WO2013014269A1

WO2013014269A1 - A method to promote growth of a newborn animal

Info

Publication number: WO2013014269A1
Application number: PCT/EP2012/064773
Authority: WO
Inventors: Holger Lehmann; Marc-Antoine Driancourt
Original assignee: Intervet International B.V.
Priority date: 2011-07-28
Filing date: 2012-07-27
Publication date: 2013-01-31

Abstract

The present invention pertains to a method to promote growth of a newborn animal, the method comprising administering anti-PYY antibodies to the newborn animal. The invention also pertains to the use of PYY to raise anti-PYY antibodies for the manufacture of a composition comprising these antibodies that after administration to a newborn animal promote growth of this animal and to the use of PYY to manufacture a composition comprising immunogenic PYY for administration to a female animal to induce anti-PYY antibodies in the said animal for passing these antibodies to a newborn animal by allowing the newborn animal to drink milk from the female animal.

Description

A METHOD TO PROMOTE GROWTH OF A NEWBORN ANIMAL

The present invention pertains to a method to promote growth of a newborn animal, i.e. any method to stimulate the growth of an animal versus a control animal (not subjected to the method), for example by increasing food efficiency or growth speed. In the art it is known to promote growth of newborn animals by various means, such as e.g. by optimising food, medicine and environmental circumstances, by reducing stress etc. Still, despite the fact that considerable costs are involved in the provision of these growth promoting means, it appears hard to obtain a direct effect on growth of the newborn animals. Surprisingly it has been found that the growth of a newborn animal, in particular a vertebrate, can be promoted by administering anti-PYY antibodies to the newborn animal. PYY (also known as Peptide YY or Peptide tyrosine-tyrosine) is a member of the pancreatic polypeptide family that also includes neuropeptide Y (NPY), and is produced by endocrine cells (called L cells) of the lower intestine and the pancreas of vertebrates (Karra et al. in: Molecular and Cellular Enodcrinology ^ S, 2010, 120-128), including mammals, poultry and fish. PYY is a peptide that in most animals contains 36 amino acids, although in chickens it is 37 amino acids long, displaying alanine (-1 position) before the first tyrosine (Conlon et al. in: FEBS letters, 313: 225-228). Two main forms of PYY have been described, viz. the full-length PYY1 -36 and a truncated form PYY3-36, each form is denoted as "PYY" in the art. The said truncated form is a 34 amino acid form produced by cleavage of the N-terminal Tyr-Pro residue (or Ala-Tyr-Pro in the case of chicken PYY) from PYY1-36 by the enzyme dipeptidyl-peptidase IV. PYY3-36 is the main circulating form of the peptide (although it is not excluded that other truncated forms, e.g. a form defining the portion that binds to the PYY receptor, may exist or can be artificially provided).

PYY has a range of biological effects (see the Karra et al. reference indicated here- above). Circulating PYY levels increase in response to nutrient ingestion. Caloric load, food consistency and nutrient composition all affect PYY concentrations. It has also been shown that circulating postprandial PYY levels correlate with postprandial energy expenditure and the thermic effect of food. A positive correlation between postprandial PYY concentrations and subjective satiety has been reported. Peripheral administration of PYY3-36 reduced food intake in rodents and normal weight humans, while, on the opposite, transgenic mice that lacked Pyy were hyperphagic when freely feeding and ate significantly more than their wild-type littermates. These PyyKO mice had increased subcutaneous and visceral adiposity. In other words, they had become obese.

Based on the prior art understanding that "decreasing the levels of circulating PYY in general stimulates food intake and ultimately may lead to obesity", it is tempting to assume that decreasing the level of circulating PYY in newborn animals, may also stimulate food intake in these particular animals, and if so, may promote the same processes in these animals that would induce obesity in an adult animal, which promotion on it's turn might lead to additional growth in these newborn animals instead of obesity, that is if the metabolism of these newborn animals is such that they are programmed to grow instead of becoming obese.

