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The existence of pathogens that escape recognition by specific vaccines, the need to improve existing vaccines and the increased availability of therapeutic (non-infectious disease) vaccines necessitate the rational development of novel vaccine concepts based on the induction of protective cell-mediated immune responses. For naive T-cell activation, several signals resulting from innate and adaptive interactions need to be integrated, and adjuvants may interfere with some or all of these signals. Adjuvants, for example, are used to promote the immunogenicity of antigens in vaccines, by inducing a pro-inflammatory environment that enables the recruitment and promotion of the infiltration of phagocytic cells, particularly antigen-presenting cells (APC), to the injection site. Adjuvants can enhance antigen presentation, induce cytokine expression, activate APC and modulate more downstream adaptive immune reactions (vaccine delivery systems, facilitating immune Signal 1). In addition, adjuvants can act as immunopotentiators (facilitating Signals 2 and 3) exhibiting immune stimulatory effects during antigen presentation by inducing the expression of co-stimulatory molecules on APC. Together, these signals determine the strength of activation of specific T-cells, thereby also influencing the quality of the downstream T helper cytokine profiles and the differentiation of antigen-specific T helper populations (Signal 3). New adjuvants should also target specific (innate) immune cells in order to facilitate proper activation of downstream adaptive immune responses and homing (Signal 4). It is desirable that these adjuvants should be able to exert such responses in the context of mucosal administered vaccines. This review focuses on the understanding of the potential working mechanisms of the most well-known classes of adjuvants to be used effectively in vaccines.

Context: Trypanosoma brucei brucei (T.b. brucei) infection causes death in cattle, while the current treatments have serious toxicity problems. However, natural products can be used to overcome the problems associated with parasitic diseases including T.b. brucei., Objective: Artemisia elegantissima Pamp (Asteraceae) was evaluated phytochemically for its constituents and antitrypanosomal potential against T.b. brucei for the first time. Scopoletin isolated from A. elegantissima has shown better potential then the standard drug suramin, used against T.b. brucei., Materials and Methods: The ethanol extract of the aerial parts of A. elegantissima was fractionated by column and preparative thin-layer chromatography into six fractions (A-F) yielding 13 compounds, these were evaluated for their antitrypanosomal activity against T.b. brucei at different concentrations., Results: Thirteen compounds were isolated from A. elegantissima: (Z)-p-hydroxy cinnamic acid, stigmasterol, β-sitosterol, betulinic acid, bis-dracunculin, dracunculin, scopoletin, apigenin, dihydroluteolin, scoparol, nepetin, bonanzin, and 3',4'-dihydroxy bonanzin. The fractions D-F were found to be active at the concentration of 20 µg/ml and three compounds isolated from these fractions, scopoletin (MIC ≤0.19 µg/ml), 3',4'-dihydroxy bonanzin (MIC = 6.25 µg/ml) and bonanzin (MIC = 20 µg/ml), were found to be highly active., Discussion and Conclusion: Artemisia elegantissima was phytochemically and biologically explored for its antitrypanosomal potential against T.b. brucei. The number and orientation of phenolic hydroxyl groups play an important role in the antitrypanosomal potential of coumarins and flavonoids. The compounds 3',4'-dihydroxy bonanzin and scopoletin with low MIC values, hold potential for use as antitrypanosomal drug leads.

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., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

Overpopulation of selected groups of animals is widely recognised as an issue that can have adverse effects on several current global problems, such as animal and human health, conservation and environmental changes. This review will, therefore, focus on recent novel contraception together with future technologies that may provide additional contraceptive methods.

