No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display
Abstract
Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5×10(11) virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2×10(12) vir per mouse in combination with a poly(lactide- co -glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems.
... Phage virions are genetically engineered to display GnRH-like peptides as fusions to the phage coat proteins. 12 Phages are highly immunogenic due to their particulate nature and nanoscale size. Methods for engineering phage-peptide constructs are well established and easy to reproduce. ...
... 16,17 Recently, we have developed several phage-based vaccines against GnRH that were tested in male mice and demonstrated promising contraceptive properties. 12 These proof of principle results in mice warranted the next logical step in evaluating the vaccines in the target species, cats. Consequently, the objectives of the study were focused on evaluation of the vaccines for immune responses and effects on reproductive parameters (testosterone, semen production and quality, testicle size and histology) in cats. ...
... GnRH antibodies were detected by ELISA using a synthetic GnRH peptide (New England Peptide) bi 879814 otinylated at C terminus (NH 2 -EHWSYGLRPG-Lysbiotin-CONH 2 ) to serve as the detector molecule. 12 Cat serum was diluted 1:100 and then tested at two-fold dilutions to endpoint titers. The endpoint titer was defined as the highest dilution providing an optical density twice that of the pre-immune sera. ...
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.
... Roehnisch et al. [37] showed that anti-phage antibody responses reached their peak at the maximum tested dose of 5x10 11 virions per mouse. Immunization of mice with a dose of 2x10 12 virions per animal resulted in a signifi-cant increase in serum antibodies against peptides displayed on phage as compared to the response of mice inoculated with 5x10 11 virions each [66]. ...
... Also a possibility is that phage immunogenicity can be improved via optimization of vaccine administration routes. Only a few studies compared delivery routes for particular phage applications [50,66]. Extensive side-by-side studies are needed to draw objective conclusions about preferable administration routes for phage-based vaccines. ...
... Samoylov et al. [66] developed multiple phage constructs displaying GnRH-like peptides and tested them in mice. Recombinant phages stimulated production of neutralizing anti-GnRH antibodies resulting in suppressed serum testosterone. ...
Background:
Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines.
Objective:
In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals.
Results:
Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines.
Conclusion:
Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents.
... An effective approach involves displaying tumor-specific antigens on the phage surface, which can be recognized and destroyed by immune cells. Animal studies have elucidated the way that phage-based vaccines can generate potent and durable antitumor immune responses [71][72][73]. In addition to their efficacy, phages are safe, cost-effective, and easy to produce, making them a feasible option for large-scale vaccine development and distribution [74,75]. ...
... In another study, phage display was used to identify mimotopes of gonadotropinreleasing hormones (GnRH) that stimulated the production of therapeutic antibodies. In mice, immunization with phages displaying the selected GnRH mimotope induced longlasting humoral responses and decreased testosterone production, which indicate that they may have the potential to treat hormone-sensitive cancers [73]. ...
Bacteriophages have emerged as versatile tools in the field of bioengineering, with enormous potential in tissue engineering, vaccine development, and immunotherapy. The genetic makeup of phages can be harnessed for the development of novel DNA vaccines and antigen display systems, as they can provide a highly organized and repetitive presentation of antigens to immune cells. Bacteriophages have opened new possibilities for the targeting of specific molecular determinants of cancer cells. Phages can be used as anticancer agents and carriers of imaging molecules and therapeutics. In this review, we explored the role of bacteriophages and bacteriophage engineering in targeted cancer therapy. The question of how the engineered bacteriophages can interact with the biological and immunological systems is emphasized to comprehend the underlying mechanism of phage use in cancer immunotherapy. The effectiveness of phage display technology in identifying high-affinity ligands for substrates, such as cancer cells and tumor-associated molecules, and the emerging field of phage engineering and its potential in the development of effective cancer treatments are discussed. We also highlight phage usage in clinical trials as well as the related patents. This review provides a new insight into engineered phage-based cancer vaccines.
... While phage penetration studies have been inconsistent, the evidence for phage entry into the bloodstream and systemic tissues is consistent in nearly every animal model and administration route. Administration routes considered to date include: oral (Delmastro et al. 1997;March et al. 2004;Zuercher et al. 2004), subcutaneous (Grabowska et al. 2000;Wu et al. 2002), intramuscular (Acton and Evans 1968;Clark and March 2004;Samoylov et al. 2015), intravenous (Inchley 1969), and intradermal injection (Roehnisch et al. 2013). Animal models studied to date include: mice (Samoylov et al. 2015;Willis et al. 1993;Wu et al. 2002), rabbits March et al. 2004), pigs (Zuercher et al. 2004), non-human primates (Chen et al. 2001;Rao et al. 2004;Trouche et al. 2009), as well human in phase I/II clinical trials for the treatment of patients with multiple myeloma (Roehnisch et al. 2013(Roehnisch et al. , 2014. ...
... Administration routes considered to date include: oral (Delmastro et al. 1997;March et al. 2004;Zuercher et al. 2004), subcutaneous (Grabowska et al. 2000;Wu et al. 2002), intramuscular (Acton and Evans 1968;Clark and March 2004;Samoylov et al. 2015), intravenous (Inchley 1969), and intradermal injection (Roehnisch et al. 2013). Animal models studied to date include: mice (Samoylov et al. 2015;Willis et al. 1993;Wu et al. 2002), rabbits March et al. 2004), pigs (Zuercher et al. 2004), non-human primates (Chen et al. 2001;Rao et al. 2004;Trouche et al. 2009), as well human in phase I/II clinical trials for the treatment of patients with multiple myeloma (Roehnisch et al. 2013(Roehnisch et al. , 2014. This section will consider the specific immune responses that inactivate phage, specifically focusing on innate responses clearing phage and the generation of specific antibodies against phage antigens. ...
Bacteriophages (phages) are well-established bacteria-specific viruses whose discovery is credited to the independent and nearly simultaneous works of Twort (1915) and d’Hérelle (1917) (Summers 1999) in the early 20th century. Each of the researchers characterized phages as the pathogens of bacteria following the hint of much phage-like phenomena from the 19th and 20th centuries. The late 1930s and early 1940s represented the most significant era for phage research and its impact on biological research (Abedon and Thomas-Abedon 2010), including the research by the “Phage Group”. This group included the work of Max Delbrück and other highly notable geneticists, including James Watson and Francis Crick (Abedon 2012). The group quickly established that phage could be used for the treatment of bacterial infections, since called “phage therapy”, and were so named. “Bacteriophage” translates to “bacteria eaters”.
... While phage penetration studies have been inconsistent, the evidence for phage entry into the bloodstream and systemic tissues is consistent in nearly every animal model and administration route. Administration routes considered to date include: oral (Delmastro et al. 1997;March et al. 2004;Zuercher et al. 2004), subcutaneous (Grabowska et al. 2000;Wu et al. 2002), intramuscular (Acton and Evans 1968;Clark et al. 2011;Clark and March 2004;Samoylov et al. 2015), intravenous (Inchley 1969), and intradermal injection (Roehnisch et al. 2013). Animal models studied to date include: mice (Samoylov et al. 2015;Willis et al. 1993;Wu et al. 2002), rabbits (Clark et al. 2011;March et al. 2004), pigs (Zuercher et al. 2004), non-human primates (Chen et al. 2001;Rao et al. 2004;Trouche et al. 2009), as well human in phase I/II clinical trials for the treatment of patients with multiple myeloma (Roehnisch et al. 2013(Roehnisch et al. , 2014. ...
... Administration routes considered to date include: oral (Delmastro et al. 1997;March et al. 2004;Zuercher et al. 2004), subcutaneous (Grabowska et al. 2000;Wu et al. 2002), intramuscular (Acton and Evans 1968;Clark et al. 2011;Clark and March 2004;Samoylov et al. 2015), intravenous (Inchley 1969), and intradermal injection (Roehnisch et al. 2013). Animal models studied to date include: mice (Samoylov et al. 2015;Willis et al. 1993;Wu et al. 2002), rabbits (Clark et al. 2011;March et al. 2004), pigs (Zuercher et al. 2004), non-human primates (Chen et al. 2001;Rao et al. 2004;Trouche et al. 2009), as well human in phase I/II clinical trials for the treatment of patients with multiple myeloma (Roehnisch et al. 2013(Roehnisch et al. , 2014. This section will consider the specific immune responses that inactivate phage, specifically focusing on innate responses clearing phage and the generation of specific antibodies against phage antigens. ...
While the natural hosts for bacteriophages are bacteria, there is growing evidence for the ability of phage to interact with mammalian cells, particularly with those of the human immune system. These interactions typically encompass two main features: (i) phage immunogenicity, or ability of phages to induce specific immune responses; and (ii) phage immunomodulation, which can be defined as the ability of phages to modify the immune system in both innate and adaptive responses. The aim of this chapter is to explore the interactions between phages and the immune system, and more specifically the implications of these interactions in the development of novel medical applications.
... It was proposed that this vaccine could inactivate endogenous gonadotropin-releasing hormones, thereby regulating their release and, by extension, fertility. 198 Bacteriophage-particle vaccines have been developed for viral infections, such as Zika, influenza A, and Norwalk, and bacterial diseases, such as brucellosis, anthrax, and bubonic plague. 104 Phage virus-like particles (VLPs) have also presented the human papillomavirus (HPV) L2 epitope, leading to the development of HPV-neutralizing antibodies. ...
