IMMUNONEUTRILIZATION OF

HORMONES

Veterinary Gynaecology and Obstetrics

GADVASU, Ludhiana
IMMUNONEUTRILIZATION AND OPSONISATION
TARGET
EPITOPES RECEPTOR
CELL
ANTIGEN

PARATOPE

ANTIBODY
Active immunization against ‘self’

• Autoimmunity to hormones exerting a suppressive effect on the expression of

genetic potential.
• Basic concept involved 
Hormone+protein immunogen+immunostimulant
(adjuvent)
( INJECT )

Formation of ANTIBODIES
• luteinizing hormone (LH) (Wakabayashi and Tamaoki, 1966; Quadri et al., 1966; Pineda et al., 1968).
• Follicle-stimulating hormone (FSH) (Torjesen and Sand, 1975; Wickings and Nieschlag, 1980; Al-Obaidi et
al., 1986)

• Human chorionic gonadotropin (hCG) (Talwar et al., 1976; 1992;1993)

• Thyroid-stimulating hormone (TSH) (Melmed et al., 1980)
• Inhibin (Henderson et al., 1984; Scanlon et al., 1993)
• Growth hormone (GH) (Beattie et al., 1992)
• Luteinizing hormone-releasing hormone (LHRH) (Fraser and Gunn, 1973; Ladd et al.,
1990; Adams and Adams, 1992; Thau, 1992; Hoskinson et al., 1990))

• Vasopressin (Kamoi et al., 1977)

• Somatostatin (Varner et al., 1980)

• Testosterone (Thomson et al., 1985; Hillier et al., 1973))

• Progesterone (Kaushansky et al., 1977)
PRACTICAL SIGNIFICANCE

• Anti-self immunity ( auto immune diseases , anti-tumour vaccines )

• Better understand endocrine regulation (Bettencourt et al., 1993; Martin et al., 1986; Ohlson et
al., 1981; Safir et al., 1987).
• Control fertility by providing new contraceptives for humans and animals
(Lincoln 1992; Talwar et al., 1992; Tallberg et al., 1985)

• Increase meat quality (Adams and Adams 1992; Finnerty et al., 1994; Hoskinson et al., 1990; Reeves et al.,
1989; Prendiville et al., 1992; Crowe et al., 1994a, 1994; Bonneau et al., 1994)
• Combat diseases and boost productivity (Tallberg et al., 1985)
• Practical way to control fertility of stray animals
LHRH
• Alternative to surgical castration of male piglets and bulls (Meloen et al., 1994; Finnerty et
al., 1994; Hoskinson et al., 1990; Bonneau et al., 1994)

• Practical way to control fertility of stray animals

• Suggested for use in men suffering from prostate cancer and as a

contraceptive, in combination with testosterone supplementation (Thau, 1992;
Ladd, 1993).
hCG
• cheap, long-lasting, easy to apply contraceptive for females (Lincoln, 1992).

• General Rules
the antigen ,
the form in which it is presented,
the vaccine formulation (dose, adjuvant) ,
the vaccination procedure,
and the evaluation of the efficacy, reversibility.
(1)The Antigen
• In contrast to ‘foreign’ antigens, ‘self antigens are poorly immunogenic
when used in a vaccine (Reeves et al., 1989). Thus, all sorts of practical
approaches have been taken to overcome this lack of immunogenicity. The
major ones are:
1. Coupling of the antigen to a carrier molecule bovine serum albumin (BSA),
keyhole limpet haemocyanin (KLH) or tetanus toxoid (TT)
2. Changing the native structure of the hormone molecule either by changing
its structure (e.g., by denaturation) or by changing its configuration (e.g.,
by the application of heterodimers or by truncation or dimerization of the
molecule)
3. Applying intact molecules from a different species.
Coupling of the antigen
• large protein molecules are used, notably KLH or TT AND the carrier
molecule should be ‘foreign’ to the target species.
• Small antigens like LHRH (ten amino acids) or vasopressin (nine amino
acids) AND ‘self antigens sufficiently ‘foreign’.
• Bifunctional chemical linkers like m-maleimidobenzoyl-N-hydroxysuccinimide
ester (MBS) (Lerner et al., 1981).
Coupling of the antigen
• The linker molecule is first attached to the free amino groups present in
protein. Then the MBS-activated protein will link to a free SH group (e.g.,
from cystein) present in the peptide or protein molecule.