The proteoliposome derived from Vibrio cholerae O1 (PLc) is a nanoscaled structure obtained by a detergent extraction process. Intranasal (i.n) administration of PLc was immunogenic at mucosal and systemic level vs. V. cholerae; however the adjuvant potential of this structure for non-cholera antigens has not been proven yet. The aim of this work was to evaluate the effect of coadministering PLc with the Vi polysaccharide antigen (Poli Vi) of S. Typhi by the i.n route. The results showed that Poli Vi coadministered with PLc (PLc+Poli Vi) induce a higher IgA response in saliva (p<0.01) and faeces (p<0.01) than Poli Vi administered alone. Likewise, the IgG response in sera was higher in animals immunised with PLc+Poli Vi (p<0.01). Furthermore, IgG induced in sera of mice immunised with PLc+Poli Vi was similar (p>0.05) to that induced in a group of mice immunised by the parenteral route with the Cuban anti-typhoid vaccine vax-TyVi, although this vaccine did not induce a mucosal response. In conclusion, this work demonstrates that PLc can be used as a mucosal adjuvant to potentiate the immune response against a polysaccharide antigen like Poli Vi.

Haloxylon salicornicum (H. salicornicum) is a plant that is frequently taken as a tea by Bedouin women in Egypt who are experiencing difficulties during pregnancy, as well as to provide relief from dysmenorrhoea. Despite its medical use, there has been no detailed evaluation of the effect of this plant on uterine tissue. Therefore, the initial aim of this study was to determine whether H. salicornicum affected the contraction of the mouse uterus in vitro. The crude aqueous extract of H. salicornicum was found to inhibit the spontaneous contractions of the uterus, with the effect being rapid in onset and completely reversible upon washout. Subsequent purification of the plant extract resulted in the identification of synephrine and N-methyltyramine, both of which were found to have inhibitory effects on the spontaneous contractions of the uterus. The EC50 for the purified constituent identified as synephrine was 0.82 ± 0.24  μ g/mL. The inhibitory activity of crude H. salicornicum, as well as the isolated constituents, could be prevented by pretreatment of the uterus with the β -adrenoceptor antagonist propranolol. In conclusion, the use of H. salicornicum during preterm labour appears to be justified, and its pharmacologic effect is consistent with it acting as a β -adrenoceptor agonist.

Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer's patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging.

In general, there are only a few vaccines administered via mucosal routes, as the mucosal immune system presents numerous hurdles, including diversity in mucosal surface structure, complexity in immune cell interaction and limitations in experimental methodology. This therefore necessitates a range of strategies to be used for each target area. With reference to the three main routes of delivery and associated mucosal surfaces (oral/intestinal, nasal/respiratory and female genital tract), this review examines how coadministration of immune-stimulatory molecules, adjuvants, delivery systems and mucoadhesives are used to improve mucosal vaccine efficacy. Key considerations to the development of next-generation mucosal vaccines include improved efficacy and safety, technological advancements in medical devices to enable convenience and better administration, as well as reduced manufacturing costs.

Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc.

Introduction: There has been no novel contraceptive development since 'the Pill', 50 years ago. Despite the subsequent steady increase in the use of contraceptives, the contraceptive needs of a significant proportion of the world population have not yet been met. The key need is for novel, effective, practical, long-lasting, affordable, non-steroidal contraceptives. Immunocontraception, based on vaccination against components of the reproductive system that do not affect other physiological systems, fulfils most of the criteria of such a contraceptive. To date, immunocontraceptives have been developed for animal use and the application to human contraception is an exciting proposition. In addition, immunocontraceptive research has provided a greater understanding of the vaccination against 'self-antigens' and has led to non-contraceptive developments for these vaccines., Areas Covered: This review provides an understanding of the historic context of immunocontraceptives and the progress that has been made. In some cases, the contraceptive aspect has been abandoned, but the knowledge gained has enabled other therapeutic advances., Expert Opinion: Reproductive research is still an important area and innovations continue to arise, which offer hope for new therapeutics in reproduction and related fields.