The effective delivery of therapeutic agents is as important as the active therapeutic agent because it may make or mar the outcome. Traditional/conventional drug delivery systems (DDSs) face many limitations, including poor bioavailability, poor specificity and targeting, inconsistent drug adsorption, short half-life, rapid drug clearance, instability, varying and suboptimal drug effects, uncomfortable administration, limited drug loading into delivery vehicles, and poor treatment adherence and compliance, opening the way for innovations, including optimising the use of nanocarriers. Nanocarriers encapsulate and deliver drug agents using nanosized vehicles to enhance drug effectiveness, bioavailability as well as specific, targeted, and controlled drug release. Examples of such nanocarriers include micelles, microemulsions, nanoemulsions, lipid nanoparticles, liposomes, niosomes, dendrimers, and exosomes, which are formulated using different vehicles. Virus-like nanoparticles are emerging, with most involving bacteriophages—the environmentally ubiquitous, abundant, and diverse group of nanosized structurally simple viral particles with the intrinsic and inherent ability to invade bacteria. Studies involving different bacteriophages, their nano drug formulation, and their application against some diseases exist; however, no current review aggregates the advances made so far, which could be attributed to the recency of the research areas. Such a review is vital because it highlights and precipitates milestones and can provide necessary basis for further advancements. Thus, this study aims to evaluate the advancing potential use of bacteriophages as nanocarriers for targeted drug delivery and their potential for enhanced therapeutic effects.
... This method has been used for many years. The target of this immunization can be GnRH [1][2][3], zona pellucida-3 (ZP3), or a combination of GnRH and ZP3 [4]. Zona pellucida, a glycoprotein that covers oocytes, especially ZP3, is a potential target of immunocontraception [5]. ...
A series of studies have explored the immunocontraceptive potential of zona pellucida-3 in local Indonesian goats. Goat zona pellucida-3 (gZP3) proteins have been identified and isolated. It has 82 kDa molecular weight and comprises 63.47% of the total goat zona pellucida protein. Immunofluorescence staining showed that gZP3 protein recognized goat sperm plasma membrane, whereas gZP3 antibodies recognized goat zona pellucida in native smear. In vitro fertilization study showed that gZP3 protein-supplemented in sperm capacitation medium and gZP3 antibody supplemented in oocyte maturation medium inhibited fertilization. It could be concluded that gZP3 was a fertilization receptor in a goat. Elisa and dot blot analysis showed that gZP3 protein was recognized by the serum of fertile unpregnant female mice. Therefore, it could be hypothesized that the gZP3 protein might be immunocontraceptive in mice. Immunization of mice with gZP3 proved that gZP3 prevented pregnancy and was dose-dependent. The contraceptive action of gZP3 was reversible after 14 estrous cycles. In vitro fertilization technique, it was proved that the antibody of gZP3 was able to prevent fertilization and decrease sperm binding rate on mice ova. The gZP3 nucleotide sequence has a homology at nearly 50% of the monkey ZP3 sequence (Macaca sp) but is foreign phylogenetically. The gZP3 protein has the potential to be developed as an immunocontraceptive substance for overpopulated monkeys to prevent agriculture disturbance in areas near forestry.
... GnRH has been used in vaccine formulations, which limit reproduction and decrease sex hormone synthesis in various mammalian species, including cats, over lengthy periods [155]. A phage-based vaccine to control fertility in cats was selected from an f8-8 landscape phage display library with neutralizing GnRH antibodies [44]. The cats vaccinated with phage-GnRH produced high levels of antibodies against GnRH, decreasing their serum testosterone levels and reducing their total testicular volume, indicating gonadal atrophy. ...
Because vaccine development is a difficult process, this study reviews aspects of phages as vaccine delivery vehicles through a literature search. The results demonstrated that because phages have adjuvant properties and are safe for humans and animals, they are an excellent vaccine tool for protein and epitope immunization. The phage genome can easily be manipulated to display antigens or create DNA vaccines. Additionally, they are easy to produce on a large scale, which lowers their manufacturing costs. They are stable under various conditions, which can facilitate their transport and storage. However, no medicine regulatory agency has yet authorized phage-based vaccines despite the considerable preclinical data confirming their benefits. The skeptical perspective of phages should be overcome because humans encounter bacteriophages in their environment all the time without suffering adverse effects. The lack of clinical trials, endotoxin contamination, phage composition, and long-term negative effects are some obstacles preventing the development of phage vaccines. However, their prospects should be promising because phages are safe in clinical trials; they have been authorized as a food additive to avoid food contamination and approved for emergency use in phage therapy against difficult-to-treat antibiotic-resistant bacteria. Therefore, this encourages the use of phages in vaccines.
... In contrast, one of the phage constructs after immunization of mice (with a dose of 2 × 10 12 virions per mouse) in combination with poly(lactide-co-glycolide) (PLGA) adjuvant was responsible for a multiple increase in the production of GnRH antibodies and a significant reduction in serum testosterone levels. The studies confirmed that the antibodies generated in response to phage-GnRH immunization had neutralizing properties [85]. Further research of the above phage constructs revealed their contraceptive properties in vaccinated (sexually mature male) cats. ...
The constantly growing number of people suffering from bacterial, viral, or fungal infections, parasitic diseases, and cancers prompts the search for innovative methods of disease prevention and treatment, especially based on vaccines and targeted therapy. An additional problem is the global threat to humanity resulting from the increasing resistance of bacteria to commonly used antibiotics. Conventional vaccines based on bacteria or viruses are common and are generally effective in preventing and controlling various infectious diseases in humans. However, there are problems with the stability of these vaccines, their transport, targeted delivery, safe use, and side effects. In this context, experimental phage therapy based on viruses replicating in bacterial cells currently offers a chance for a breakthrough in the treatment of bacterial infections. Phages are not infectious and pathogenic to eukaryotic cells and do not cause diseases in human body. Furthermore, bacterial viruses are sufficient immuno-stimulators with potential adjuvant abilities, easy to transport, and store. They can also be produced on a large scale with cost reduction. In recent years, they have also provided an ideal platform for the design and production of phage-based vaccines to induce protective host immune responses. The most promising in this group are phage-displayed vaccines, allowing for the display of immunogenic peptides or proteins on the phage surfaces, or phage DNA vaccines responsible for expression of target genes (encoding protective antigens) incorporated into the phage genome. Phage vaccines inducing the production of specific antibodies may in the future protect us against infectious diseases and constitute an effective immune tool to fight cancer. Moreover, personalized phage therapy can represent the greatest medical achievement that saves lives. This review demonstrates the latest advances and developments in the use of phage vaccines to prevent human infectious diseases; phage-based therapy, including clinical trials; and personalized treatment adapted to the patient’s needs and the type of bacterial infection. It highlights the advantages and disadvantages of experimental phage therapy and, at the same time, indicates its great potential in the treatment of various diseases, especially those resistant to commonly used antibiotics. All the analyses performed look at the rich history and development of phage therapy over the past 100 years.
... Immunization of mice with EHPSYGLA-phage, even at the highest doses, resulted in no adverse effect. While the main purpose of this research group remains animal contraception, an approach similar to the one described in this paper-as suggested by the authors themselves-might be useful in the treatment of human hormone-sensitive cancers of the reproductive trait [105]. ...
Phage display is a nanotechnology with limitless potential, first developed in 1985 and still awaiting to reach its peak. Awarded in 2018 with the Nobel Prize for Chemistry, the method allows the isolation of high-affinity ligands for diverse substrates, ranging from recombinant proteins to cells, organs, even whole organisms. Personalized therapeutic approaches, particularly in oncology, depend on the identification of new, unique, and functional targets that phage display, through its various declinations, can certainly provide. A fast-evolving branch in cancer research, immunotherapy is now experiencing a second youth after being overlooked for years; indeed, many reports support the concept of immunotherapy as the only non-surgical cure for cancer, at least in some settings. In this review, we describe literature reports on the application of peptide phage display to cancer immunotherapy. In particular, we discuss three main outcomes of this procedure: (i) phage display-derived peptides that mimic cancer antigens (mimotopes) and (ii) antigen-carrying phage particles, both as prophylactic and/or therapeutic vaccines, and (iii) phage display-derived peptides as small-molecule effectors of immune cell functions. Preclinical studies demonstrate the efficacy and vast potential of these nanosized tools, and their clinical application is on the way.
... 13,14 Development of a DNA vaccine to generate antibodies to prevent GnRH signaling is challenging due to its weak immumogeniciy. 11 To overcome the limitation, various strategies have been employed, including coupling of GnRH to ligands such as heat shock protein 65, 12 keyhole limpet hemocyanin 13 and stronger T helper cell epitopes. 14 Similarly, we attempted to elongate the length of time the antigen was present within the animal by developing a hydrogel-based pDNA nanoparticulate vaccine. ...
