Use of a growth hormone secretagogue peptide as a vaccine adjuvant

Abstract

The present invention is related to the use of the GHRP-6 and one structural analogue as molecular adjuvants for vaccines. Among other applications, these vaccines may be employed for preventing diseases caused by infectious agents, like viruses, bacteria and ectoparasites, that affect mammals, birds and aquatic organisms. The GHRP-6 and its analogue A233 are effectives as adjuvants when they are combine with a given antigen, since they enhance the specific immune response against it.

Claims

1. A vaccine composition comprising a growth hormone secretagogue peptide consisting of SEQ ID No. 1 or SEQ ID No. 2, at least one vaccine antigen, and pharmaceutically acceptable vehicles or diluents, wherein the growth hormone secretagogue peptide is in an amount sufficient to stimulate host immune response to the vaccine antigen.

2. The composition of claim 1 wherein the at least one vaccine antigen is selected from the group consisting of peptides, proteins, viruses and attenuated bacteria.

3. The composition of claim 2, wherein said composition is administered in mammals, birds or fish, orally or by injection.

4. The composition of claim 3 wherein the peptide of sequence SEQ ID No. 1 or SEQ ID No. 2 is in a concentration of 50-600 μg/Kg of formulated feed when the composition is orally administered to fish.

5. A method for increasing an immune response against a vaccine antigen, wherein an effective amount of a growth hormone secretagogue peptide consisting of SEQ ID No. 1 or SEQ ID No. 2 is administered as a molecular adjuvant of said antigen.

6. The method of claim 5 wherein the vaccine antigen is used for the prevention of diseases caused by infectious agents.

7. The method of claim 5 wherein the peptide of sequence SEQ ID No. 1 or SEQ ID No. 2 is used at 50-600 μg/Kg of formulated feed when the vaccine antigen and the molecular adjuvant peptide are administered by oral route.

8. The method of claim 5 wherein the peptide of sequence SEQ ID No. 1 or SEQ ID No. 2 is employed at 0.1-40 μg/g of animal body weight, when the vaccine antigen and the molecular adjuvant peptide are administered by injections.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Antibody response in mice immunized with OVA, GHRP-6 and Freund's adjuvant, or with OVA and Freund's adjuvant. A. Titers of immunoglobulin G (IgG) in groups immunized by subcutaneous route (s.c). B. Titers of IgG in groups immunized by intraperitoneal route (i.p). The median and the standard deviation within each group are represented. ** indicates p<0.0014; *** indicates p<0.001.

(2) FIG. 2. Antibody response in mice immunized with OVA, GHRP-6 and Freund's adjuvant, by s.c and i.p routes. (Groups 3 and 4, respectively); or with OVA and Freund's adjuvant, by s.c and i.p routes (Groups 5 and 6 respectively). A. Titers of IgG1. B. Titers of IgG2a. The median and the standard deviation within each group are represented. ** indicates p<0.0014; *** indicates p<0.001.

(3) FIG. 3. IgG titers in mice immunized with A233 peptide, OVA and Freund's adjuvant; or OVA and Freund's adjuvant, by i.p route. The levels of antibodies detected in serum extracted on day 22 of the immunization schedule are shown. The median and the standard deviation within each group are represented. ** indicates p<0.0014.

(4) FIG. 4. IgG titers (detected on day 28 of the immunization schedule) in groups immunized with C2 and aluminum; or with C2, GHRP-6 and aluminum, by s.c route. The median and the standard deviation within each group are represented. * indicates p<0.05.

(5) FIG. 5. IgG titers in mice immunized with P0 and Freund's adjuvant; or with P0, GHRP-6 and Freund's adjuvant by i.p route, on day 36 of the immunization schedule. The median and the standard deviation within each group are represented. * indicates p<0.05.

(6) FIG. 6. Immunoglobulin M (IgM) titers in tilapias immunized with P0-my32 or with P0-my32 combined with GHRP-6 or with the peptide A233, detected on day 28 of the experiment, the immunogens were administered by i.p. route. A. and C. Titers of IgM against my-32. B and D. Titers of IgM against P0. In A and B the titers in animals treated with or without GHRP-6 are shown, while in C and D the titers in animals treated with or without A233 are presented. The median and the standard deviation within each group are represented. * indicates p<0.01; ** indicates p<0.0014; *** indicates p<0,001.

