The use of therapeutic vaccines in medicine is already in practice for treatment of human melanoma and prostate cancer. Following these novel products are other vaccines for treating various pathological conditions and metabolic diseases [15]. A therapy to treat and manage human obesity by vaccination would afford physicians with a common and accepted medical procedure to treat a globally widespread human disease condition. This therapy can be accomplished without drugs or surgical procedures.
Our approach in designing a therapeutic vaccine targeting the counter-regulatory hormone somatostatin is based on the highly antigenic carrier protein chloramphenicol-acetyl transferase (CAT) which has been made inactive by double site mutations [13, 16]. Coupled with the enhanced antigenicity of the chimeric-somatostatin, adjuvants have been added which contain metabolizable oils, a polyacrylic polymer and vegetable polysaccharides [14]. These vaccines have been previously tested in normal, non-obese mice for safety and efficacy. The JH17 and JH 18 adjuvants were demonstrated to be safe via intraperitoneal injection in a 0.5 mL dose. The chimeric-somatostatin protein was demonstrated to be highly antigenic by measuring mouse weight gain over a 7 day period and demonstrating a 110% to 130% weight increase compared pre-vaccination weights (data not shown).
The use of somatostatin vaccines for treatment of obesity is based upon previous studies using GH therapy [5, 6] and the accepted endocrine model of somatostatin to down-regulate growth hormone releasing hormone (GHRH), GH and IGF-1 [17]. By reversing the effects of somatostatin by immuno- regulation, increased levels of GHRH, GH and IGF-1 were anticipated with increased metabolism.
The DIO mouse model was chosen as it is easily adaptable for vaccination studies and is well characterized for obesity treatments. Since the effective dose in the C57BL/6 J male mouse was not previous determined, we utilized the same dose as we had proven effect in non-obese mice (1 mg protein/mL in a 0.5 mL dose). In this study, this dose volume would represent approximately 1.6% of the mouse body weight. This dose volume would translate to 1.6 liters of vaccine for a 100 kg human, if volume were the determining factor of the vaccine’s effectiveness. In a recently completed pig study, a 1 mL dose containing 0.5 mg of vaccine antigen demonstrated effectiveness in 91 kg pigs as assessed by positive seroconversion and enhanced IGF-1 levels (unpublished data). Therefore, the vaccine’s effectiveness appears to be more related to immunogen content rather than the volume of the injection. This specific antigen dose relationship was previously described by Lunin [18].
Mice were vaccinated and observed for physiological responses to vaccination with the somatostatin vaccines, presented as weight loss and reduction in body weight gain. The amount of food intake was also monitored as it was important to determine if any continued weight loss was due to lack of eating the 60% Kcal diet. Although there was an initial reduction in food consumption during the first 2 days post-1st vaccination, JH17 and JH 18 mice food intake was not statistically significant from the food consumption of PBS control for the duration of the study.
Previous experience with the chimeric-somatostatin vaccines indicated antibody responses are first demonstrable at 4 to 10 days post-vaccination (IgM and IgG subclasses). At these time periods, specific antibody binds to somatostatin and attenuates, but do not completely eliminate the counter-inhibitory effects of the hormone. The main increase of GH and IGF-1 follows this initial antibody production and expected metabolic changes are observed [13]. Since the biological activity of the anti-somatostatin antibodies are short-lived, and are not re-stimulated by endogenous somatostatin, the vaccine effectiveness is similar to repeated drug administration and is not cumulative.
Memory cells responses, as are normally observed with infectious disease vaccines, have not been observed with the chimeric-somatostatin vaccines and each vaccination is likened to a primary dose [18]. Based on these inherent immunological traits of the vaccine, the 2nd vaccination with either JH 17 or JH 18, did not produce a memory response, but rather effected a lesser control on weight gain. In response to the lessened effect, the vaccine-treated mice continued to gain less weight, while consuming the same amount of food as the PBS controls. The second vaccination of 1/5th the amount of protein of the 1st vaccination, can be viewed as a maintenance dose.
Reduction in weight gain can be linked to high anti-somatostatin antibody levels and enhanced IGF-1 quantities in the plasma. Since the samplings of these two markers were conducted at the end of the study, their maximal effectiveness would be anticipated to be during the first week post-vaccination. The demonstration of high levels of anti-somatostatin antibodies at day 39 is indicative of the continued presence of down regulation of somatostatin which is demonstrated by the continued weight gain differentials observed through the study. The antibody titers measured at this one time point, are residual titers and represent the downward part of the antibody effectiveness curve. A similar argument can be made for IGF-1 levels in plasma. It could be postulated the maximal IGF-1 would be seen in the first week following vaccination or revaccination. Even in the presence of these elevated anti-somatostatin antibodies, plasma insulin levels in vaccinated mice were similar to control mice, and demonstrated no statistically significant differences.
The two adjuvants reported in this study were chosen for usefulness in human vaccines. Another adjuvant, approved for use in livestock and containing mineral oil, was also tested in the DIO mouse model (data not shown). The latter adjuvant produced a much more heightened weight loss result accompanied by dehydration, lethargy and death in 8/10 mice treated. Based upon the comparison of these vaccines, it was determined that the chimeric-somatostatin vaccine effects are related to both antigen dose and adjuvant type.
The differences between the JH17 and JH18 adjuvants reside only in their plant polysaccharide content, tragacanthin and arabinogalactan. The results obtained in this study were not statistically significant in any tested parameter between the two vaccine groups. Comparing the two adjuvants, the observation of JH18 producing percent of baseline body weight and enhanced IGF-1 are indicative but not predictive of a superior adjuvant effect of the two polysaccharide additions. In future studies, only a single adjuvant will be utilized and sham vaccinated controls will receive the same adjuvant, but without the chimeric protein included. In this design, the observable food reduction for 2 days after the 1st vaccination can be determined to be a property of the adjuvant or the total vaccine compound.
In summary, DIO mice treated with 2 vaccine formulations, containing the same dose of chimeric-somatostatin protein, were effective in reducing weight gain and reducing final body weight percentage versus baseline weights, when compared to PBS control mice. The vaccine effect was observable even while the mice were continuously fed a 60% Kcal fat diet. The vaccination effects did not significantly reduce cumulative food consumption and was confirmed by residual anti-somatostatin antibodies in mouse plasma at the study’s end. Measured levels of Insulin in vaccinates were similar to controls, further adding to the vaccine’s safety profile. The final result of the study is the demonstration of the usefulness of treating obesity with vaccination and warrants additional studies and parameter monitoring in other animal models (normal pigs, obese minipigs and obese dogs).