... GnRHR antibodies were detected by ELISA using a synthetic 29 amino acid peptide (AnaSpec, Fremont, CA, USA) representing N-terminal extracellular domain of the feline GnRH receptor protein (MASAPPEQNQNHCSAINNSI-PLMQGNLP-K-Biotin) to serve as the fGnRHR antibody detector molecule. The step-by-step protocol was described previously [27]. To measure serum testosterone, a Mouse and Rat Testosterone ELISA kit (ALPCO, Salem, NH, USA) was used as directed by the manufacturer. ...
Overpopulation of free-roaming and wildlife animals negatively affects economy and public health in many parts of the world. Contraceptive vaccines are viewed as a valuable option for reducing numbers of unwanted animals. This study develops vaccines for potential use in animal contraception exploiting a DNA platform. Objectives of the study were to generate DNA constructs directed against gonadotropin-releasing hormone receptor (GnRHR), a crucial molecular player in animal reproduction, and characterize them for ability to promote immune responses and suppression of reproductive parameters in vivo. DNA constructs were created to encode for a recombinant protein composed of two domains: GnRHR, the target antigen, and ubiquitin (Ub), a support protein. Ub-GnRHR constructs administered intramuscularly or intradermally or containing different promoters were compared. CMV and EF1α promoters were shown to be superior to CAG. In fertility trials, mice immunized intradermally with Ub-GnRHR construct driven by EF1α had a significantly lower number of fetuses. Importantly, the impaired fertility was achieved with a single DNA immunization and without the use of adjuvants. The study demonstrated for the first time that targeting the GnRH receptor with DNA-based vaccines could be a viable option for animal contraception.
... For comprehensive reviews on phage-based pathogen biosensors, one can be referred to [21,58]. Data on the performance of landscape-based interfaces in different biosensor platforms are summarized in Table 2. Streptavidin [49] Antibodies against gonadotropin-releasing hormone (GnRH) in patient sera [76] Lyme disease patient sera [77] Free prostate-specific antigen (f-PSA) 0.16 ng/mL, 0.825-165 ng/mL [78] Total prostate-specific antigen (t-PSA) 1.6 ng/mL, 3-330 ng/mL [79] Differential pulse voltammetry (DPV) analytical platform ...
The development of phage engineering technology has led to the construction of a novel type of phage display library—a collection of nanofiber materials with diverse molecular landscapes accommodated on the surface of phage particles. These new nanomaterials, called the “landscape phage”, serve as a huge resource of diagnostic/detection probes and versatile construction materials for the preparation of phage-functionalized biosensors and phage-targeted nanomedicines. Landscape-phage-derived probes interact with biological threat agents and generate detectable signals as a part of robust and inexpensive molecular recognition interfaces introduced in mobile detection devices. The use of landscape-phage-based interfaces may greatly improve the sensitivity, selectivity, robustness, and longevity of these devices. In another area of bioengineering, landscape-phage technology has facilitated the development and testing of targeted nanomedicines. The development of high-throughput phage selection methods resulted in the discovery of a variety of cancer cell-associated phages and phage proteins demonstrating natural proficiency to self-assemble into various drug- and gene-targeting nanovehicles. The application of this new “phage-programmed-nanomedicines” concept led to the development of a number of cancer cell-targeting nanomedicine platforms, which demonstrated anticancer efficacy in both in vitro and in vivo experiments. This review was prepared to attract the attention of chemical scientists and bioengineers seeking to develop functionalized nanomaterials and use them in different areas of bioscience, medicine, and engineering.
... The findings of that study revealed the vaccine inhibited ovarian function, but also altered reproductive behaviours that are integral to the maintenance of the complex social structure of herd animals such as horses. - Eidne et al. (2000), Meloen et al. (2000), Aitken (2006), Samoylova et al. (2010Samoylova et al. ( , 2012Samoylova et al. ( , 2012Samoylova et al. ( , 2015 Proteins found on spermatozoa, oocyte and blastocyst ...
Feral horses are a significant pest species in many parts of the world, contributing to land erosion, weed dispersal and the loss of native flora and fauna. There is an urgent need to modify feral horse management strategies to achieve public acceptance and long-term population control. One way to achieve this is by using non-surgical methods of sterilisation, which are suitable in the context of this mobile and long-lived species. In this review we consider the benefits of implementing novel mechanisms designed to elicit a state of permanent sterility (including redox cycling to generate oxidative stress in the gonad, random peptide phage display to target non-renewable germ cells and the generation of autoantibodies against proteins essential for conception via covalent modification) compared with that of traditional immunocontraceptive approaches. The need for a better understanding of mare folliculogenesis and conception factors, including maternal recognition of pregnancy, is also reviewed because they hold considerable potential in providing a non-surgical mechanism for sterilisation. In conclusion, the authors contend that non-surgical measures that are single shot and irreversible may provide a sustainable and effective strategy for feral horse control.
Purpose: To compare a novel, sustained release formulation and a bolus injection of a targeted nanocarrier for the ability to specifically deplete cells responsible for the development of posterior capsule opacification (PCO) in week-long, dynamic cell cultures. Methods: A novel, injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymer hydrogel was engineered for the sustained release of targeted, nucleic acid nanocarriers loaded with cytotoxic doxorubicin (G8:3DNA:Dox). Human rhabdomyosarcoma (RD) cells were used due to their expression of brain-specific angiogenesis inhibitor 1 (BAI1), a specific marker for the myofibroblasts responsible for PCO. Under constant media flow, nanocarriers were injected into cell cultures as either a bolus or within the hydrogel. Cells were fixed and stained every other day for 7 days to compare targeted depletion of BAI1+ cells. Results: The formulation transitions to a gel at physiological temperatures, is optically clear, noncytotoxic, and can release G8:3DNA:Dox nanocarriers for up to 4 weeks. In RD cell cultures, G8:3DNA:Dox nanocarriers specifically eliminated BAI1+ cells. The bolus nanocarrier dose showed significantly reduced cell depletion overtime, while the sustained release of nanocarriers showed increased cell depletion over time. By day 7, <2% of BAI1+ cells were depleted by the bolus injection and 74.2% BAI1+ cells were targeted by the sustained release of nanocarriers. Conclusions: The sustained release of nanocarriers from the hydrogel allows for improved therapeutic delivery in a dynamic system. This method can offer a more effective and efficient method of prophylactically treating PCO after cataract surgery.
Polyester–polyether block copolymers with thermogelling properties have been widely used in pharmaceutical and biomedical applications for their biocompatibility and degradation to nontoxic components. The biodegradable polymers, such as poly(lactide‐co‐glycolide) and poly(lactide‐co‐caprolactone) (PLCL), have been used as the polyester block. The most commonly used polyether has been poly(ethylene glycol) (PEG). The thermogelling polymers dissolve in cold water, but become a semisolid gel at higher temperatures, for example, body temperature. This thermogelling property, however, occurs only within very narrow molecular weight ranges of the polyester at given polyether blocks. Only a few hundred Dalton differences in the polyester block can turn a thermogelling polymer into a water‐insoluble polymer. This study describes the hydrophilic and hydrophobic block sizes for endowing the thermogelling property and a simple, water‐free, synthesis of new thermogelling PLCL–PEG–PLCL triblock polymers. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48673.
Nanomedicine is the application of nanotechnology in medicine. This review is focused on the use of both lytic and temperate bacteriophages (phages) nanoparticles in nanomedicine, in particular, in the context of developing nano-probes for precise disease diagnosis and nano-therapeutics for targeted disease treatment. Phages as bacteria-specific viruses do not naturally infect eukaryotic cells and are not toxic to them. They not only can be genetically engineered to bear the capability of targeting nanoparticles, cells, tissues, and organs, but also can be integrated with functional abiotic nanomaterials to gain physical properties that enable disease diagnosis and treatment. Therefore, they are ideal for many applications in precision nanomedicine. This review will summarize the current use of the great diversity of phage structures in many aspects of precision nanomedicine including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nano-therapeutics for targeted disease treatment. It will also propose future directions in the area of phage-based nanomedicines as well as the state of phage-based clinical trials.
Nanomedicine is the application of nanotechnology in medicine. This review is focused on the use of both lytic and temperate bacteriophages (phages) nanoparticles in nanomedicine, in particular, in the context of developing nano-probes for precise disease diagnosis and nano-therapeutics for targeted disease treatment. Phages as bacteria-specific viruses do not naturally infect eukaryotic cells and are not toxic to them. They not only can be genetically engineered to bear the capability of targeting nanoparticles, cells, tissues, and organs, but also can be integrated with functional abiotic nanomaterials to gain physical properties that enable disease diagnosis and treatment. Therefore, they are ideal for many applications in precision nanomedicine. This review will summarize the current use of the great diversity of phage structures in many aspects of precision nanomedicine including ultrasensitive biomarker detection, enhanced bioimaging for disease diagnosis, targeted drug and gene delivery, directed stem cell differentiation, accelerated tissue formation, effective vaccination, and nano-therapeutics for targeted disease treatment. It will also propose future directions in the area of phage-based nanomedicines as well as the state of phage-based clinical trials.