(7) FIG. 7. Percent of tilapias with IgM titers greater than 1:1000, after the immunization with a P0-my32 formulation including Montanide ISA 50 or P0-my32 including GHRP-6 and Montanide ISA 50, at day 28 of the experiment. A. Percentage of anti-my32 responder animals. B. Percentage of anti-P0 responder animals.

(8) FIG. 8. Response of IgM antibodies in claria immunized with P0-TT or with P0-TT in presence of GHRP-6 or peptide A233, on day 28 of the experiment, where the fish were immunized by i.p. route. A. Anti-P0 titers in the groups of the evaluation experiment with GHRP-6 as an adjuvant. B. Anti-P0 titers in the groups of the evaluation experiment with A233 as an adjuvant. All immunogens were combined with Montanide ISA 50.

(9) FIG. 9. Titers of agglutinating antibodies against Aeromonas hydrophila in common carp. The Y-axis values represent median±standard error. * indicates p<0.05; ** indicates p<0.01. Group 1: Injected with PBS, Group 2: Injected with formalin inactivated A. hydrophila cells, Group 3: Injected with formalin inactivated A. hydrophila cells and GHRP-6 (20 μg per fish). All immunogens were combined with Montanide ISA 50.

EXAMPLES

Example 1

Effect of Co-Administration of GHRP-6 on the Humoral Immune Response Against OVA

(10) Thirty-six female BALB/c mice were used. Six study groups, with 6 animals each, were separated. Three groups were immunized by s.c route and the rest of them were treated by i.p route. In all groups, the immunogens were emulsified with Freund's adjuvant.

(11) Group 1: Placebo (phosphate buffered saline, abbreviated PBS). s.c route.

(12) Group 2: Placebo (PBS). i.p. route.

(13) Group 3: GHRP-6 at 10 μg/animal and OVA at 5 μg/animal. s.c. route.

(14) Group 4 GHRP-6 at 10 μg/animal and OVA at 5 μg/animal. i.p route.

(15) Group 5: OVA 5 μg/animal. s.c. route.

(16) Group 6: OVA 5 μg/animal. i.p. route.

(17) Mice received 150 μL of the immunogen in all groups, which was administered to the animals on days 1 and 15 of the immunization schedule. Blood extractions were performed on days 0 (pre-immune serum), 8, 15, 22, 36, 43 and 50. Total IgG titers, IgG1 and IgG2a were evaluated. Complete Freund's Adjuvant (CFA) was used in the first immunization and Incomplete Freund's Adjuvant (IFA) in the next one. The animals of the group immunized with GHRP-6 and OVA, by s.c route, had higher titers (with statistical significance: p<0.05) than those of the group receiving OVA without GHRP-6, by the same route, on days 22, 36, 43 and 50 of the experiment (FIG. 1A). Animals injected with the same immunogens, but by i.p. route, had the same behavior on days 36 and 43 of the experiment (FIG. 1 B).

(18) IgG1 and IgG2a titers were determined in the serum extracted on day 36, in groups immunized with OVA with or without GHRP-6, by s.c and i.p routes. (FIGS. 2A and 2B). No significant differences were observed in IgG2a titers between the groups treated with GHRP-6 and those that did not receive said secretagogue, for both routes of administration (FIGS. 2A and 2B). Significant differences were observed for IgG1 in both administration routes, being higher in the groups vaccinated in the presence of GHRP-6.

(19) The ratio IgG1/IgG2a, as a measure of the differencial reactivity towards a Th2 or Th1 response, respectively, was significantly superior in the group immunized by i.p route, with OVA in the presence of GHRP-6 and Freund's adjuvant, compared to the group injected with OVA and Freund's adjuvant only. This ratio is shown in Table 1, where data represent the mean of the IgG1/IgG2a ratio corresponding to the six animals in the group.

(20) TABLE-US-00001 TABLE 1 Ratio IgG1/IgG2a in both groups immunized by intraperitoneal route. Immunogen of the group IgG1/IgG2a GHRP-6 + OVA + Freund's adjuvant 981.33* OVA + Freund's adjuvant 172.33 *indicates significant differences p < 0.05.