Oral vaccination using protein antigens is complicated by the degradative effects of the inhospitable conditions in the gastrointestinal tract, such as low pH and digestive enzymes, nescessitating protection and effective delivery of the antigen. The bilosome is a lipid-based, vesicle delivery system incorporating bile salts, which is believed to protect the antigen from degradation, and has been shown to induce significant antibody responses when delivered orally with various vaccines. In translational research, from laboratory bench to industrial scale-up, it is necessary to optimise the manufacturing process in order to improve efficiency and simplify production, giving a more economical end-product. In this study we tested two simplified production methods (3-step and 1-step) along with two different storage methods (lyophilised and non-lyophilised), as well as looking at the effect of buffer pH. The formulations were assessed in a murine system for immunogenicity, alongside characterisation in terms of size and antigen entrapment, with the stability of these aspects assessed with respect to time. Both lyophilised and non-lyophilised 3-step formulations induced significant IgG1, IgG2a and IgA titres, with the lyophilised version showing stable size and antigen entrapment up to 9months.

Previous studies, using parenteral administration of antigen in lipid vesicles, have indicated a possible role for vesicle size in determining the Th1/Th2 bias of the resulting immune response. We have also demonstrated that the incorporation of bile salts into lipid vesicles (bilosomes) allows successful induction of mucosal and systemic immunity via the oral route. The following study was therefore carried out to determine whether size could also influence the Th1/Th2 bias in the immune response to bilosome entrapped influenza A antigen containing haemagglutinin administered by the oral route in the mouse and whether this could influence the disease process in the classical ferret model of disease. Consequently we produced two formulations of influenza A antigen entrapped in bilosomes: BV3 which contained a single population (range 10-100 nm, Z-average diameter 250 nm) and BV which had two populations (60-350 and 400-2,500 nm, Z-average 980 nm). Following oral vaccination of BALB/c mice, BV was found to generate an immune response that had a significantly greater Th1 bias than BV3 as measured by serum IgG2a production and antigen-induced spleen cell IFN-gamma production. In the traditional infection challenge model (ferrets) vaccination with BV (large) vesicles resulted in greater protection in terms of symptom-score and a higher responder number. However, both oral vaccine formulations were an improvement on intramuscular administration in terms of higher antibody production, lower temperatures, and reduced symptoms over time, post-infection. The results presented here demonstrate that oral vaccine formulations can be physically modified to manipulate resultant immune responses following vaccination and consequently can be designed to enhance the effectiveness of candidate vaccine antigens.

Cyclic GMP (cGMP) mediates various physiological functions of nitric oxide (NO) synthesized by nitric oxide synthase (NOS). A crude peel extract and purified fraction of Flemingia vestita, as well as a crude rhizome extract of Stephania glabra and fractions were tested with respect to the activity of NOS, NO efflux and cGMP concentration in the cestode Raillietina echinobothrida in order to find out the possible mode of anthelmintic action of these plant-derived components. For comparison purposes, the parasites were also treated with pure genistein, sodium nitroprusside (SNP-a known NO donor), and the reference drug, praziquantel (PZQ). At the time of onset of paralysis in the parasites, a significant increase (32%-87%) in the NOS activity and a two to three fold increase of NO efflux into the incubation medium were observed in the treated parasites in comparison to their respective controls. The cGMP concentration in the treated parasites' tissue was also increased by 44%-103%. However, in the presence of NG-nitro-L-arginine methyl ester, a potent inhibitor of NOS, there was no increase in the cGMP concentration in the parasite tissue. This study indicates that the phytochemicals, in particular genistein and tetrahydropalmatine, from F. vestita and S. glabra, respectively, disturb the downstream signalling pathway of NO, as indicated by the change in cGMP concentration in the parasite tissue.

Antigens administered via the oral and, to a lesser extent, the nasal route are potentially able to invoke tolerance, resulting in a nonreactive immune response. This has been a hurdle for mucosal vaccine development and yet the desire to induce protective local and systemic responses, with pain-free and more convenient products, has been the impetus driving mucosal vaccine R&D. Nevertheless, few mucosal vaccines have reached the marketplace and products are still treated with caution, particularly where live organisms are utilized. In this review, we examine the use of delivery systems with adjuvant properties as key components in a vaccine strategy that does not require the use of live vectors to overcome tolerance and have exemplified their success in mucosal vaccines, concentrating on the nasal and oral routes of administration.