A fundamental understanding of the in vivo biodegradation phenomenon as well as an appreciation of cellular and tissue responses which determine the biocompatibility of biodegradable PLA and PLGA microspheres are important components in the design and development of biodegradable microspheres containing bioactive agents for therapeutic application. This chapter is a critical review of biodegradation, biocompatibility and tissue/material interactions, and selected examples of PLA and PLGA microsphere controlled release systems. Emphasis is placed on polymer and microsphere characteristics which modulate the degradation behaviour and the foreign body reaction to the microspheres. Selected examples presented in the chapter include microspheres incorporating bone morphogenetic protein (BMP) and leuprorelin acetate as well as applications or interactions with the eye, central nervous system, and lymphoid tissue and their relevance to vaccine development. A subsection on nanoparticles and nanospheres is also included. The chapter emphasizes biodegradation and biocompatibility; bioactive agent release characteristics of various systems have not been included except where significant biodegradation and biocompatibility information have been provided.
Background
Multiple myeloma is characterized by clonal expansion of B cells producing monoclonal immunoglobulins or fragments thereof, which can be detected in the serum and/or urine and are ideal target antigens for patient-specific immunotherapies.
Methods
Using phage particles as immunological carriers, we employed a novel chemically linked idiotype vaccine in a clinical phase I/II trial including 15 patients with advanced multiple myeloma. Vaccines composed of purified paraproteins linked to phage were manufactured successfully for each patient. Patients received six intradermal immunizations with phage idiotype vaccines in three different dose groups.
Results
Phage idiotype was well tolerated by all study participants. A subset of patients (80% in the middle dose group) displayed a clinical response indicated by decrease or stabilization of paraprotein levels. Patients exhibiting a clinical response to phage vaccines also raised idiotype-specific immunoglobulins. Induction of a cellular immune response was demonstrated by a cytotoxicity assay and delayed type hypersensitivity tests.
Conclusion
We present a simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible patient-specific therapy for patients with advanced multiple myeloma and produced promising anti-tumor activity in a subset of patients.
The goal of this study was to improve the pharmacokinetic properties and specificity of an ERBB2-targeted peptide for SPECT imaging.
Bacteriophages (phages) displaying the ERBB2 targeting sequence, KCCYSL, flanked by additional random amino acids were used for in vivo selections in mice-bearing ERBB2-expressing MDA-MB-435 human breast xenografts. Phage-displayed peptides were evaluated for ERBB2 and cancer cell binding affinity and specificity in vitro, and one peptide was radiolabeled with (111)In-DOTA and biodistribution and SPECT imaging properties were compared to the first generation peptide, (111)In-DOTA-KCCYSL.
In vivo phage display selected two peptides, 1-D03 (MEGPSKCCYSLALSH) and 3-G03 (SGTKSKCCYSLRRSS), with higher breast carcinoma cell specificity and similar ErbB2 affinity (236 and 289 nM, respectively) to the first generation peptide. The corresponding radiolabeled probes bound with higher affinity to target cancer cells than (111)In-DOTA-KCCYSL; however, only (111)In-DOTA-1-D03 demonstrated higher specificity for MDA-MB-435 cells. Biodistribution analysis demonstrated that although (111)In-DOTA-1-D03 had slightly reduced tumor uptake (0.661 % ID/g) in comparison to (111)In-DOTA-KCCYSL (0.78 %/ID/g), its dramatic improvement in blood clearance led to a significantly higher tumor/blood ratio (6.02:1). Non-specific uptake was also reduced in most organs including heart, lung, muscle, bone, and kidneys. SPECT imaging revealed tumor-specific uptake of (111)In-DOTA-1-D03, which was confirmed by blocking with unlabeled 1-D03 peptide.
This is the first evidence that SPECT imaging peptides with improved tumor specificity and pharmacokinetics can be obtained by in vivo phage display affinity maturation. The combination of ERBB2-specific binding, rapid clearance, and tumor specificity may make 1-D03 a viable candidate for clinical imaging studies.
Thirty goats were randomly allocated in five groups of six animals each, for immunization with 1 × 10(14) phage particles of clones 11, 13, and 13 with Quil A adjuvant and wild-type M13KE phage at the beginning and 4 weeks later. The control group received phosphate-buffered saline. All groups were challenged with 200 metacercariae at week 6 and slaughtered 14 weeks later. The mean worm burdens after challenge were reduced by 46.91 % and 79.53 % in goats vaccinated with clones 13 and 13 with Quil A (P < 0.05), respectively; no effect was observed in animals immunized with clone 11 and M13KE phage. Animals receiving clones 11, 13, and 13 with Quil A showed a significant reduction in eggs output. Vaccinated animals produced parasite-specific total IgG antibody which were boosted after challenge with metacercariae of F. hepatica. Furthermore, levels of anti-phage total IgG increased rapidly within 2 weeks of the first vaccination and were always significantly higher in all vaccinated goats than in the infected control group. The fluke burden of goats immunized with clones 13 and 13 with Quil A was significantly correlated with IgG2 and total IgG. Goats vaccinated with phage clones produced significantly high titres of IgG1 and IgG2 antibodies indicating a mixed Th1/Th2 response. These data indicate that cathepsin L1 mimotopes has a potential as a vaccine candidate against Fasciola hepatica, whose efficacy will be evaluated in other host species, including those of veterinary importance.
B cell malignancies are characterized by clonal expansion of B cells expressing tumor-specific idiotypes on their surface. These idiotypes are ideal target antigens for an individualized immunotherapy. However, previous idiotype vaccines mostly lacked efficiency due to a low immunogenicity of the idiotype. The objective of the present study was the determination of the feasibility, safety and immunogenicity of a novel chemically linked phage idiotype vaccine.
In the murine B cell lymphoma 1 model, tumor idiotypes were chemically linked to phage particles used as immunological carriers. For comparison, the idiotype was genetically expressed on the major phage coat protein g8 or linked to keyhole limpet hemocynanin. After intradermal immunizations with idiotype vaccines, tolerability and humoral immune responses were assessed.
Feasibility and tolerability of the chemically linked phage idiotype vaccine was demonstrated. Vaccination with B cell lymphoma 1 idiotype expressing phage resulted in a significant survival benefit in the murine B cell lymphoma 1 protection model (60.2 +/- 23.8 days vs. 41.8 +/- 1.6 days and 39.8 +/- 3.8 days after vaccination with wild type phage or phosphate buffered saline, respectively). Superior immunogenicity of the chemically linked phage idiotype vaccine compared to the genetically engineered phage idiotype and keyhole limpet hemocynanin-coupled idiotype vaccine was demonstrated by significantly higher B cell lymphoma 1 idiotype-specific IgG levels after vaccination with chemically linked phage idiotype.
We present a novel, simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible therapy and may produce a superior immune response compared to previously employed idiotype vaccination strategies.
Antibodies to the epitope EFRH, representing residues 3-6 within the -amyloid (A) sequence, were previously shown to affect the solubility and disaggregation of A fibrils in vitro. Here, we describe a novel method of immunization, using as antigen the EFRH peptide displayed on the surface of the filamentous phage. The EFRH phage evoked effective auto-immune antibodies in amyloid precursor protein (V717I) (APP(V717I)) transgenic mice that recapitulate the amyloid plaques and vascular pathology of Alzheimer's disease (AD). The immunization provoked a considerable reduction in the number of A amyloid plaques in the brain of the transgenic mice and may serve as the basis for anti-A vaccine. © 2002 Elsevier Science Ltd. All rights reserved.
The synthetic peptide GK-1, derived from Taenia crassiceps, enhances the protection induced by human influenza vaccine in both young and aged mice. Herein, the adjuvant properties of GK-1 fused to the pVIII protein of a heat-inactivated phagemid vector (FGK1) when co-administered with the influenza vaccine were assessed, to evaluate its feasibility as a low-cost adjuvant. In mice, FGK1 significantly increased the expected IgG and IgA anti-influenza antibody levels both in sera and in bronchoalveolar fluids when intranasally or subcutaneously co-administered with influenza vaccine. Single-dose pig co-immunization with FGK1 and influenza vaccine induced serum levels of IgG anti-influenza antibodies similar to those elicited by a two-dose immunization with the influenza vaccine alone. Preclinical evaluation of FGK1 with the influenza vaccine is currently in progress, in order to recommend its use for veterinary purposes.
Peptides displayed on the surface of filamentous bacteriophage fd are able to induce humoral as well as cell-mediated immune responses, which makes phage particles an attractive antigen delivery system to design new vaccines. The immune response induced by phage-displayed peptides can be enhanced by targeting phage particles to the professional antigen presenting cells, utilizing a single-chain antibody fragment that binds dendritic cell receptor DEC-205. Here, we review recent advances in the use of filamentous phage fd as a platform for peptide vaccines, with a special focus on the use of phage fd as an antigen delivery platform for peptide vaccines in Alzheimer's Disease and cancer.
