Treatment with 3.4 mg CpG ODN 2006 resulted in higher production of LHRH antibodies compared to the same dose of ovaLHRH in FCA. Among all treatment groups that received CpG ODN/w-o as an adjuvant, the treatment group that resulted in optimal production of LHRH antibodies was that receiving a dose of 3.4 mg ova-LHRH. Compared to the untreated control group, all treatment combinations resulted in decreased reproductive tract weight at slaughter and decreased proportions of cyclic heifers as measured by serum progesterone concentrations. All heifers in the control group were cycling at the end of the study (week 27). Treatment groups 2, 4, 7 had 8 (out of 8) acyclic heifers at week 27. Treatment groups 3, 5, and 6 had one cyclic heifer at week 27. In summary, treatment with 3.4 mg ova-LHRH in CpG ODN 2006/w-o per immunization is recommended for LHRH immunization in heifers. A third study, independent study was conduct with the following objectives: 1) to validate a protocol for noninvasive assessment of changes in corticoid concentrations due to acute stress in jaguars by measuring fecal corticoid metabolite concentrations in males and females before and after ACTH injection; and 2) to investigate the relationship between fecal corticoid and androgen metabolite concentrations in male jaguars. Eight adult jaguars were used in this study: 3 intact males, 1 vasectomized male, 3 intact females, and 1 ovariectomized female. All animals, with the exception of the vasectomized male, which was used as an untreated control, were chemically restrained and treated with 500 IU/animal of ACTH gel to simulate the effects of acute stress (day 0). Fecal samples were collected for 20 consecutive days (days - 10 through 10). Samples were frozen, lyophilized, and extracted in 90% ethanol. Extracts were assayed for corticoid (males and females) and androgen metabolites (males) by radioimmunoassay. Overall, there was a significant (P <0.01) increase in fecal corticoid metabolite concentrations after ACTH injection (pre-ACTH: 924.79 157.67 ng/g dry feces; post-ACTH: 2613.37 443.54 ng/g). No significant effect of sex on corticoid metabolite concentrations was detected (P> 0.05). Androgen metabolite concentrations increased in males 1 and 2 in the post-ACTH injection period. In male 2 only, a (positive) correlation between corticoid a

CpG motif-based adjuvant enhances immunogenicity of a recombinant LHRH vaccine and noninvasive monitoring of adrenal and gonadal function in the jaguar (Panthera onca)

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