Slide 1: Scott Coonrod,
Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University Viral Particle-Based Display of Multiple Antigens for Companion Animal Immunosterilization Slide 2: Spike Trish Coonrod Slide 3: Immunocontraception – a reversible birth control method that uses the body's immune response to prevent pregnancy
Immmunosterilization – Use of immune system to permanently sterilize animal.
How does immunocontraception/immunosterilization work? Molecules from the reproductive axis that are essential for fertility are identified
Recombinant (synthetic) forms of these molecules are produced using bacterial or eukaryotic systems
Inject reproductive antigen into host animal to generate an immune reaction that either neutralizes antigen or removes cell type that produces the antigen Slide 4: FSH LH Estrogen GnRH Zona Pellucida
encased oocyte Primary target for immunocontraception: The hypothalamic-pituitary-gonadal axis Hypothalamus
Many other functions
Secretes FSH LH
which stimulates follicular
maturation and ovulation
Produces germ cells
Synthesizes estrogen and progesterone with estrogen feeding back and suppressing GnRH release Hypo Pit + - Slide 5: Antibody-based T-cell based Two main immunological approaches to removing fertility antigens from animal to induce contraception. Antibody removes target antigen (and possibly associated cells) by binding to the molecule rendering it functionally inactive. This technique can potentially be reversible once antibody titers go down T-cells (such as Macrophages)
attack and destroy cells containing
target antigen. Technique can be
permanent. Immune response can be pushed toward antibody (B-cell) or
T-cell based response depending on how the antigen is presented to the
immune system. Slide 6: Adaptive Immune Response Humoral (Antibody mediated) Cellular (T-cell mediated Extracellular antigens Intracellular antigens Helper
T-cells Neutralize antigen Antigen Presenting cells Pro-inflammatory
Cytokines Cytotoxins Phagocytize
infected cell Memory T and B cells Slide 7: GnRH Generation of antibody (humoral) response Inject reproductive
antigen formulation As long as high antibody titer (concentration)
is maintained animals are infertile Slide 8: GnRH Low antibody titer animals become
fertile again + FSH LH Generation of antibody (humoral) response Slide 9: Inject antigen
formulation Generation of T-cell mediated response GnRH Slide 10: X Hypothalamus X X X Generation of T-cell mediated response Good Not Good Slide 11: Cellular Response Hypo X X GnRH Antibody Response Ideal immunosterilant would be one that targets GnRH with antibodies (so you don't ablate the hypothalamus) and also targets the ovary with a T cell response (so you do ablate the germ cells). Slide 12: How would such a immunosterilant be produced
State of the art method for targeting GnRH will be described
in next talk
Here I will focus on some strong candidate ovarian antigens
for T cell targeting and also discuss how that antigens could be delivered. Slide 13: Tolerance, Regulatory T-cells (T Regs), and Autoimmunity Tolerance - The ability of the immune system to distinguish self from non-self and not mount a response to self antigens. Autoreactive T cells are deleted and autoreactive T cells that escape deletion are controlled by Tregs
Regulatory T-cells - Tregs prevent immune responses from becoming too strong by “training” immune system to tolerate self antigens.
Autoimmunity – Induced by an overactive immune response which mistakes certain self molecules as foreign pathogens. Tregs are “overwhelmed” by response and cannot prevent attack of tissues expressing these self antigens by the immune system.
During autoimmune response, proinflammatory T cells , macrophages, and proinflammatory cytokines (TNF, IL-1B, GM-CSF) can be activated leading to recruitment of inflammatory cells to self antigen expression tissues leading to tissue destruction. Proof of principle studies demonstrating that T-cell-mediated response targeting the ovary can render animals sterile : Proof of principle studies demonstrating that T-cell-mediated response targeting the ovary can render animals sterile Slide 15: Removal of thymus gland from 3 day old female mice (d3tx) resulted in potent and specific autoimmune responses directed at the ovary at sexual maturity leading to germ cell depletion and infertility. Murine autoimmune oophoritis (AOD) identifies germ cell antigens that induce potent sterilizing autoimmune response. Science Vol 166, 1969, pp 753 36 of 48 d3tx females were
sterile. Ovaries small and
atrophic Slide 16: Kenneth Tung Immunological Reviews Ovaries from day 3 thymectomy mice have heavy monocytic
(T cell) inflammation and are devoid of follicles and oocytes d3tx normal Slide 17: What is the cause of this potent autoimmune response. Removal of thymus gland at day 3 also removed resident Treg cells thus making female mice incapable of suppressing autoimmune attack when females became sexually mature.
d3tx phenotype caused by endogenous antigen(s) required for generation and maintenance of the ongoing autoimmune response.