A fundamental understanding of the in vivo biodegradation phenomenon as well as an appreciation of cellular and tissue responses which determine the biocompatibility of biodegradable PLA and PLGA microspheres are important components in the design and development of biodegradable microspheres containing bioactive agents for therapeutic application. This chapter is a critical review of biodegradation, biocompatibility and tissue/material interactions, and selected examples of PLA and PLGA microsphere controlled release systems. Emphasis is placed on polymer and microsphere characteristics which modulate the degradation behaviour and the foreign body reaction to the microspheres. Selected examples presented in the chapter include microspheres incorporating bone morphogenetic protein (BMP) and leuprorelin acetate as well as applications or interactions with the eye, central nervous system, and lymphoid tissue and their relevance to vaccine development. A subsection on nanoparticles and nanospheres is also included. The chapter emphasizes biodegradation and biocompatibility; bioactive agent release characteristics of various systems have not been included except where significant biodegradation and biocompatibility information have been provided.
Endotoxin contamination of protein solutions was reduced by a phase separation technique using the detergent, Triton X-114. Protein solutions containing endotoxin were treated with Triton X-114 on ice. The solution was then warmed to 37°C, whereupon two phases formed. The Triton X-114 phase, containing the endotoxin, was precipitated by centrifugation. The first cycle of phase separation produced a 1000-fold reduction of endotoxin from contaminated preparations of cytochrome c, catalase and albumin. Complete removal of endotoxin could be achieved by further cycles of phase separation. Each cycle of phase separation resulted in only a 2% loss of protein, and could be completed within 15 min. The small amount of detergent (0.018%) that persisted in protein solution could be removed by gel filtration or absorption. Proteins treated by this procedure retained normal functions. This phase separation technique provides a rapid and gentle method for removing endotoxin from protein solutions.
We recently reported that administration of tumor-specific bacteriophages initiates infiltration of neutrophilic granulocytes with subsequent regression of established B16 tumors. The aim of the current study was to investigate the mechanism of action of bacteriophage-induced tumor regression and to examine possible stimulatory effects of bacteriophages on macrophages. We observed that the mechanism of phage-induced tumor regression is TLR dependent as no signs of tumor destruction or neutrophil infiltration were observed in tumors in MyD88(-/-) mice in which TLR signaling is abolished. The microenvironment of bacteriophage-treated tumors was further analyzed by gene profiling through applying a low-density array preferentially designed to detect genes expressed by activated APCs, which demonstrated that the M2-polarized tumor microenvironment switched to a more M1-polarized milieu following phage treatment. Bacteriophage stimulation induced secretion of proinflammatory cytokines in both normal mouse macrophages and tumor-associated macrophages (TAMs) and increased expression of molecules involved in Ag presentation and costimulation. Furthermore, mouse neutrophils selectively migrated toward mediators secreted by bacteriophage-stimulated TAMs. Under these conditions, the neutrophils also exhibited increased cytotoxicity toward B16 mouse melanoma target cells. These results describe a close interplay of the innate immune system in which bacteriophages, located to the tumor microenvironment due to their specificity, stimulate TAMs to secrete factors that promote recruitment of neutrophils and potentiate neutrophil-mediated tumor destruction.
A billion-clone library of filamentous phage with different surface structures (‘landscapes’) was generated by fusing random
octapeptides to the N-terminus of all 4000 copies of the major coat protein. Such a ‘landscape library’ might include clones
exhibiting emergent properties that inhere in the entire surface architecture, not in the peptides by themselves. Because
the diverse surface landscapes are displayed on viable phage, they can be surveyed for exceedingly rare functions using microbiological
selection methods. Clones with several emergent properties of the sort envisioned were successfully selected, suggesting that
landscape libraries have promise as a novel source of nanomaterials with exploitable surface properties.
Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.
D17DT consists of the GnRH decapeptide linked to diphtheria toxoid. The aim of this pilot study was to assess the tolerance of D17DT and the production of anti-GnRH antibodies from two doses, 30 and 100 microg, in patients with locally advanced prostate cancer. Twelve patients with histologically proven prostate cancer in whom hormonal therapy was indicated were recruited. Patients received either 30 or 100 microg given intramuscularly on three separate occasions over six weeks. Patients were followed up and blood was taken for estimation of serum testosterone, PSA and anti-GnRH antibody titre. Overall the drug was well tolerated. In 5 patients a significant reduction in serum testosterone and PSA was seen. Castrate levels of testosterone were achieved in 4 and maintained for up to 9 months. Patients with the highest antibody titre had the best response in terms of testosterone suppression. This study shows that it is possible to immunize a patient with prostate cancer against GnRH to induce castrate levels of testosterone. This state appears to be reversible. This novel form of immunotherapy may have advantages over conventional forms of hormonal therapy and further studies are warranted in order to try and increase the proportion of responders.
Peri- and postpubertal boars accumulate substances (e.g., androstenone and skatole) in their fatty tissue that are responsible for boar taint in pork. The objective of this study was to assess the efficacy of a GnRH vaccine, Improvac, in eliminating boar taint. Three hundred male (200 intact boars, 100 barrows) crossbred (Large White x Landrace) pigs were used in a 2 x 3 factorially arranged experiment. The respective factors were sex group (barrows, boars treated with placebo, or boars treated with Improvac) and slaughter age (23 or 26 wk). Vaccines were administered 8 and 4 wk before slaughter. All Improvac-treated pigs exhibited anti-GnRH titers. Testes and bulbo-urethral gland weights in treated pigs were reduced by approximately 50% (P < 0.001) and serum testosterone levels were below 2 ng/mL in the majority of treated boars (94 and 92% across both age groups at 2 and 4 wk, respectively). Boar taint, as assessed by the concentration of androstenone and skatole in s.c. fat, was suppressed to low or undetectable levels in 100% of Improvac-treated boars. No Improvac-treated pigs had significant concentrations of either androstenone (> 1.0 microg/g) or skatole (> 0.20 microg/g). In contrast, 49.5% of placebo-treated controls had significant androstenone and 10.8% had significant skatole levels, resulting in 10% of the control boars with high concentrations of both compounds. The mean concentrations of taint compounds in the Improvac-treated pigs were not significantly different from those in barrows. Improvac-treated boars grew more rapidly (P = 0.051 and < 0.001 for pigs slaughtered at 23 and 26 wk of age, respectively) than control boars over the 4 wk after the secondary vaccination, possibly because of reduced sexual and aggressive activities. Compared with barrows, Improvac-treated boars were leaner and had superior feed conversion efficiency. The vaccine was well tolerated by the pigs, and no observable site reactions could be detected at the time of slaughter. Vaccination of boars with Improvac allows production of heavy boars with improved meat quality through prevention and control of boar taint.
Biodegradable microspheres (MS) consisting of poly(D,L-lactide-co-glycolide) (PLGA) represent a promising alternative to conventional adjuvants. The adjustable pulsatile release of encapsulated material from such MS offers the potential to mimic the priming and boosting injections of conventional immunization regimens. In this paper, we demonstrate that MS can serve as antigen reservoirs in antigen presenting cells (APC), so that antigen is presented for extended periods of time (up to 9 days). In particular, we could show by measurement of IFN-gamma production that encapsulated peptides were presented to cytotoxic T lymphocytes (CTL) by mouse and human macrophages as well as by human dendritic cells in vitro for a longer time period as compared to soluble peptides. The extended antigen presentation may thus improve the CTL response in vivo. These results may be of paramount importance in cancer vaccination therapy since MS may serve as antigen reservoirs to extend the presentation time by APC used to boost the patient's immune response to tumor antigens.
The development of non-surgical contraceptives for cats may facilitate population control of the species. The purpose of this study was to investigate the utility of GnRH for immunocontraception of male cats. Male cats (n ¼ 12) were divided into groups of three and were immunized once with 0 (sham), 50, 200, or 400 mg synthetic GnRH coupled to keyhole limpet hemocyanin and combined with a mycobacterial adjuvant to enhance immunogenicity. GnRH antibody titer, serum testosterone concentration, and scrotal size were determined monthly. At 6 months, semen was collected by electroejaculation and testes were examined histologically. GnRH antibodies were detected in all cats receiving GnRH vaccine by 1 month post-treatment and persisted throughout the study. No dose effect of GnRH was observed; titers were not different among cats treated with 50, 200, or 400 mg GnRH (P ¼ 0:5). Six of nine treated cats were classified as responders based on high GnRH antibody titers (>32,000). By 3 months post-treatment, responder cats had undetectable testosterone concentrations and testicular atrophy. Non-responder cats had GnRH titers of 4000–32,000 and testosterone concentrations intermediate between responder and sham-treated cats. At 6 months, total sperm counts were similar for sham-treated cats (3:1 _ 1:8 _ 106 sperm) and non-responder cats (3:4 _ 1:6 _ 106 sperm; P ¼ 0:7). Only one of the six responder cats produced sperm, none of which were motile. Combined testicular weights of responder cats (1:3 _ 0:1 g) were lower than sham-treated controls (5:3 _ 1:3 g; P ¼ 0:02) and non-responder cats (2:9 _ 0:3 g; P ¼ 0:02). Histologic evaluation of the testes revealed that in responder cats, the interstitial cells that were present were pale and shrunken compared to the plump, polyhedral eosinophilic cells in sham-treated cats.