Example 2

Effect of the Co-Administration of the Peptide A233 on the Humoral Immune Response Against OVA

(21) To evaluate whether the peptide A233 exerts an adjuvant effect on OVA, 18 female BALB/c mice were used. Mice received 150 μL of immunogen in all groups, by i.p route, which was given to the animals on days 1 and 15 of the immunization schedule. Blood samples were taken at days 0 (preimmune serum), 8, 15, 22, 36, 43 and 50. The titers of total IgG present in the sera were evaluated. The animals were immunized with 5 μg OVA/animal and 10 μg A233/animal (Group 2), or with 5 μg of OVA/animal (Group 3). The control group was injected with PBS (Group 1). All immunogens were emulsified with Freund's adjuvant.

(22) In animals injected with OVA and the peptide A233 there was a significant increase in anti-OVA antibody titers, in comparison with those immunized with OVA alone, at day 22 of the immunization schedule (FIG. 3).

Example 3

Effect of the Co-Administration of GHRP-6 on the Humoral Immune Response Against C2 Antigen

(23) The C2 antigen, a Dengue virus capsid protein, was obtained as a recombinant protein in Escherichia coli, with a molecular weight of 15 kDa. To evaluate the humoral and cellular immune response in mice vaccinated with C2, in presence or absence of GHRP-6, 24 female BALB/c mice, 6-weeks old, were selected. They were distributed into 3 groups. Each animal was given an immunogen, according to the experimental group:

(24) Group 1:10 μg C2

(25) Group 2: 10 μg C2 co-administrated with 10 μg GHRP-6

(26) Grupo 3: PBS

(27) In all groups, the immunogens further comprised aluminum hydroxide, also known as alum. Immunizations were performed by s.c route, on days 0, 15 and 30 of the immunization schedule. Blood samples were taken at days 0 (pre-immune), 7, 16, 21, 28 and 35, to assess total IgG titers.

(28) FIG. 4 shows that, on day 28 of the immunization schedule, Group 2, treated with C2 and GHRP-6, showed a significant increase in anti-C2 IgG titer, compared to Group 1, treated with C2 and the adjuvant aluminum hydroxide, without GHRP-6.

Example 4

Effect of the Co-Administration of GHRP-6 on the Humoral Immune Response Against the Peptide P0 of R. sanguineus

(29) The peptide P0 is a fragment corresponding to the region of less sequence identity between the ribosomal protein P0 of the tick R. sanguineus and its mammalian host. To evaluate the humoral immune response stimulated by the co-administration of GHRP-6 and P0, 24 female, 6-week old BALB/c mice were selected and divided into three groups of 8 mice. Each animal received 150 μL of immunogen, by i.p route, on days 1, 15 and 29 of the immunization schedule. In the first immunization FCA was employed, and FIA was used in the other two administrations.

(30) Group 1. Control (PBS)

(31) Group 2. 100 μg of P0.

(32) Group 3. 100 μg of P0 co-administrated with 200 μg of GHRP-6.

(33) Blood samples were taken on days 0 (pre-immune serum), 8, 16, 21, 28, 36, 43, 50 and 58, to determine the levels of total IgG. In the animals immunized with P0, in the presence of GHRP-6, there was a significant increase in the IgG titers, if compared to those that did not receive GHRP-6, as seen in FIG. 5, which shows the anti-P0 antibody levels at 36 days of the schedule.

Example 5

Effect of the Co-Administration of GHRP-6 or the Peptide A233 on the Humoral Immune Response Against the P0-my32 Protein in Tilapia (Oreochromis sp.)

(34) The chimeric protein P0-my32 was generated by cloning the complementary deoxyribonucleic acid (cDNA) encoding a 35 amino acid peptide of the L. salmonis P0 ribosomal protein fused to the N-terminal end of the cDNA encoding the my32 protein of the same ectoparasite (Carpio et al., 2013; Exp. Parasitol 135: 188-199), in a vector designed for the induced expression of genes of interest in the host bacterium E. coli. This protein was produced in the bacterium and purified, as a fusion protein with a tail of histidine, by metal chelate affinity chromatography.