Endogenous antigens that induce this potent germ-cell depleting immune response would likely make ideal contraceptive vaccine antigens. Slide 18: Ooplasm Zona Ovarian sections from normal mice
probed with serum from d3tx mice Number of d3tx mice with sera reactive to: Slide 19: Immunohistochemistry:
cross section of mouse
ovary probed with
pooled sera zona Western blot:mouse egg
proteins probed with sera
from 4 different mice oocyte Sera from day 3 thymectomized mice specifically reacts with
three different mouse oocyte proteins: MW ~ 76, 90, and 125kDA 1 2 3 4 mouse # Pascale et al, and Tung J. Immunology, 2001, 166: 4363-4369. Most reactive mouse
oocyte protein is 125 kDA Slide 21: MATER – Maternal Antigen That Embryos Require.
MATER is an oocyte and embryo abundant protein thatis required for oocyte-to embryo transition. Slide 22: MII GV MII Ovary Terminally
differentiated Totipotent – Maternal to Zygotic transition
Time dependent - 24 hours Transcriptional
arrest Maternal products Zygotic products The oocyte-to-embryo transition. Accumulation of
mRNA and protein Embryonic genome
reprogramming Slide 23: PADI6 Used proteomics to identify abundant oocyte-restricted factors in the mature mouse oocyte that may play a role in the OET
PADI6 – Peptidylarginine deiminase 6. Slide 24: PADI6 is maternally derived and expression is restricted to oocyte and preimplantation embryo. Levels Oocyte
maturation protein RNA Oocyte
development Slide 25: IIF Ph. GV Fertilized. Morula Blastocyst PADI6 subcellular localization in oocytes and early embryos. GV MII PN 2 cell 4 cell Mo. Bl. PI Western
Blot Slide 26: Characterization of the PADI6 -/- Phenotype. Alejandra Vitale/Piraye Yurttas Slide 27: A. B. 0 10 20 30 40 50 60 70 80 90 0 50 100 PADI6 -/- PADI6 +/+ % pronuclear development % development beyond 2 - cell * PADI6 -/- PADI6 +/+ PADI6 -/- developmental arrest occurs at
the two-cell stage. PADI6-null two-cell developmental arrest likely due to failure to activate embryonic genome. : PADI6-null two-cell developmental arrest likely due to failure to activate embryonic genome. MII GV MII Ovary Embryonic genome
reprogramming PADI6-null arrest MATER and PADI6-null two-cell developmental arrest likely due to failure to activate embryonic genome. : MATER and PADI6-null two-cell developmental arrest likely due to failure to activate embryonic genome. MII GV MII Ovary PADI6 and MATER-null arrest PADI6 MATER and PADI6 are part of the same
complex in mouse oocytes Slide 31: Is MATER expressed in companion animals. Dog ovary : MATER RNA expression is restricted to the ovary in the dog Dog ovary preimmune rabbit serum anti-mouse-MATER peptide antiserum PCR with MATER primers Slide 33: How can MATER, GnRH, and possibly other autoimmunogenic maternal genes be incorporated into an immunosterilant formulation for companion animals. Slide 34: Animal viruses:
Small packages of protein or membrane.
DNA or RNA genome.
Replicate only inside cells.
Damage host cells and tissues.
Many controlled by vaccines – induce host immunity. Canine parvovirus (CPV) : Canine parvovirus (CPV) Non-enveloped, 26nm diameter capsid
Single-stranded DNA genome ~5kb
2 genes – 4 overlapping proteins
Capsid - 60 copies of VP2 and VP1 (90% VP2; 10% VP1).
VP2 forms capsids; VP1 required for cell infection
Infects rapidly dividing cells
Evolved from feline panleukopenia virus
Binds the transferrin receptor
Infects young dogs and wild carnivores bigcatrescue.org
wolf.org Production of CPV virus-like particles (VLPs) : Production of CPV virus-like particles (VLPs) Contains only the VP2 capsid protein
Can be generated in mammalian or insect cells
Can bind and enter permissive cells (but are non-infectious)
Have similar stability and morphology to complete viruses w/o pathology Singh, P. et al. J Nanobiotech. 2006 Yuan and Parrish, Virology 2001 Slide 37: The CPV-derived VLPs present two opportunities to generate enhanced immune responses.
They are multivalent carriers: An incorporated antigen can be displayed as 60 copies/capsid with spacing sufficient to cross-link receptors on lymphocytes, thus effectively inducing activation.
Most dogs and cats of reproductive age have been exposed to CPV/FPV, thus existing immune surveillance system will mount a classical immunological memory response to the immunosterilant vaccine. Therefore, even though the specific attached antigen may not have been the target of an immune response before, this still results in an enhanced immune response. Slide 38: (Courtesy of C. Parrish) VLP containing GnRH and MATER epitopes. Model of VLP structure– GnRH and MATER peptides
could potentially be inserted at the loop sites. These
sites are antigenic and recognized by antibodies Final Immunosterilant Vaccine : Final Immunosterilant Vaccine A combination of GnRH-VPL and MATER-VLP
Would likely rapidly reduce GnRH levels, leading to reduced hormone levels and infertility
Would get slower cellular immune attack against MATER thus permanently eliminating oocytes and follicular cells Acknowledgements : Acknowledgements Baker Institute, Cornell University
Collin Parrish What if a dog or cat has not been exposed to CPV? : What if a dog or cat has not been exposed to CPV? Juvenile animals may not have been exposed to CPV infection or vaccination
For these, we may need to give a booster dose of vaccine after the initial dose
But ideally we would like a single-injection vaccine Slide 42: How does immunocontraceptive vaccination formulation “trick” immune system into destroying reproductive antigens.