Immunocastration is a considerable alternative to a surgical castration method especially in male animal species for alleviating unwanted male behaviors and characteristics. Induction of high titer of antibody specific for gonadotropin-releasing hormone (GnRH) correlates with the regression of testes. Fusion proteins composed of canine GnRH and T helper (Th) cell epitope p35 originated from canine distemper virus (CDV) F protein and goat rotavirus VP6 protein were produced in E. coli. When these fusion proteins were injected to male dogs which were previously immunized with CDV vaccine, the fusion protein of GnRH-CDV Th cell epitope p35 induced much higher antibody than that of GnRH-rotavirus VP6 protein or GnRH alone. The degeneration of spermatogenesis was also verified in the male dogs immunized with the fusion protein of GnRH-CDV Th cell epitope p35. These results indicate that canine GnRH conjugated to CDV Th cell epitope p35 acted as a strong immunogen and the antibody to GnRH specifically neutralized GnRH in the testes. This study also implies a potential application of GnRH-based vaccines for immunocastration of male pets.
The application of combinatorial approaches in conjunction with phage display techniques might be critical for development of vaccines against various infective and cancer diseases. Phage technique allows the generation of novel immunogens representing structural/molecular mimics of pathogen-derived immunodominant epitopes, or protein domains displayed on phages capable of inducing protective antibodies, or construction of novel vaccines based on incorporation of antigenic/genetic variability of pathogens or cancer cells in the context of phage particles. The diversity of applications and success of phage display are due to its simplicity and flexibility along with the possibilities of very cheap large-scale production of phage particles by recovering them from infected bacterial culture supernatants as nearly 100% homogenous preparations. Phages are easy to manage, they resist heat and many organic solvents, chemicals, or other stresses, and, importantly, phage particles are highly immunogenic and do not require adjuvant. Furthermore, phages do not require the cold chain (requirement to store vaccines at refrigerated or frozen temperatures), which equates to lower transport and storage costs. Considering these points, recombinant phages should be viewed as promising vaccine discovery tools and vaccine delivery vectors, and it is worth even considering the possibility of replacing the delivery systems of known vaccines currently in use with phage particles as vaccine carriers. The chapter outlines the current advances in phage vaccine development and analyses possible advances of phages as engineered immunogens.
Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.
The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18–38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.
Unlabelled:
Vaccines administered parenterally have been developed against gonadotrophin-releasing hormone (GnRH) for anti-fertility and anti-cancer purposes. The aim of this study was to demonstrate whether mucosal delivery using GnRH immunogens entrapped in lipid nanoparticles (LNP) could induce anti-GnRH antibody titers. Immunogens consisting of KLH (keyhole limpet hemocyanin) conjugated to either GnRH-I or GnRH-III analogues were entrapped in LNP. Loaded non-ionic surfactant vesicles (NISVs) were administered subcutaneously, while nasal delivery was achieved using NISV in xanthan gum and oral delivery using NISV containing deoxycholate (bilosomes). NISV and bilosomes had similar properties: they were spherical, in the nanometre size range, with a slightly negative zeta potential and surface properties that changed with protein loading and inclusion of xanthan gum. Following immunization in female BALB/c mice, systemic antibody responses were similar for both GnRH-I and GnRH-III immunization. Only nasal delivery proved to be successful in terms of producing systemic and mucosal antibodies.
From the clinical editor:
The main research question addressed in this study was whether mucosal delivery using gonadotrophin-releasing hormone immunogens entrapped in lipid nanoparticles could induce anti-GnRH antibody titers. Only nasal delivery proved to be successful in terms of producing systemic and mucosal antibodies with this approach.
Stimuli triggered polymers provides a variety of applications related with the biomedical fields. Among various stimuli triggered mechanisms, thermoresponsive have been extensively investigated, as they are relatively more convenient and effective stimuli for biomedical applications. In contemporary for achieving the sustained action of proteins, peptides and bioactives, injectable depots and implants have been always remain the thrust area of research. In the same series, Poloxamer based thermogelling copolymers have its own limitation regarding its biodegradability. Thus, there is a need to have an alternative biomaterial for formulation of injectable hydrogel, which must remain biocompatible along with safety and efficacy. In the same context, Poly (ethylene glycol) (PEG) based copolymers plays a crucial role as a biomedical material for biomedical applications, because of its biocompatibility, biodegradability, thermosensitivity and easy controlled characters. This review, stresses on the physicochemical property, stability and compositions prospects of smart PEG/ poly (lactic-co-glycolic acid) (PLGA) based thermoresponsive injectable hydrogels, recently utilized for biomedical applications. Manuscript also highlights synthesis scheme and stability characteristics of these copolymers, which will surely help the researchers working in same area. We have also emphasized the applied use of these smart copolymers along with their formulation problems, which could help to understand the possible modifications related with these, to overcome their inherent associated limitations.
Raising gonad-intact farm animals has many advantages over conventional castrates. Intact animals are generally more efficient in the conversion of feed into lean meat. In addition to decreasing production costs, better feed efficiency also leads to a reduction in the output of various biological pollutants, particularly nitrogen. Moreover, traditional surgical castration is perceived by many people to be ethically questionable. However, intact animals tend to cause problems, mostly in terms of management for cattle, mostly in terms of meat quality for pigs. Among the various methods envisaged as alternatives to surgical castration, the present review is focused on active immunisation against gonadotrophin-releasing hormone (GnRH). Pilot studies have demonstrated that, providing a good immune response is achieved, this technique is very efficient in preventing aggressive and sexual behaviour in bulls, oestrus behaviour in heifers and boar taint in male pigs. Performance of the immunocastrated animals are generally intermediates between those of intact and surgically castrated animals. Immunisation against GnRH appears to be a very attractive approach to castration of farm animals. However, these results still need to be confirmed with sufficient animal numbers. In addition, much work remains to be done to try to reduce animal variation in response to active immunisation and to reduce the adverse side effects associated with traditional adjuvants. The problems related to the immunocastration of male pigs should be easier to solve than those for cattle, because only short -term biological responses are required.
Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of (439)QAAG(442) or (443)AVYT(446) of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.
Multiple phage-peptide constructs, where the peptides mimic sperm epitopes that bind to zona pellucida (ZP) proteins, were generated via selection from a phage display library using a novel approach. Selections were designed to allow for identification of ZP-binding phage clones with potential species-specific properties, an important feature for wildlife oral vaccines as the goal is to control overpopulation of a target species while not affecting non-target species' reproduction. Six phage-peptide antigens were injected intramuscularly into pigs and corresponding immune responses evaluated. Administration of the antigens into pigs stimulated production of anti-peptide antibodies, which were shown to act as anti-sperm antibodies. Potentially, such anti-sperm antibodies could interfere with sperm delivery or function in the male or female genital tract, leading to contraceptive effects. Staining of semen samples collected from different mammalian species, including pig, cat, dog, bull, and mouse, with anti-sera from pigs immunized with ZP-binding phage allowed identification of phage-peptide constructs with different levels of species specificity. Based on the intensity of the immune responses and specificity of these responses in different species, two of the antigens with fusion peptide sequences GEGGYGSHD and GQQGLNGDS were recognized as the most promising candidates for development of contraceptive vaccines for wild pigs.
Problem An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations.
Method of study Two GnRH conjugates, GonaCon™ (GnRH–KLH) and GonaCon-B™ (GnRH–blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH–KLH protein conjugate was lyophilized and suspended in AdjuVac™ adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments.
Results The longevity of the contraceptive response (2–5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine.
Conclusion One-injection and two-injection formulations of GonaCon™ and GonaCon-B™ produced multi-year contraception in adult female white-tailed deer. GonaCon-B™ provided a longer lasting contraceptive effect.
The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines.
With the exception of the provision of clean water supplies, vaccination remains the most successful public health intervention strategy for the control of infectious diseases. However, the logistics of delivering at least two to three doses of vaccines to achieve protective immunity are complex and compliance is frequently inadequate, particularly in developing countries. In addition, newly developed purified subunit and synthetic vaccines are often poorly immunogenic and need to be administered with potent vaccine adjuvants. Microparticles prepared from the biodegradable and biocompatible polymers, the poly(lactide-co-glycolides) or (PLG), have been shown to be effective adjuvants for a number of antigens. Moreover, PLG microparticles can control the rate of release of entrapped antigens and therefore, offer potential for the development of single-dose vaccines. To prepare single-dose vaccines, microparticles with different antigen release rates may be combined as a single formulation to mimic the timing of the administration of booster doses of vaccine. If necessary, adjuvants may also be entrapped within the microparticles or, alternatively, they may be co-administered. The major problems which may restrict the development of microparticles as single-dose vaccines include the instability of vaccine antigens during microencapsulation, during storage of the microparticles and during hydration of the microparticles following in vivo administration. In the present review, we discuss the adjuvant effect of PLG microparticles, and also their potential for the development of single-dose vaccines through the use of controlled-release technology.