Research into active immunisation against gonadotrophin releasing hormone (GnRH-I) has gained widespread acceptance as a means of controlling reproduction and behaviour of farm, companion and wild animals. Many studies describe the use of multiple copies of the self-peptide in linear alignment and conjugation with a large carrier protein to increase the immune response to the peptide. However, problems resulting from carrier protein epitope suppression have seen a diversion of interest into the use of genetic materials to elicit an optimum immune response. In this study, a 533-bp long DNA vaccine was constructed in pcDNAV5-HisB coding for 18.871 kDa GnRH-I-T-helper-V5 epitopes fusion protein. COS1 cells transfected with the vaccine construct were found to release fusion protein into culture supernatant. The vaccine construct (100 microg/mice) in saline solution administered into the anterior quadriceps muscle of ICR male and female mice stimulated antigen-specific IgG antibody responses. Testosterone levels in the vaccinated male mice were significantly (p = 0.021) reduced. A significant reduction in uterine implants were noted following mating between immunised males and control females (p = 0.028), as well as between immunised females and control males (p = 0.004). Histological examination of both the male and female gonads in study week 13 showed atrophy of the seminiferous epithelium and suppression of folliculogenesis.

The present study was carried out to examine the anti-inflammatory activity of the inner leaf gel component of Aloe barbadensis Miller. A simple in vitro assay was designed to determine the effect of the inner gel on bacterial-induced pro-inflammatory cytokine production, namely TNF-alpha and IL-1 beta, from peripheral blood leukocytes stimulated with Shigella flexneri or LPS. This report describes the suppression of both cytokines with a freeze-dried inner gel powder and a commercial health drink from the same source. Comparison was made with a human monocytic cell-line (THP-1 cells) and a similar trend in responses was demonstrated.

The emergence of antibiotic resistant bacterial strains is a growing problem and is an important concern for patients, physicians, healthcare managers, and policymakers as it results in poorer health and economic outcomes. This has led to an urgent global call for new antimicrobial drugs, particularly from natural resources. We have been studying the antimicrobial properties of the inner leaf gel component of Aloe barbadensis Miller and have used a number of different, simple in vitro assays to establish a scientific basis for the potential use of Aloe vera on a range of clinically relevant bacteria. The bacteria used include Shigella flexneri, Methicillin-Resistant Staphylococcus aureus (MRSA), Enterobacter cloacae and Enterococcus bovis. In this paper, we compare standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI) with a microtitre assay using a metabolic colour indicator Alamar blue. All the techniques described have shown that Aloe vera has an antimicrobial effect, however, the microtitre assay enables high throughput screening, under similar conditions and is less wasteful of plant material.

Immunisation against mammalian gonadotrophin releasing hormone (GnRH-I) linked to large carrier proteins has been shown to disrupt fertility. However, various studies have shown that the carrier protein causes epitope suppression of the hapten response, resulting in short-lived immunoneutralisation, followed by a return of fertility. A range of strategies has been used to resolve this, with limited success. The aim of this study was to construct a plasmid DNA vaccine encoding GnRH-I and T-helper epitopes. A 498 bp long vaccine construct in pcDNA3.1+ was administered to male mice in conjunction with a Hemagglutinating Virus of Japanese Envelop (HVJ-E) vector or in saline solution. The vaccine efficacy was evaluated in terms of GnRH-I specific IgG antibody response, serum testosterone levels, testicular spermatogenesis and the ability to produce offspring. The vaccine appeared to induce higher anti-GnRH-I IgG antibody response and insult the fertility axis, which was characterised by a drop of epididymal sperm counts, reduction of serum testosterone levels, suppressed testicular spermatogenesis and a significant decrease in litter numbers compared to control animals. The end-point vaccine efficacy was much higher in the HVJ-E vector mediated immunisation, than in saline alone.