• Instead of chemical linkage the coupling can be effected genetically using

recombinant methods as shown for inhibin and ovalbumine (Geary and
Reeves, 1994).
 Changing the native
structure
• Denaturation or conformational change may sufficiently alter antigenic sites
of ‘self’ molecules to induce the immune system to respond with the
production of antibodies which cross react with the native antigen .
• For small peptide molecules it has been reported that dimerization further
increases its immunogenicity.
• obtained very good results applying a tandem repeat of the amino acid
sequence of LHRH
Changing the native structure
• For large molecules like hCG or FSH, which contain an (alpha- and beta-
subunit).

• ‘hetero dimers’ or ‘hybrid molecules’ have been applied, i.e., an artificial

combination of an alpha-subunit from one species combined with the beta-
subunit of another species
• Use only small peptides which inactivates the biological activity of the
intact hormone.
• Allows specific targeting of the immune response
• FSH specific anti beta-subunit antibodies which will not cross-react with
LH and specifically neutralize FSH, while leaving LH active.
• FSH is neutralized, spermatogenesis is blocked while libido is maintained
due to the activity of LH.
• The real problem of this approach is to define the amino acid sequence of
the peptide(s) which can be used in anti- FSH vaccines.
• For pathogens including foot-and-mouth disease virus (FMDV) , HIV, corona
viruses and herpes virus
• First synthetic peptide vaccine which gives full protection against a viral
disease (canine parvovirus) in the target animal itself.
Applying intact molecules to heterologous species
• This approach is used when the amino acid sequence of the hormone
differs between species For example, human FSH in rabbits readily induces
anti-FSH antibodies which are specific for human FSH.

• Amino acid sequences of human FSH and rabbit FSH differ by approx. 15%
which is apparently sufficient to induce antibodies.
(2) Vaccine formulation

• Antigens (and especially ‘self’ antigens) without any adjuvants will only
rarely induce a useful immune response.

• Require larger doses and multiple injections.

• Better results can be obtained by using adjuvants (i.e., immunostimulants) .

• Adjuvants provide two major functions.

One is the reservoir function: most adjuvants store the antigen and release it
slowly into the circulation, thus continuously stimulating the immune system

The second is by producing a local inflammation which activates, in a non

specific way, the immune system (for instance induction of cytokines,
recruitment of cells,etc.)
• The best known adjuvant is Freund’s Complete Adjuvant (FCA)

• Mineral oil-based adjuvant (containing cell wall extract of mycobacterium

tuberculosis) in which the aqueous antigen solution is emulsified to form a
water-in-oil emulsion.

• It is thought to provide a good reservoir and also to activate the immune

system well
• However, it often cause unwanted side reactions at the injection site and, in
the case of cattle, causes the animal to appear positive when tested for
tuberculosis.
• Despite this, it is still used very often in laboratory animals because it
forms more or less the ‘golden standard’ for adjuvants: it provides the
upper range of effectiveness of adjuvants, it is well defined, and easy to
obtain and use.
• Thus all adjuvants normally fall in the range between aluminum hydroxide
(lower level) and FCA (upper level).
IMMUNIZATION PROCEDURE
• The number of vaccinations should be kept preferably one.