The efficacy of a new vaccine-delivery vector, based on the filamentous bacteriophage fd displaying a single-chain antibody fragment known to bind the mouse DC surface molecule DEC-205, is reported. We demonstrate both in vitro and in vivo an enhanced receptor-mediated uptake of phage particles expressing the anti-DEC-205 fragment by DCs. We also report that DCs targeted by fd virions in the absence of other stimuli produce IFN-α and IL-6, and acquire a mature phenotype. Moreover, DC-targeting with fd particles double-displaying the anti-DEC-205 fragment on the pIII protein and the OVA(257-264) antigenic determinant on the pVIII protein induced potent inhibition of the growth of the B16-OVA tumor in vivo. This protection was much stronger than other immunization strategies and similar to that induced by adoptively transferred DCs. Since targeting DEC-205 in the absence of DC activation/maturation agents has previously been described to result in tolerance, the ability of fd bacteriophages to induce a strong tumor-specific immune response by targeting DCs through DEC-205 is unexpected, and further validates the potential employment of this safe, versatile and inexpensive delivery system for vaccine formulation.
Gonadotrophin-releasing hormone (GnRH) is the prime decapeptide hormone in the regulation of mammalian reproduction. Active immunization against GnRH has been a good treatment option to fight against hormone-dependent disease such as breast cancer. We designed and purified a novel protein vaccine Hsp65-GnRH(6) containing heat shock protein 65 (Hsp65) and six copies of GnRH in linear alignment. Immunization with Hsp65-GnRH(6) evoked strong humoral response in female mice. The generation of specific anti-GnRH antibodies was detected by ELISA and verified by western blot. In addition, anti-GnRH antibodies effectively neutralized endogenous GnRH activity in vivo, as demonstrated by the degeneration of the ovaries and uteri in the vaccinated mice. Moreover, the growth of EMT-6 mammary tumor allografts was inhibited by anti-GnRH antibodies. Histological examinations have shown that there was increased focal necrosis in tumors. Taken together, our results showed that immunization with Hsp65-GnRH(6) elicited high titer of specific anti-GnRH antibodies and further led to atrophy of reproductive organs. The specific antibodies could inhibit the growth of EMT-6 murine mammary tumor probably via an indirect mechanism that includes the depletion of estrogen. In view of these results, the protein vaccine Hsp65-GnRH(6) appears to be a promising candidate vaccine for hormone-dependent cancer therapy.
Phage display is based on expressing peptides as a fusion to one of the phage coat proteins. To date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. In recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as DNA delivery vehicles to mammalian cells. In this study, recombinant filamentous phage whole particles were used for vaccination of mice. BALB/c mice were inoculated with filamentous phage particles containing expression cassette of Herpes simplex virus 1 (HSV-1) glycoprotein D that has essential roles in the virus attachment and entry. Both humoral and cellular immune responses were measured in the immunized mice and compared to conventional DNA vaccination. A dose-response relationship was observed in both arms of immune responses induced by recombinant filamentous phage inoculation. The results were similar to those from DNA vaccination. Filamentous phages can be considered as suitable alternative candidate vaccines because of easier and more cost-effective production and purification over plasmid DNA or bacteriophage lambda particles.
A random constrained hexapeptide phage display library (Cys-6aa-Cys) was screened with purified neutralizing human anti-rabies virus IgG antibodies (hRABVIgG) to identify peptides that correspond to or mimic natural epitopes on rabies virus glycoprotein (RABVG) and to investigate their immunogenicities in vivo. After four rounds of biopanning, 20 phage clones randomly selected for their specificity to hRABVIgG, effectively blocked the binding of the inactive rabies virus (RABV) to hRABVIgG. The phage clones were sequenced and the deduced amino acid sequences were derived (C-KRDSTW-C; C-KYLWSK-C; C-KYWLSR-C; C-KYWWSK-C; C-KYAWSR-C; C-KYSMSK-C). Alignments to the amino acid sequence of RABVG showed good match with the antigenic site III (at 330-338 aa), indicating that the hRABVIgG antibodies most likely recognize preferentially this antigenic site. The selected mimotopes were able to inhibit the interactions of the hRABVIgG antibodies with RABV in a dose-dependent manner. Subcutaneous administration of phageKRDSTW expressing the RABVG site III mimotope induced an RABVG-specific IgG response in BALB/c mice. The results indicated that peptide mimotopes when displayed on phages, are accessible to the mice immune system to trigger a humoral response and to induce IgG production. The RABVG site III mimotope (C-KRDSTW-C) would provide a new and promising concept for the development of rabies vaccine.
We have examined the range of mucosal and systemic immune responses induced by oral or parenteral immunization with ovalbumin (OVA) entrapped in poly(D,L-lactide-co-glycolide) (PLG) microparticles. A single subcutaneous immunization with OVA-PLG primed significant OVA-specific IgG and delayed-type hypersensitivity (DTH) responses. The DTH responses were of similar magnitude to those obtained using immunostimulating complexes (ISCOMS) as a potent control adjuvant, although ISCOMS stimulated higher serum IgG responses. Both vectors also primed OVA-specific in vitro proliferative responses in draining lymph node cells following a single immunization and strong OVA-specific CTL responses were found after intraperitoneal (i.p.) immunization. ISCOMS were more efficient in inducing cytotoxic T lymphocytes (CTL), requiring much less antigen and only ISCOMS could stimulate primary OVA-specific CTL responses in the draining lymph nodes. Multiple oral immunizations with OVA in PLG microparticles or in ISCOMS resulted in OVA-specific CTL responses and again ISCOMS seemed more potent as fewer feeds were necessary. Lastly, multiple feeds of OVA in PLG microparticles generated significant OVA-specific intestinal IgA responses. This is the first demonstration that PLG microparticles can stimulate CTL responses in vivo and our results highlight their ability to prime a variety of systemic and mucosal immune responses which may be useful in future oral vaccine development.
We sequenced the complementary DNA (cDNA) encoding guinea pig LHRH from an expression library derived from the preoptic area-anterior hypothalamus. Data from in situ hybridization and RNase protection assays verified that the cloned cDNA was complementary to guinea pig LHRH messenger RNA. The architecture of the deduced precursor resembles that of LHRH precursors identified in other species. In contrast, the predicted sequence of the decapeptide differs from mammalian LHRH by two amino acid substitutions in positions 2 and 7. This is a novel finding, because the amino acid sequence that comprises LHRH decapeptide is identical in all mammals studied to date. Moreover, the predicted substitution in amino acid position 2 is unique among vertebrates. A second observation of potential significance is the existence of two subspecies of LHRH messenger RNA differing only in the length of their 3' untranslated regions. These two transcripts were verified by sequence analysis of positive clones from the cDNA library and by RNase protection analysis of preoptic area-anterior hypothalamus extracts, and their presence is consistent with the two polyadenylation signals identified in the untranslated regions of the LHRH gene. Future studies will examine LHRH gene expression in guinea pigs, which like primates but unlike rats, have a true luteal phase as a component of their reproductive cycle.
Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.
Active immunization against gonadotropin-releasing hormone (GnRH) was recognized in the 1970s as a potential means by which the reproductive system of mammals might be shut down for various practical and clinical reasons. Numerous studies in males have been performed since that time to determine the applicability of the technique as an alternative to surgical removal of the testes. Reasons for such immunocastration include improvement of meat and carcass characteristics for cattle, sheep, goats, and swine; improvement in feed efficiency relative to castrates in those same species; reduction in male aggressive behavior; reduction in male-associated odors in goats and swine; and fertility neutralization in pet species. Although application as a fertility control agent in men is unlikely, there is renewed interest in active immunization against GnRH as a means of treating prostate cancers and related steroid-dependent pathologies.
Immunocastration targeting gonadotropin releasing hormone (GnRH) can be obtained in male piglets using native GnRH conjugates. However, due to insufficient efficacy of these conjugates, improved GnRH antigens, like peptides existing of repeats of the GnRH amino acid sequence, have been designed. We previously reported about a dimerised GnRH-tandem peptide with a D-Lys at position 6 of the native GnRH sequence (G6k-TD) being highly effective. To evaluate the contribution of each individual amino acid of the GnRH decapeptide to the efficacy of the G6k-TD peptide, each amino acid was replaced consecutively by alanine (Ala-scan). The G6k-TD peptides were conjugated to ovalbumin, used for immunisation and tested for their ability to elicit GnRH antibodies and to immunocastrate male piglets. The results show that four out of nine amino acids (pGlu-1, Ser-4, Arg-8 and Gly-10) can be replaced by alanine without negatively affecting immunocastration efficacy. Replacement of amino acids in other positions (Tyr-5, Leu-7 and Pro-9) gave partial decrease of efficacy, respectively, five, six and six out of seven piglets were immunocastrated. Replacements at two other positions (His-2 and Trp-3) completely negated immunocastration activity. Thus, seven out of nine amino acid positions in the basic unit of G6k-TD can be substituted by alanine without affecting immunocastration efficacy.
The follicle-stimulating hormone is one of the two pituitary hormones that control fertility in both sexes. In the male, receptors for FSH (FSHR) are only expressed on testicular Sertoli cells. FSH plays different roles during the male life; it functions as a growth factor during development and sustains spermatogenesis in adults. However, the exact role of this hormone as an initiator of male fertility is not fully understood and few data are available concerning its involvement during the peripubertal period. We recently produced filamentous phages displaying FSHR fragments overlapping residues 18-38, which, if injected in animals, induced anti-FSH receptor immunity capable of inhibiting hormone binding. We employed this strategy to transiently inhibit FSH activity in male mice and male goats of the Saanen and the Mongolian Alpas Cashmere breeds at the prepubertal stage. Anti-FSHR peptide immunization from the age of 3 wk delayed the acquisition of fecundity in male mice by up to 1 wk. Once fertile, progeny sizes produced by mating immunized males and untreated females were found to be reduced by up to 60%. In two different breeds of goats, FSHR peptide vaccines were able to maintain circulating testosterone at low prepubertal levels for several months despite no alteration in LH levels, reflecting their ability to delay the onset of puberty. These results support the conclusion that FSH may play a central role in the male at puberty through the control of testosterone production.
