Phage constructs targeting gonadotropin-releasing hormone for fertility control: evaluation in cats

Abstract

Objectives: Phage-gonadotropin-releasing hormone (GnRH) constructs with potential contraceptive properties were generated in our previous study via selection from a phage display library using neutralizing GnRH antibodies as selection targets. In mice, these constructs invoked the production of antibodies against GnRH and suppressed serum testosterone. The goal of this study was to evaluate this vaccine against GnRH for its potential to suppress reproductive characteristics in cats.

Methods: Sexually mature male cats were injected with a phage-GnRH vaccine using the following treatment groups: (1) single phage-GnRH vaccine with adjuvant; (2) phage-GnRH vaccine without adjuvant and half-dose booster 1 month later; or (3) phage-GnRH vaccine with adjuvant and two half-dose boosters with adjuvant 3 and 6 months later. Anti-GnRH antibodies and serum testosterone, testicular volume and sperm characteristics were evaluated monthly for 7-9 months.

Results: All cats developed anti-GnRH antibodies following immunization. Serum antibody titers increased significantly after booster immunizations. In group 3, serum testosterone was suppressed 8 months after primary immunization. Total testicular volume decreased in group 1 by 24-42% and in group 3 by 15-36% at 7 months after immunization, indicating potential gonadal atrophy. Vacuolation of epididymides was observed histologically. Although all cats produced sperm at the conclusion of the study, normal morphology was decreased as much as 38%. Phage alone produced no local or systemic reactions. Immunization of phage with AdjuVac produced unacceptable injection site reactions.

Conclusions and relevance: Our phage-based vaccine against GnRH demonstrated a potential for fertility impairment in cats. Future research is required to optimize vaccine regimens and identify animal age groups most responsive to the vaccine. If permanent contraception (highly desirable in feral and shelter cats) cannot be achieved, the vaccine has a potential use in zoo animals or pets where multiple administrations are more practical and/or reversible infertility is desirable.

Keywords: Fertility control; GnRH; filamentous phage; gonadotropin-releasing hormone.

Figure 1

Study design. Group 1 received one-time immunization. A total phage dose of 8 × 10¹² vir was split into two equal injections. The injections were given subcutaneously (SC) in the left and right shoulders. One of the injections contained phage–gonadotropin-releasing hormone (GnRH) in phosphate buffered saline (PBS) and the other had phage mixed with AdjuVac. Group 2 received two immunizations, a prime immunization followed by a booster immunization 1 month later. The prime immunization was a total phage dose of 4 × 10¹³ vir split into two equal injections given SC in the left and right shoulders. The booster immunization was 2 × 10¹³ vir split between two shoulders. All injections contained phage in PBS (no adjuvant). Group 3 received three immunizations, a prime immunization followed by two boosters 3 months apart. The prime immunization was a total phage dose of 8 × 10¹² vir split between two injections. The injections were given intramuscularly (IM) in the left and right rear legs (semitendinosus muscle). The first booster was 4 × 10¹² vir combined with AdjuVac given IM on the site of ‘phage in PBS’ primary immunization. The second booster was 4 × 10¹² vir combined with Thermogel (adjuvant) given IM in the left shoulder. Blood was collected from each cat for three times prior to immunization to obtain negative control samples for GnRH antibody and to establish baseline testosterone values. This was followed by monthly post-immunization blood collections for the rest of the study. Testicular measurements and sperm collections were performed prior to immunization and then monthly post-immunization

Figure 2

Gonadotropin-releasing hormone (GnRH) antibodies in cat sera following immunization with phage–GnRH. (a) Group 1; (b) group 2; (c) group 3. The immunization schedule is given in Figure 1. Samples were collected prior to immunization followed by monthly post-immunization collections for 7 (groups 1 and 2) or 9 (group 3) months. The antibodies were detected in sera by ELISA. All measurements were performed in duplicate. The data are presented as endpoint titers (defined as the highest dilution providing an optical density twice that of the pre-immune sera). Each bar represents an individual cat. Lines connecting monthly data points with symbols represent means of antibody titers ± SD for each group

Figure 3

Testosterone in cat sera following immunization with phage–gonadotropin-releasing hormone. Serum samples were collected prior to immunization followed by post-immunization collections performed monthly. All samples were evaluated in duplicate using ELISA. Data are presented as group means ± SD. (a) Group 1. Data analysis was performed for five cats. (b) Group 2. Black line is data analysis for five cats. Red line is data analysis for four cats, excluding cat 2.1 with abnormally high testosterone. (c) Group 3. Black line is data analysis for five cats. Red line is data analysis for four cats, excluding cat 3.1 with abnormally high testosterone. P <0.05 is noted with an asterisk. P value refers to post-treatment vs pretreatment

Figure 4

Total testicular volume in cats immunized with phage–gonadotropin-releasing hormone. Testicular size was measured via scrotal ultrasound prior to immunization and then monthly post-immunization for the rest of the experiment. The procedure was carried out by placing an ultrasound probe on the external surface of scrotum and measuring length (L), width (W) and height (H) of each testicle. Testicular volume was calculated by the formula L × W × H × 0.5233, and the total testicular volume for each cat was found by adding the volume of both testicles. All measurements of L, W and H were performed in duplicate. Data are presented as group means ± SD. P <0.05 is noted with an asterisk. P value refers to post-treatment vs pretreatment

Figure 5

Histologic evaluation of cat epididymis (representative images). (a) Corpus of epididymis of a treated cat in group 3. Note the vacuolation shown by the arrows. (b) Corpus of epididymis of an age-matched cat from the same cat colony housed under identical conditions (not treated). Hematoxylin and eosin staining, magnification × 10

Figure 6

Relative quantity of morphologically normal sperm cells in cats immunized with phage–gonadotropin-releasing hormone. Semen collection was accomplished by electroejaculation under general anesthesia. The morphologic analysis was performed using eosin–nigrin stain evaluating 100 cells/sample for defects. Relative numbers (%) of normal cells for individual cats prior to and post-immunization (monthly) are shown. Data are presented as group means ± SD. P <0.05 is noted with an asterisk. P value refers to post-treatment vs pretreatment. (a) Group 1; (b) Group 2; (c) Group 3

Figure 7

Injection site reaction in cats immunized with phage–gonadotropin-releasing hormone combined with AdjuVac. (a) Physical appearance of an injection site in cat 1.3, representative of the nodules that developed in group 1 cats (subcutaneous immunization). (b) Representative histologic image of soft tissue nodules in cat 1.3 (group 1) biopsied from the injection site. (c) Physical appearance of an injection site in cat 3.1 (group 3, intramuscular immunization). The reaction appeared at the site of the initial vaccination 7 months after injection. The sterile abscess was observed. (d) Ultrasound image of the injection site nodule in cat 3.1. The reaction is characterized by hyperechogenicity (area between two crosses)