It is clear that the above reasoning is based on many assumptions for which no proof is available. More importantly, in addition to the fact that the above reasoning is based on many assumptions, prior art specifically relating to newborn animals at least strongly suggests that in this particular group of animals high (at least above normal) PYY levels are needed for optimal growth (see i.a. the handbook: Physiology of the Gastrointestinal Tract, Volume 2, 2006, by Leonard R. Johnson et al., in particular page 141 ). In particular, it is known that digestive and absorptive processes are not fully developed in newborn animals. It has been suggested in the art that the expression of PYY has an important positive role in the adaptation of the bowel to the rapid changes in dietary intake during the first few weeks of life. In newborn cord blood, plasma PYY levels are greater when compared to healthy fasting adults, and plasma PYY levels increase during the first 12 days after birth in neonates fed with either breast milk or formula. Also in rats, there is a significant stimulation of PYY levels by ingestion of fat contained in the mother's milk. Only after weaning, there is a coordinated decrease in PYY levels to become normal. Apparently, for newborn animals high PYY levels are needed. Indeed, Coles et al. showed that in-ovo administration (thus to pre-newborn animals) of PYY to broiler eggs resulted in increased body weight and feed efficiency during the first week post-hatch (in: Poultry Science, 78, 1999, 1320-1322; and in: The Journal of Applied Poultry Research, 10, 2001 , 380-384). In short, for the specific group of newborn animals it seems that high levels of PYY are necessary for successful development and growth of the newborn animal. Contrary to the above public knowledge however, it was found that by administering anti-PYY antibodies to a newborn animal, growth of this animal is promoted. In particular, it was found that upon application of the current method with piglets, it appeared that before as well as after weaning newborn animals that received anti-PYY antibodies grew faster than the control animals. Given the fact that PYY and its function are highly conserved in vertebrates, it is believed that the current method can be successfully applied in all vertebrates. With regard to the in-ovo administration results as indicated here-above (Coles et al.), which on first glance seem contradictory to the present finding, it is believed that these prior art results are in line with the current invention: Coles et al. chose human recombinant PYY based on the fact that it was shown before that such human PYY can evoke a biological response in poultry.

However, this human PYY is now believed to have evoked an anti-PYY immunological response in the eggs (cf the common application of in-ovo vaccination), in particular since it has been described years after the Coles et al. reference that non-conjugated PYY may in itself already evoke an immune response given its considerable length (see i.a. WO 2005/1 13600, page 15, lines 27-29).

The invention also pertains to the use of PYY to raise anti-PYY antibodies for the manufacture of a composition comprising these antibodies that after administration to a newborn animal promote growth of this animal. Also, the invention pertains to the use of PYY to manufacture a composition comprising immunogenic PYY for administration to a female animal to induce anti-PYY antibodies in the said animal for passing these antibodies to a newborn animal by allowing the newborn animal to drink milk from the female animal.

Definitions

A newborn animal is an animal right after birth, i.e. right after exposure to the exterior environment, until it reaches about 10% of its adult weight, in particular about 5% of its adult weight. For poultry and fish, the moment of birth is defined as the moment when the animal leaves its egg.

Growth is the process of an increase in body weight by a balanced increase in the size of all cells of an animal. Obesity is the presence of excess body fat, particularly present under the skin and around internal organs, as a result of an uneven increase in adipose tissue with respect to other tissue such as bone and muscle.

Embodiments

In an embodiment of the present invention the antibodies are administered during the first 4 weeks after birth of the animal. During this stage the newborn animals are very susceptible for passive immunisation, in particular when the antibodies are administered during the first week after birth of the animal, preferably even during the first day after birth of the animal. Given the fact that the immune system of newborn animals has to mature after birth, while being exposed to many pathogens, may explain why early immunisation with antibodies is very effective.

In an embodiment the antibodies are administered orally. Oral administration of antibodies may be convenient and safe. In particular for newborn animals, more in particular during the first 24-48 hours after birth, it is known that the gut allows almost free passing of antibodies into the circulatory system. In a particular embodiment, the antibodies are administered through suckling of milk from a lactating animal that has circulating anti-PYY antibodies. This method has shown to be very convenient and effective for application of the present invention. Indeed, colostrum (the milk produced during the first 24-48 hours after giving birth) is known to comprise a high concentration of antibodies.

In yet a further embodiment, the anti-PYY antibodies in the lactating animal are induced via administration of a composition comprising immunogenic PYY (i.e. PYY in a form such that the immune system of a subject animal may raise anti-bodies directed against PYY upon administration of the composition to the animal). In this method, the composition comprising the immunogenic PYY is administered to a female animal before it is lactating. As soon as the animal is lactating, one or more newborn animals are allowed to drink milk from the said female animal, for example by simply laying the one or more animals to the female animal, or by leaving the one or more animals to the female animal when she has given birth to the one or more newborn animals.

Surprisingly this embodiment has shown to be very effective in raising antibodies. Given the fact that healthy animals such as humans and rats already have autoantibodies against PYY in the circulatory system (Fetissov et al. in: Nutrition 24, 2008, 854-859), it was not beforehand expected that sufficient levels of anti-PYY antibodies could be raised to pass an effective amount to newborn animals via the milk of a lactating animal. Raising antibodies this way appears to be very effective and inexpensive when compared to for example recombinant expression of antibodies. In order to make sure that the lactating animal has adequate levels of ant-PYY antibodies in the milk right after birth, it is preferred that the lactating animal has received the immunogenic composition during pregnancy, preferably multiple times. Typical dosage of the immunogenic PYY lies in a range between 50 and 1000 μg, administered via a volume typically between 0.1 and 5 ml, preferably between 0.2 and 2 ml.