Antibodies to the epitope EFRH, representing residues 3-6 within the beta-amyloid (Abeta) sequence, were previously shown to affect the solubility and disaggregation of Abeta fibrils in vitro. Here, we describe a novel method of immunization, using as antigen the EFRH peptide displayed on the surface of the filamentous phage. The EFRH phage evoked effective auto-immune antibodies in amyloid precursor protein [V717I] (APP[V717I]) transgenic mice that recapitulate the amyloid plaques and vascular pathology of Alzheimer's disease (AD). The immunization provoked a considerable reduction in the number of Abeta amyloid plaques in the brain of the transgenic mice and may serve as the basis for anti-Abeta vaccine.
Virions of filamentous bacteriophage fd are capable of displaying multiple copies of peptide epitopes and generating powerful immune responses to them. To investigate the antigen processing mechanisms in human B cell lines used as antigen presenting cells, the major coat protein (pVIII) in intact virions was fluorescently labeled, and its localization in various intracellular compartments was followed using confocal microscopy. We show that the virions were taken up and processed to yield peptides that reach both the major histocompatibility complex (MHC) class II compartment and the endoplasmic reticulum. Moreover, when exposed to bacteriophages displaying a cytotoxic T lymphocyte (CTL) epitope from the reverse transcriptase of human immunodeficiency virus type-1 (HIV-1), B cells were lysed by specific cytotoxic lymphocytes. This confirms that filamentous bacteriophage virions are capable of being taken up and processed efficiently by MHC class I and class II pathways, even in nonprofessional antigen presenting cells. These remarkable features explain, at least in part, the unexpected ability of virions displaying foreign T-cell epitopes to prime strong T-helper-dependent CTL responses. These findings have important implications for the development of peptide-based vaccines, using filamentous bacteriophage virions as scaffolds.
Two non-pathogenic scaffolds (represented by the filamentous bacteriophage fd and the dihydrolipoyl acetyltransferase E2 protein of the Bacillus stearothermophilus pyruvate dehydrogenase (PDH) complex) able to deliver human immunodeficiency virus (HIV)-1 antigenic determinants, were designed in our laboratories and investigated in controlled assay conditions. Based on a modification of the phage display technology, we developed an innovative concept for a safe and inexpensive vaccine in which conserved antigenic determinants of HIV-1 reverse transcriptase (RTase) were inserted into the N-terminal region of the major pVIII coat protein of bacteriophagefd virions. Analogously, we developed another antigen delivery system based on the E2 component from the PDH complex and capable of displaying large intact proteins on the surface of an icosahedral lattice. Our data show that both of these systems can deliver B and T epitopes to their respective presentation compartments in target cells and trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo.
Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.
A modified GnRH peptide (CHWSYGLRPG-NH2) was conjugated to tetanus toxoid and formulated with different adjuvants (non-ionic surfactant vesicles, aluminium hydroxide, Quil A, PLGA (poly(lactide-co-glycolide)/triacetin), and Quil A/PLGA). A comparison of the anti-fertility efficacy of the formulations was made by examining specific antibody levels, antibody subclasses, endocrine ablation and gonadal atrophy. The production of IgG2b antibody provided the best correlation for castration. PLGA was considered the most effective adjuvant as it produced a consistent anti-fertility response in all the treated animals.
B lymphocyte stimulator (BLyS) is a tumor necrosis factor (TNF) family member and a key regulator of B cell responses. We employed a phage display-based approach to identify peptides that bind BLyS with high selectivity and affinity. Sequence analysis of first-generation BLyS-binding peptides revealed two dominant peptide motifs, including one containing a conserved DxLT sequence. Selected linear peptides with this motif were found to bind BLyS with K(D) values of 1-3 microM. In order to improve the binding affinity for BLyS, consensus residues flanking the DxLT sequence were seeded into a second-generation, BLyS affinity maturation library (BAML). BAML phage were subjected to stringent binding competition conditions to select for isolates expressing high-affinity peptide ligands for BLyS. Post-selection analysis of BAML peptide sequences resulted in the identification of a core decapeptide motif (WYDPLTKLWL). Peptides containing this core motif exhibited K(D) values as low as 26 nM, approximately 100-fold lower than that of first-generation peptides. A fluorescence anisotropy assay was developed to monitor the protein-protein interaction between BLyS labeled with a ruthenium chelate, and TACI-Fc, a soluble form of a BLyS receptor. Using this assay it was found that a BAML peptide disrupts this high-affinity protein-protein interaction. This demonstrates the potential of short peptides for disruption of high affinity cytokine-receptor interactions.
Injectable biodegradable polymeric particles (usually microspheres) represent an exciting approach to control the release of vaccine antigens to reduce the number of doses in the immunization schedule and optimize the desired immune response via selective targeting of antigen to antigen presenting cells. After the first couple of decades of their study, much progress has been made towards the clinical use of antigen-loaded microspheres. Poly(lactide-co-glycolic acids) (PLGAs) have been studied most commonly for this purpose because of their proven safety record and established use in marketed products for controlled delivery of several peptide drugs. PLGA microspheres have many desirable features relative to standard aluminum-based adjuvants, including the microspheres' ability to induce cell-mediated immunity, a necessary requirement for emergent vaccines against HIV and cancer. This review examines several impediments to PLGA microparticle development, such as PLGA-encapsulated antigen instability and deficiency of animal models in predicting human response, and describes new trends in overcoming these important issues. PLGA microparticles have displayed unprecedented versatility and safety to accomplish release of one or multiple antigens of varying physical-chemical characteristics and immunologic requirements, and have now met numerous critical benchmarks in development of long-lasting immunity after a single injected dose.
A major challenge in vaccine design has been to identify antigen presentation systems that elicit strong T- and B-cell responses. In the authors' laboratory, two new delivery vehicles derived from nonpathogenic prokaryotic organisms were recently designed and investigated. Conserved antigenic determinants were inserted into the N-terminal region of the major pVIII coat protein of bacteriophage fd virions or on the surface of an icosahedral scaffold formed by the acyltransferase component (E2 protein) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus. The data indicate that the antigenic determinant displayed by either fd virions or on the surface of the E2 lattice are accessible to the immune system, and are able to trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo. These systems offer the potential for safe and inexpensive vaccines to elicit full-spectrum immune responses.
In this study we describe our attempts to improve the immunogenicity of a synthetic epitope-based vaccine. The vaccine consists of an epitope (P25) that is recognised by CD4+ helper T cells and the target epitope luteinising hormone releasing hormone (LHRH). We show that replacement of the single cysteine residue within P25 with amino acids such as alanine, aminobutyric acid, serine or with carboxymethylated cysteine leads to diminished immunogenicity of the vaccine and only the oxidised dimeric form of the peptide retains the full immunogenicity of the vaccine. Secondly, by measuring the serum antibody response and the number of the antigen secreting cells in spleen and bone marrow we found that three doses of 20 nmol per mouse induced the more consistent and higher immune responses than those induced by three doses of either 2 nmol or 80 nmol per mouse. A greater variation in antibody titre was observed in mice that received the 2 mol or 80 nmol dose regimes. Last, by administering the vaccine in its lipidated form in the presence or absence of additional adjuvant we found that either inoculation regime elicited similar antibody responses. Only at low doses of antigen was a synergistic effect observed when lipopeptide was co-administered with additional adjuvant.
The specificities of immunoglobulin G antibodies obtained from the sera of dogs inoculated with totally synthetic immunocontraceptive vaccine candidates based on luteinising hormone releasing hormone (LHRH: amino acid sequence HWSYGLRPG) were examined using peptides expressed in a phage display library. The three vaccine candidates each contained a different T helper-cell epitope chemically linked with the same LHRH amino acid sequence HWSYGLRPG and all of them elicited high antibody titres against the hormone. Delineation of epitopes recognised by sera from vaccinated dogs using a phage display library indicated that two of the three vaccine candidates induced antibody directed to the consensus sequence xHWSxxLxxx whereas the third vaccine candidate induced antibody against the consensus sequence xxxxxxxRPx. Two of the three vaccine candidates elicited antibodies against B cell epitopes present within the helper T-cell epitope component of the vaccine whereas the third vaccine did not. The occurrence of anti-T helper cell epitope antibodies appeared to have little or no effect on the generation of the anti-LHRH responses indicating that carrier-induced epitope suppression was not operating here. Our results also demonstrated that with animal sera of high quality, it is possible to delineate immunodominant epitopes recognised by polyclonal antibodies with high efficiency using phage display library. The approach has utility in the definition of immunodominant epitopes, which may "decoy" antibody responses away from other epitopes, which may be more useful in prophylaxis or therapy.
It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.