• However, even at best and, in contrast to ‘foreign’ antigens, vaccines

against ‘self need to be applied atleast twice and often many more times
to obtain full efficacy. However, recently effective one shot vaccines (for
GnRH in heifers and bulls and for PGF in heifers) have been developed (W.J. Enright,
personal communication) .
• Furthermore, recent data suggest that one shot vaccines may be possible by
using a mixture of slow release particles to deliver a primary and booster
vaccination at the same time. Fast ‘dissolving’ particles provide the primary
antigenic dose while slowly ‘dissolving’ particles can be timed to release their
contents when a booster immunization is required (Stevens, 1993).
EVALUATION OF EFFICACY
• In the case of immunocastration of male piglets, it was reported that ‘all’
male swine had fully regressed testicles using an LHRH vaccine (Meloen et
al.,1994).
• Using another LHRH vaccine, 80% of pregnancies of heifers were
prevented (Hoskinson et al., 1990).
• Reversible; i.e .the response disappears after a while (Ladd et al.1989).
Although this is desirable, as in the case of contraceptive vaccines in
humans, it also poses another serious problem, because reversibility is likely
to vary with each individual and, therefore, is difficult to control.
•  The experimental observations in the early 1970's indicates that
increased ovulation rates in sheep could be produced by immunization
against some oestrogens or androgens, opened up a completely new
area for improving reproductive efficiency.
(Cox et al. 1976; Scaramuzzi 1976; Scaramuzzi et al. 1977).
SOMATOSTATIN and ACTH

• Immunization has potential, no need of exog. Adm

• inc. GH and growth rate in lambs(Varner et al1980)

• Objective is growth promoting effect of I.N.

Roeder and Garber (USA)

Methodology

24 Cross bred steers with body

weight 335 kg

SS immunised group(SST) Control group(ssc)

SS + HSG HSG
Methodology

24 Cross bred steers with body

weight 337 kg

ACTH immunised group

(AT) Control group(AC)
ACTH + HSG HSG
Methodology

• Homobifunctional crosslinker glutaraldehyde

• Freund’s Adjuvent

• Injected at day 0,21,42,63,130 during 180 growing and finishing period.

RESULTS

• ADG 11% greater in SST than SSC

• Inc. in feed intake and feed efficiency does not differ

• AT group does not differ much from AC

• Active immunisation may improve growth performance

INHIBIN

• Objective examine the role of endogenous inhibin in the regulation of

FSH, LH, and testosterone secretion and sperm production in bulls.

• Bulls were actively immunized against bovine inhibin alpha 26 gly-tyr

(bINH) conjugated to human alpha globulin (HAG) or HAG alone
(controls) and emulsified in Freund’s complete adjuvant.

• Primary immunization was at 14 wk of age, followed by booster

immunizations in Freund’s incomplete adjuvant at 28. 30, and 34 wk of
age.

Martin et al (1991)
• Ten days after each booster immunization, scrotal circumferences and body
weights were measured, and blood was sampled for determination of blNH
antibody titer.

• Ten days after the third booster, blood was sampled at 1-h intervals for 8 h to
quantify serum concentrations of FSH, LH, and testosterone. After this blood
sampling period, bulls were castrated and testicular sperm production was
determined.
RESULTS
• Serum diluted 1:4000 from bINH-immunized bulls bound 36%, 52%, and 53% of
radioiodinated blNH after the first, second, and third boosters, respectively.
Serum from controls bound less than 1% radioiodinated bINH.

• After the third booster, serum concentrations of FSH and testosterone were
increased and LH concentrations were decreased in blNH-immunized bulls
compared with controls.
• After the third booster, daily sperm production per gram of testicular
parenchyma was increased in bINH-immunized bulls compared with
controls. Scrotal circumferences and body weights were similar between
treatment groups throughout the experiment.
• We concluded that inhibin has a role in regulation of secretion of
gonadotropins and testosterone and testicular sperm production, but
not testicular growth, in bulls.
• The results clearly indicate that the active immunization against
inhibin peptides can induce multiple ovulations in Malpura ewes and
its effect on multiple ovulations is sustained for a prolonged period of
time after the initial immunization.

SMK NAQVI ( NDRI )

• Active immunization against insulin BETA subunits affects sleep
variables, particularly slow-wave sleep during the dark period, increasing
significantly this stage of sleep and decreasing waking. Feeding behavior
and body weight remained unchanged.
• These results are compatible with previous studies showing a positive
correlation between decrease of insulin secretion and sleep
disturbances.

(Zara et al 1996)
THANKS