(35) For the accomplishment of the immunization experiment, 6 study groups of male juveniles of Oreochromis niloticus, were formed, with 15 animals each. In all groups, the immunogen was administered by i.p. route, adjuvanted with Montaide ISA 50.

(36) The experimental groups received the following immunogens:

(37) Group 1: PBS

(38) Group 2: P0-my32 (1 μg/g of fish weight)

(39) Group 3: P0-my32 (1 μg/g of fish weight) co-administrated with 20 μg GHRP-6

(40) Group 4: P0-my32 (1 μg/g of fish weight) co-administrated with 20 μg A233

(41) Group 5: P0-my32 (1 μg/g of fish weight). The fish in this group were fed with feed formulated with 100 μg of GHRP-6 per kg of feed, twice daily, for one week before and one week after the administration of my32 in injections.

(42) During the course of the experiment, the fish in groups 1 to 4 were fed twice daily with a balanced unmedicated commercial formula, at the rate of 1% of their body weight. Immunizations were performed on days 0 and 14 of the schedule. Blood samples were taken at days 0, 21, 28 and 35 from the start of the experiment.

(43) In tilapias injected with P0-my32 in the presence of GHRP-6 there was an increase in anti-my32 IgM antibody titers, compared to the group injected with P0-my32. This increase was statistically significant, as shown in FIG. 6A, which shows the levels of antibodies in sera extracted on day 28. The same effect was observed for anti-P0 IgM titer (FIG. 6B). Co-administration of A233 with the chimeric antigen also allowed an increase in IgM titers against the two components, as compared to the control group (FIGS. 6C and 6D). The group injected with P0-my32 and simultaneously fed with feed containing GHRP-6 also showed an increase in the titers, if compared to the group injected with the chimeric protein, which was fed with an unmodified feed (Table 2).

(44) TABLE-US-00002 TABLE 2 Effect on anti-my32 and anti-P0 IgM titers of the co-administration of GHRP-6 in feed IgM titers Group 2 Group 5 Anti-my32 601.0 ± 484.8 5760 ± 1866* Anti-P0 200.0 ± 150.0 6600 ± 2229* *indicates significant differences between the groups p < 0.05.

(45) In addition, in the group injected by i.p route with immunogen containing GHRP-6 (Group 3), the number of responder animals, with IgM titers greater than 1:1000, was higher for both components of the chimeric protein, compared to the group treated with P0-my32 that did not receive the peptide GHRP-6 (Group 2). It can be seen in FIGS. 7A and 7B.

Example 6

Effect of the Co-Administration of GHRP-6 or A233 and the P0-TT Protein on the Humoral Immune Response in Clarias (Clarias gariepinus)

(46) The chimeric protein P0-TT is based on: a) the peptide named pP0, composed by 35 amino acids, corresponding to the region less conserved between the L. salmonis P0 ribosomal protein and the same protein in one of its hosts, Salmo salar and b) two T cell epitopes, coming from the measles virus and tetanus toxoid, respectively. For the experiment, 4 study groups of C. gariepinus were formed, with 12 animals in each one. These animals were immunized by i.p. route with 1 μg of the P0-TT protein per gram of animal weight (Group 2) or with the same dose of the chimeric protein P0-TT (1 μg/g of animal weight) co-administered with 20 μg of either GHRP-6 or A233 per animal (Groups 3 and 4, respectively), in a total volume of 120 μL. The control group (Group 1) received the same volume of PBS. The immunogens corresponding to all groups were emulsified with Montanide ISA 50 oily adjuvant. The immunization was performed under the same conditions, on days 0 and 14 of the administration schedule. Blood samples were taken at days 0, 21, 28 and 35 counted from the start of the experiment.

(47) In clarias immunized with P0-TT and GHRP-6 there was an increase in anti-P0 IgM antibody titers, when it as compared to the group injected with P0-TT without the GHRP-6 peptide, which had statistical significance at day 28 (FIG. 8A). The same effect was observed for the titers obtained after administration of P0-TT and A233 (FIG. 8B).