Immunology 101. Innate immune response – A non-specific response to infection. Does not confer immunity to host. Provides immediate cellular defense against infection
Adaptive (acquired) immune response – A learned response by the immune system (B and T cells with defined antigenic specificity) whereby resistance to pathogen is achieved by previous exposure (vaccination). Slide 43: Previous companion animal immunocontraception research: Contraceptive vaccines targeting the Zona Pellucida oocyte antigens.
Native Porcine Zona Pellucida preparation.
Hamster (Mahi Brown)
Females immunized with soluble pZP generated antibodies reactive with hamster ZP.
Females became permanently infertile.
Ovarian follicles completely destroyed.
SpayVac is a contraceptive vaccine formulation containing native porcine ZP and CFA
packaged in liposomes
SpayVac induced long-term contraception in seals and deer (and other wildlife species).
In cats, SpayVac induced long-lasting high-titer anti-pZP antibodies but the antisera was
not reactive with feline ZP.
Likewise similar vaccines using native ZP isolated from a range of species did not generate
an immune response towards feline ZP and did not affect fertility. Slide 44: Dogs (Mahi Brown)
Immunization of bitches with crude and purified native pZP with CFA or Alum induced infertility, abnormal estrous cycles, follicular cysts and degenerating oocytes.
Recombinant ZP2 and ZP3
Studies by Tung showed that either B or T cell response can lead to either neutralizing response or oophoritis.
Immunization of female dogs with recombinant ZP2 and ZP3 conjugated to diphtheria toxin led to the generation of canine ZP-specific antibodies. All females that were immunized with ZP2 became pregnant while 3 of 4 females immunized with ZP3 did not conceive. Ovarian histology revealed inhibition of follicular development in treated animals
Several other studies using recombinant ZP proteins showed negative results.
Study in rabbits showed that while native porcine ZP induced infertility in rabbits when the porcine zona prep was deglycosylated the immunized females did not respond and were fertile. (Jones) Slide 45: GnRH GonaCon, synthetic GnRH bound to adjuvant
In swine and deer, infertility has lasted up to five years after a single treatment.
GonaCon induces GnRH antibodies in cats and a recent publication demonstrated the induction of GnRH antibodies in female dogs. However, problems with adjuvant mediated side effects. Slide 46: Ideal immunosterilant would be one that targets GnRH with an antibody response and the ovary with a T cell response Previous Research : Previous Research Slide 48: Summary
Results with GnRH suggest that, once optimized, immunization with this reproductive antigen could generate a long term B-cell mediated immunocontraceptive response (more later on this from Dr. Schiller)
Results with native zona pellucida preparations provide a proof of principle that immunization of animals with this reproductive antigen can generate both a B and T cell mediated response neutralizing the zona pellicida and also stimulating follicular degeneration /oophoritis
However, results from zona deglycosylation and recombinant zona studies suggest that carbohydrate configuration is important for immunoreactivity. Slide 49: The prospect of an immunological approach to contraception that would disrupt the process of fertilisation itself has resulted in a considerable interest into research in this area. It has been known for some time that antibodies raised against the zona pellucida (ZP) can suppress fertility very effectively. However, the initial optimism of this approach has been marred by the appearance of an ovarian pathology characterized by disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have been observed in the ovaries of mice after the induction of immunity with mouse ZP3 epitopes. However, this was associated with lymphocytic infiltration of the ovarian stroma, which could be circumvented by careful selection of B-cell epitopes to induce reversible infertility. In order to identify similar epitopes on primate ZP3, epitope-mapping studies were performed and incorporated into chimeric vaccines that included a promiscuous T-helper cell epitope. Both single and triple peptide vaccines have been evaluated in vivo and no detrimental effects on ovarian function were observed. The resulting high titre antibodies bound exclusively to the ZP of marmoset and human ovarian sections and could suppress in vitro human sperm-egg binding by approximately 60%, but did not prevent pregnancy in actively immunised female marmosets. Thus, considerable research is still required to identify a combination of ZP3 epitopes that will induce infertility free of any unwanted side effects. Immunocontraception with Zona pellucida Proteins: Review Aitken, Cells Tissue Organs 166 Human Immunocontraception Research: Persistent problem with the immune system becoming “overstimulated” generating a T-cell mediated inflammatory response targeting ovary.
What's bad for people may be good for companion animal sterilization!!