In mammals, the hypothalamic decapeptide, gonadotrophin releasing hormone (GnRH-I), is regarded as the major fertility regulating peptide. However, a range of isoforms also exists, varying only in the core region between amino acids 5-8. The physiological role of two of these, GnRH-II and GnRH-III, remains controversial, particularly with regard to fertility. The basis of the present study was to examine whether there is potential for GnRH-II and GnRH-III to be developed into highly specific vaccines, and to determine what the impact of their neutralisation would be on fertility. Computer modelling was used to predict how many common amino acids could be sequentially removed from the N-terminus, without loss of conformational structure. Sequences predicted to retain structure, were synthesised and conjugated to tetanus toxoid. Male mice were actively immunised, in study weeks 0, 2, 4 and 6 and peptide specific ELISA carried out. Mice immunised with TT-GnRH-I, TT-GnRH-II and TT-GnRH-III conjugates induced high antibody titres to the respective peptide. However, serum from TT-GnRH-I treated mice showed cross-reactivity to GnRH-II and GnRH-III peptides, and serum from TT-GnRH-II immunised mice showed cross-reactivity to GnRH-III. On the other hand, serum from only two of the TT-GnRH-III treated animals showed cross-reactivity to GnRH-II. Histological examination of the testes enabled comparative quantification of the disruption to spermatogenesis. Immunisation against TT-GnRH-I and TT-GnRH-III caused 66% and 68%, respectively, of seminiferous tubules viewed to show evidence of spermatogenesis, compared with 82% and 92% against TT-GnRH-II and untreated controls, respectively. Endocrine analysis revealed that only the TT-GnRH-I immunised animals showed significant reduction (p<0.05) in follicle stimulating hormone, while testosterone levels were reduced in the TT-GnRH-I and TT-GnRH-III treated animals. Taken together, our data suggests that GnRH-I and GnRH-III are implicated in spermatogenesis, unlike GnRH-II.

Background and Purpose: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action., Experimental Approaches: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFkappaB pathway, at the level of IkappaBalpha degradation and phosphorylation of NFkappaB. Also its effects on NFkappaB and GAS/GAF-DNA binding were assessed by EMSA., Key Results: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IkappaBalpha or phosphorylation of NFkappaB. However, it inhibited NFkappaB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNgamma-stimulated iNOS induction and GAS/GAF-DNA binding., Conclusions and Implications: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFkappaB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified., (Published online 7 August 2006.)

A modified GnRH peptide (CHWSYGLRPG-NH(2)) was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and formulated with Quil A saponin or a sustained release injectible PLGA (poly(lactide-co-glycolide)/triacetin). For the Quil A formulations, two administrations of TT conjugate at 3-weekly intervals were followed by two booster injections with the DT conjugate in entire ram lambs. With the PLGA formulations, only two injections were administered; the first containing TT and the second DT at 6-weekly intervals. Evaluation was carried out by comparing the specific antibody levels produced in relationship to hormone profiles and testicular changes. The Quil A formulation was considered the most effective, as it caused significant reduction in testosterone and follicle stimulating hormone levels, resulting in marked suppression of spermatogenesis.

Protein antigens administered via the oral route are exposed to a hostile environment in the gastrointestinal tract, consisting of digestive enzymes and a range of pH (1-7.5). Using a delivery system can afford protection to entrapped components against degradation and permit delivery of antigen to the cells responsible for generating local and systemic immune responses. In this comparative study, mice were immunised orally with tetanus toxoid (40 or 200 microg dose/mouse, four doses in total) entrapped in non-ionic surfactant vesicles formulated with bile salts (bilosomes). The higher entrapped dose (BV-TT, 200 microg) induced IgG1 by study week 3 to similar levels to those observed with subcutaneous un-entrapped TT at the lower (<50 microg) dose. However, both bilosome formulations (BV-TT, low, and high doses), though not un-entrapped TT, caused a rise in the numbers of IgA positive plasma cells observed in the small intestine, primarily in the first 15 cm of the small intestine.