In an embodiment the composition comprises PYY 3-36. This truncated form of PYY has proven to be effective as an immunogen to raise antibodies against PYY. In order to increase the immunogenic properties it is preferred that the PYY (either full length, truncated 3-36 form or any other form) is conjugated to a carrier, such as for example keyhole limpet hemocyanine (KLH). There are several techniques commonly known for conjugating molecules to peptides via functional groups. General appropriate techniques can be found i.a. in Hermanson, G.T. (1996) Bioconjugate Techniques, Academic Press, San Diego. PYY-KLH has been described i.a. in Surgery 1994, 1 16, 1 153-1 157 (Bilchik et al).

In an embodiment the newborn animal is a monogastric, in particular a swine. The invention will be explained in more detail based on the following examples.

Examples Example 1 describes polypeptide YY.

Example 2 describes a study to assess the effect of anti-PYY antibodies in a vertebrate. Example 3 describes an experiment to assess the passing of anti-PYY antibodies via milk of lactating animals to newborn animals. Example 1

An NMR structure study of PYY (Waegele et al. in: Biochemistry 49, 2010, 7659-7664) revealed that the peptide can be described as a stable juxtaposition between residues 3-8 and 25-33, maintained by a twist around residues 10-16 and helical segments between residues 17-33. Site directed mutagenesis replacing Ser 13 and Pro 14, located at the twist between the two sides of the PYY molecule by Ala, strongly disrupted PYY folding. It thus seems that these two amino acids are key for proper folding. Based on studies done by Pedersen et al (Journal of Peptide Science 15, 2009, 753-759) and Keire {Peptides 23, 2002, 305-321 ) it may be concluded that Gin 34 as well as Tyr 36 play important roles in receptor selectivity. Also, the amino acids of the C terminal end of PP (amino acids 27-36) are important for the ability of the peptide to bind to its receptor.

Sequence identifier 1 (SEQ ID No 1 ) shows the amino acid sequence of pig PYY1 -36 (it is noted that other forms of PYY may exist, such as for example PYY3-36, or may be artificially produced taken account of the above described information about the structure of full-length PYY). The amino acid sequence of rat PYY is 100% identical to SEQ ID No 1. Human PYY1-36 (SEQ ID No 2) is slightly different and has an overall identity of 94%. Chicken PYY1 -36 (SEQ ID No 3) has an identity of 69% to pig PYY. This is a clear indication of the high degree of conservation of PYY in vertebrates.

Indeed, it has been shown in the art that the 69% structural homology between human and poultry PYY is enough to evoke a biological response in turkeys by the

administration of human PYY (Croom et al. in : The Journal of Applied Poultry

Research, 8, 1999, 242-252). Indeed, the term "PYY" at least covers the two known forms of PYY according to SEQ ID No 1 , viz. PYY1 -36 and PYY3-36, and homologs having at least 69% identity with these forms.

Example 2

This example describes a study to asses the effects of anti-PYY antibodies, raised by active immunisation in sows, on the sows themselves and on their progeny. To perform this study 6 pregnant gilts were allocated either to a placebo group (n=3) or to a PYY group (n=3). Between these groups there was no difference in mean body weight (approx. 106 kg) or age (approx. 172 days) at entrance. The PYY group was actively immunized with a vaccine containing PYY antigens (called the "PYY vaccine"). This vaccine contained per ml 250μg of PYY3-36 conjugated to KLH (obtained from Eurogentec, Seraing, Belgium), 0.5 ml W/O adjuvant (water dispersed in Marcol 52 mineral oil; w/o = 45/55 w/w) and PBS buffer. A first injection (1 ml; i.m.) with the PYY vaccine was given at inclusion, immediately after a positive pregnancy diagnosis (i.e. around day 28 of pregnancy). Three additional (1 ml; i.m.) vaccinations were given 3 weeks apart, with the last one administered around one week before farrowing. Gilts from the placebo group were injected with a placebo vaccine (the same as the PYY vaccine, but no PYY3-36-KLH present) at the same times.

After farrowing (day 0) the litter were adjusted to 10 piglets, and feeding of the sows was increased gradually from 3 kg/d (at day 0) to 12 kg/d during the last two weeks of lactation. The piglets were able to suckle freely from the moment of birth until weaning, which took place four weeks (day 28) after birth.

Anti-PYY antibody levels, changes in body weight and dorsal fat thickness were monitored during pregnancy and lactation.

The results were as follows.