Example 7

Demonstration of the Adjuvant Effect of GHRP-6 on the Humoral Immune Response of Common Carp (Cyprinus carpio) Against the Bacterium Aeromonas hydrophila

(48) The experiment was carried out with carps (C. carpio) of 40±10 g. These animals were kept in 600 L aquaria, at a temperature of 28±2° C. Three experimental groups, of 10 carps each, were established and injected by i.p. route with the following immunogens:

(49) Group 1: PBS+Montanide ISA 50.

(50) Group 2: Inactivated A. hydrophila cells (1×10.sup.8 Colony Forming Units, abbreviated CFU)+Montanide ISA 50.

(51) Group 3: Inactivated A. hydrophila cells (1×10.sup.8 CFU)+20 μg GHRP-6/fish+Montanide ISA 50.

(52) Fish were injected on days 0 and 14, and blood samples were collected from the caudal vein on days 0 and 21. The results showed that the titers of agglutinating antibodies against A. hydrophila were significantly higher in the group immunized with the bacterium and GHRP-6, compared to the group immunized with the bacterium without the GHRP-6 peptide (FIG. 9). These results corroborate the effect of GHRP-6 as a molecular adjuvant in fish. The preparation of A. hydrophila cells and the measurement of antibody titers were performed according to previous reports (Yin et al. ((1996) Fish & Shellfish Immunol. 6, 57-69).

Example 8

Controlled Challenge Experiment in Fish Immunized with the Inactivated Aeromonas hydrophila Bacterium and the Inactivated Bacterium Co-Administered with the GHRP-6 peptide

(53) The experiment was carried out with common carp (C. carpio) of 30±5 g. These animals were kept in 250 L aquaria, at a temperature of 30±2° C. Three experimental groups, of 20 carps each, were established and the animals were injected by i.p. route with:

(54) Group 1: PBS+Montanide ISA 50

(55) Group 2: Inactivated A. hydrophila cells (1×10.sup.8 CFU)+Montanide ISA 50

(56) Group 3: Inactivated A. hydrophila cells (1×10.sup.8 CFU)+20 μg GHRP-6/fish+Montanide ISA 50.

(57) Fish were injected on day 0 and 14. The challenge was performed on day 21, by i.p injection of the lethal dose 50 (LD50) of the bacterium, and the mortality in each group was recorded for 7 days. The relative survival rate (RSR) was calculated as:
RSR(%)=(% mortality of controls−% mortality of treated animals)/(% mortality of controls)×100.

(58) As a result, 65% of RSR was obtained in Group 2, and 95% of RSR in Group 3, demonstrating that administration of GHRP-6 increases the survival rate in fish vaccinated and challenged with the pathogen.

Example 9

Demonstration of the Adjuvant Effect of GHRP-6 on the Humoral Immune Response Of Chickens Against Bovine Serum Albumin

(59) Fifteen neonatal broiler chickens, hybrid EB34 Cronish×White Plymoth Rock-type, were used, and they were divided into three experimental groups of 5 animals each. Animals were immunized by i.p route, on days 12 and 18 of the immunization schedule, with 5 μg of BSA per animal (Group 2), or with 5 μg of BSA and 20 μg of GHRP-6 per animal (Group 3). The control group (Group 1) was immunized with PBS. The levels of IgY antibodies in serum were determined, on day 25 of the experiment. There were statistically significant differences between the antibody titers in the animals of the BSA-immunized group and in the one that received BSA and GHRP-6 (Table 3).

(60) TABLE-US-00003 TABLE 3 Response of antigen-specific IgY antibodies in the serum of chickens immunized with BSA. Group Immunogen IgY 1 PBS 0.10 ± 0.08 2 BSA 0.47 ± 0.06 3 BSA + GHRP-6 0.65 ± 0.07

(61) The data show the mean and the standard deviation of the maximum values of absorbance determined by indirect ELISA.

INCORPORATION OF SEQUENCE LISTING

(62) Incorporated herein by reference in its entirety is the Sequence Listing for the application. The Sequence Listing is disclosed on a computer-readable ASCII text file titled, “SequenceListing_976-103PCTUS.txt”, created on May 6, 2020. The file is 1018 kb in size.