An effective experimental vaccine may fail to become a therapeutic reality for a number of scientific, regulatory or commercial reasons. In this review, we share some of our personal experiences as University-based researchers and provide an account of some of the problems that we have encountered during preliminary scale-up and assessment of an oral influenza vaccine formulation. Many of the problems we have faced have been non-scientific and related to identifying project-funding sources, finding suitable contract manufacturing companies that are GMP compliant, and protecting intellectual property generated from the scientific studies. The review is intended as a practical guide that will allow other researchers to adopt effective strategies to permit the translation of an effective experimental formulation to a viable commercial product.

A N-terminal modified gonadotrophin releasing hormone (GnRH-I, tetanus toxoid-CHWSYGLRPG-NH2) conjugate was evaluated histologically in a number of male animal species (mice, dogs and sheep). The immunogen has previously been shown to be highly effective in rats, by suppressing both steroidogenesis and spermatogenesis. However, cross-species efficacy of peptide vaccines is known to be highly variable. Therefore, a comparative evaluation of reproductive tissues from animals immunized against this immunogen adsorbed onto an alum-based adjuvant was made. The sheep and dogs were chosen, as use of anti-fertility vaccines in these species is important in farming and veterinary practice. Changes in testicular size were measured during the immunization period and the greatest alteration (attributed to gonadal atrophy) was observed in the rat. Following euthanasia, the testicular tissue was evaluated for spermatogenesis. The most susceptible species to GnRH-I ablation was the rat, which showed significant (P < 0.0001) arrest in spermatogenesis compared with untreated controls. Testicular sections taken from treated animals were completely devoid of spermatozoa or spermatids, in comparison with 94% of the untreated controls showing evidence of spermatogenesis. The immunized mice and rams also showed significant arrest (P < 0.0001). There was a 30-45% decrease in spermatogenesis and total azoospermia was not apparent. However, the least responsive were the dogs, which showed little significant variation compared to untreated animals and only a 5% decrease in activity. A comparison of the specific IgG response to GnRH-I indicated that in sheep and dogs the response was not maintained, unlike in rodents, suggesting that suppression of fertility may be due to differences in immune responses in different animal species.

Vaccine antigens administered by the oral route are often degraded by gastric secretions during gastrointestinal transit. This necessitates larger and more frequent doses of antigen for vaccination. A delivery system, which overcomes this, is a lipid vesicle containing bile salts (bilosome), which prevents antigen degradation and enhances mucosal penetration. The effect of bilosome formulation modification on vaccine transit efficacy across the mucosa was determined. Specific antibody levels were assessed by end-point titre ELISA and the subclasses determined. Significant IgG1 titres were induced when the protein loading was doubled from 15 to 30 microg (P=0.009) and was equivalent to antigen administration by the subcutaneous route. No IgG2a was induced, indicating the generation of a TH2 response. Significant mucosal IgA levels were also observed with this treatment group (P=0.05).

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.

Vaccines have been used successfully to treat and eradicate infectious diseases; this has encouraged a major drive towards development of contraceptive vaccines (immunocontraception) as an additional method against rising human and animal populations. Despite three decades worth of research in this field, there are no licensed human contraceptive vaccines and less than ten for use in animals. The development of peptide antifertility vaccines has had a substantial impact on improving safety and specificity of immunogens, but has resulted in efficacy problems. Innovative strategies have been developed to overcome these problems.

Problem: Recent evidence shows the existence of numerous isoforms of gonadotropin releasing hormone (GnRH), with high sequence homology and a core variable region. This raises the issue that previous GnRH distribution studies may have identified a variety of isoforms. This investigation was carried out to confirm the distribution and binding activity of GnRH-I only., Method of Study: A monoclonal antibody (7B101D10), with specificity for the core region of GnRH-I was used to stain formalin-fixed tissue sections from adult male Sprague-Dawley rats, while a biotinylated GnRH-I sequence was used with avidin-labelled HRP to evaluate regions of GnRH-I binding., Results and Conclusions: GnRH-I expression was only found in the hypothalamus, cerebellum, anterior/fore brain and in Sertoli cells, while, binding activity was only present in the pituitary, subendocardium and subepicardium, thymic lymphocytes, peripheral blood lymphocytes and neutrophils. There was overlap in the olfactory neurons, liver (Kupffer macrophages and hepatocytes), spleen (lymphocytes and dendritic cells), myocardium and testes (spermatozoa and Leydig cells) and this may be further evidence of the paracrine/autocrine activity of a neuropeptide.