At farrowing, the number and weight of the piglets was the same for each group (no statistical differences). In the placebo group, an average of 12.3 (±3.2) piglets was born with a weight of 1380 (± 318) grams. In the PYY group, an average of 1 1 (±5.3) piglets was born with a weight of 1481 (± 359) grams.

The mother animals of the two groups had similar body weights (both around 205 kg at farrowing and around 180 kg at weaning) and dorsal fat thickness (both around 25 mm at farrowing and around 21 mm at weaning), despite the fact that the PYY group displayed circulating anti-PYY antibodies, whereas the placebo group had not.

Apparently in these animals, the vaccine had raised significant levels of PYY antibodies, but the presence of these antibodies had no apparent effect on the body weight and dorsal fat thickness of the pregnant animals.

Growth of the piglets however was different in the two groups. This is shown in Table 1 , summarising the changes in body weight from farrowing (day 0) until 4 weeks after weaning. It is noted that no differences in dorsal fat thickness were observed for the

PYY group piglets vs the placebo group piglets, from which it is concluded that the extra weight was the result of additional growth of the newborn animals. Table 1 Average weight of piglets per group, and the difference between the groups

Statistical analysis (ANOVA) showed that there was a significant treatment effect (P=0.0001 ) on the changes in body weight with time. As the piglets born from PYY immune sows were already heavier at weaning than those born from placebo treated sows, a final step of analysis assessed whether treatment altered post weaning growth when expressed as a percentage of weight at weaning. Again, a significant time by treatment interaction (P<0.001 ) was detected with ANOVA, demonstrating an improved growth pattern post weaning of the animals born from PYY immune mothers, independently of their weaning weight.

Example 3

A next experiment was done to assess the presence of antibodies against PYY in newborn animals that receive milk from PYY immunised mother animals. For this experiment, four sows were used which were immunized as described under example 2. Following farrowing, blood from two piglets from each litter was collected (three times between day 1 and day 16 post natal to assess its content in anti-PYY antibodies. This study clearly established that the PYY antibodies produced by the sows following active immunization against PYY were transferred to the piglets via the milk. In piglets born from PYY immunized sows, changes in antibody concentrations throughout time (between day 1 and 16 post farrowing) showed that piglets were exposed to significant amounts of PYY antibodies for at least 16 days following birth: the OD index (a measure for the amount of anti-PYY antibodies in the serum) on day 1 was approx 1.7, and decreased to about 1.2 at day 16. Piglets born from placebo treated sows had undetectable amounts of antibodies, the OD index of the serum was below 0.1 .

Surprisingly, some sows with moderate antibody titers in their blood (OD index 1 .0 at farrowing vs an average index for all sows at farrowing of approx 1.5), apparently concentrated these antibodies in their milk and transfered large amounts of antibodies to their piglets: the OD index was 1 .4 for these piglets on day 1 vs an 1.0 for the corresponding sows on day 0.

Claims

1. A method to promote growth of a newborn animal, characterised in that the method comprises administering anti-PYY antibodies to the newborn animal.

2. A method according to claim 1 , characterised in that the antibodies are administered during the first 4 weeks after birth of the animal.

3. A method according to claim 1 , characterised in that the antibodies are administered during the first week after birth of the animal.

4. A method according to claim 2, characterised in that the antibodies are administered during the first day after birth of the animal.

5. A method according to any of the preceding claims, characterised in that the antibodies are administered orally.

6. A method according to claim 5, characterised in that the antibodies are administered through suckling of milk from a lactating animal that has circulating anti-PYY antibodies.

7. A method according to claim 6, characterised in that the anti-PYY antibodies in the lactating animal are induced via administration of a composition comprising

immunogenic PYY.

8. A method according to claim 7, characterised in that the lactating animal has received the composition during pregnancy.

9. A method according to claim 8, characterised in that the composition was

administered multiple times.

10. A method according to any of the claims 7 to 9, characterised in that the

composition comprises PYY 3-36.

1 1. A method according to any of the claims 7 to 10, characterised in that the composition comprises PYY conjugated to a carrier.

12. A method according to claim 1 1 , characterised in that the composition comprises PYY conjugated to keyhole limpet hemocyanine.

13. A method according to any of the preceding claims, characterised in that the newborn animal is a monogastric.

14. A method according to claim 13, characterised in that the monogastric animal is a swine.

15. Use of PYY to raise anti-PYY antibodies for the manufacture of a composition comprising these antibodies that after administration to a newborn animal promote growth of this animal.

16. Use of PYY to manufacture a composition comprising immunogenic PYY for administration to a female animal to induce anti-PYY antibodies in the said animal for passing these antibodies to a newborn animal by allowing the newborn animal to drink milk from the female animal.