Aloe barbadensis Miller (or Aloe vera) has widespread use in health products, and despite numerous reports on the whole plant, little work has been performed on the inner gel, which has been used extensively in these products. This report describes the in vitro susceptibilities of two bacteria to this component.

The search for effective antifertility vaccines has been actively pursued for decades in clinical applications as contraceptives or in the treatment of hormone-dependent conditions. More recently, innovations have seen immunocontraception being employed successfully in veterinary and farming applications, as well as in population control of a number of different wildlife species. Although the basic principle behind these vaccines is the same, design considerations and criteria for commercialization are very different. This review will examine current advances in this field and evaluate the future scope of antifertility vaccines.

Problem: In the previous paper, we described how the tetanus toxoid (TT) conjugated monomer, CHWSYGLRPG-NH2, induced high neutralizing antibody titres, which resulted in decreased levels of testosterone and subsequent antifertility. However, its counterpart HWSYGLRPGC, induced low avidity antibody titres. We wanted to know whether peptide dimerization would improve the efficacy of both peptides., Method of Study: Male Sprague-Dawley rats were immunized with modified dimerized GnRH-I peptides (HWSYGLRPGCCGPRLGYSWH and GPRLGYSWHCCHWSYGLRPG-NH2), with or without conjugation to TT., Results: The unconjugated dimers were not effective in causing castration, although the first peptide dimer did induce production of antibodies. When conjugated to TT, both dimers showed the same level of efficacy in causing castration as each other. However, there were differences in antibody binding to native GnRH., Conclusions: Dimerization and conjugation to a carrier improved the antifertility efficacy of HWSYGLRPGC, whereas the conjugated monomer CHWSYGLRPG-NH2 showed a greater level of consistent castration than its conjugated dimer.

Problem: We previously immunoneutralized gonadotrophin releasing hormone (GnRH), using an analogue of GnRH (des-1 GnRH-I), conjugated to tetanus toxoid via a carbodiimide reaction. The castration effect on the reproductive system was not consistent in all the treated animals. Therefore, we examined the possibility that conjugation to the carrier protein via the N- or C-terminal could have an effect on efficacy., Method of Study: GnRH analogue sequences were synthesized consisting of an additional cysteine at either terminal and specific conjugation was carried out using a bifunctional linker agent., Results: Conjugation of the monomer through the N-terminal proved to be a highly effective means of causing immunocastration in terms of decreased gonadotrophin and testosterone concentrations and testicular size, whereas conjugation through the C-terminal proved to be ineffective. This was reflected in the ability of the antibodies to bind native GnRH, but not the levels of the anti-GnRH antibodies., Conclusion: Immunoneutralization efficacy was attributed to the importance of preserving the GnRH C-terminal.

In recent years, several forms of gonadotrophin releasing hormone (GnRH) molecules have been isolated from primate brain. These molecules are very similar in sequence and this raises the question of whether previously developed neutralisation vaccines based on GnRH (now termed GnRH-I) would remove other forms of GnRH (namely GnRH-II) as well. As the function of these other molecules has not yet been clearly defined, potential health risks could exist by their ablation. In view of the high sequence homology between the molecules, this paper describes the production of highly specific polyclonal antibodies against GnRH-I and GnRH-II, with negligible cross-reactivity. The ultimate aim of this is to develop an anti-fertility vaccine which does not present any inappropriate side-effects, caused by neutralisation of a GnRH molecule which may or may not be directly involved in reproduction. Several formulations were investigated, based on analogues of the following molecules, conjugated to tetanus toxoid: 1. GnRH-I pGlu-His-Trp-Ser-Try-Gly-Leu-Arg-Pro-Gly-NH2 and 2. GnRH-II pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. The specificity of the antibodies produced was examined, together with effects on fertility and any inappropriate side-effects. Immunostaining of hypothalamic sections was carried out, using the generated antisera, to determine the regional distribution of GnRH-I and GnRH-II neurones, as well as to further evaluate the specificity of the antibodies.

Gonadotropin-releasing hormone (GnRH) and its analogues have been used clinically to treat a range of hormone-dependent conditions. It is often necessary for large, toxic and expensive drug doses to be administered. Improvements in drug delivery have necessitated new developments in formulation, but these in turn can induce new adverse effects. Immunological neutralisation of GnRH has been examined as a less toxic and cheaper replacement therapy, and has been studied closely in different animal species. However, only a few clinical trials have been carried out with respect to hormone-dependent cancers. Based on clinical trials of the free peptide drug in cancer patients, it would appear that there is an increasing trend towards using GnRH and its analogues in adjuvant therapy and that antibody-based GnRH neutralisation will have a role in this treatment regimen.

Problem: Hormone immunoneutralization is hampered by immunologic cross-reactivity caused by close-sequence homology between related molecules. One solution is to use smaller fragments to induce antibodies of greater specificity., Method of Study: A number of peptides selected from beta-follicle-stimulating hormone (FSH) were conjugated to tetanus toxoid and were used to immunize female rats. The antisera were examined for FSH cross-reactivity by immunoassays and in an in vitro bioassay., Results: In the immunoassays, the antisera did not react with FSH but did react with their respective peptides. In the bioassay, sera from VYKDPARPC- and CDSLYTYP-immunized animals inhibited FSH-receptor interaction by 73% and 68%, respectively. These animals also showed reduced estradiol levels. Sequences were synthesized around VYKDPARPC and were tested on a FSH-receptor-bearing Chinese hamster ovary cell line. LVYKDPARPC, VYKDPARPC, YKDPARPIC, CLVYKDPARP, and LVYKDPARP inhibited FSH-receptor interaction by greater than 50%. In female mice, TRDLVYKDPARPKI and LVYKDPARP disrupted estrous cycling in all animals; LVYKDPARPC and CLVYKDPARP disrupted cycling in three of five animals, whereas VYKDPARPC disrupted cycling in one of four animals., Conclusions: Peptides from two areas of beta-FSH (VYKDPARP and DSLYTYP) were shown to raise FSH-neutralizing antibodies, which were able to suppress estradiol levels. An additional leucine residue to VYKDPARP greatly enhanced the peptide's ability to inhibit FSH-receptor binding and caused fertility disruption in vivo.

Gonadal function can be controlled through immunoneutralisation of gonadotrophin releasing hormone (GnRH), with an analogue, GnRH-glycys, linked to a carrier molecule and an appropriate adjuvant. In this study, four different types of carrier molecule were investigated: (a) single and branched amino acid polymers--[poly-(D-glu, D-lys) and poly-(phe, glu)-poly(DL-ala)-poly(lys)]; (b) bacterial toxoids--diphtheria (DT) and tetanus (TT); (c) synthetic T-helper epitopes--derived from malarial circumsporozite protein (CS) and measles virus fusion protein (MVF); and (d) thyroglobulin (Thy)--a large protein. The effect of non-ionic surfactant vesicles (NISV) and an aluminum hydroxide based adjuvant (alum), was also examined. Although good antibody responses were achieved with GnRH-glycys-DT, GnRH-glycys-TT and GnRH-glycys-Thy, adsorbed onto alum and the dimerised synthetic T-helper epitope constructs, incorporated into NISV, a critical antibody titre was necessary to result in morphological changes in the gonads and complete suppression of spermatogenesis. This was only achieved with tetanus toxoid and the dimerised T-helper epitopes.

The aim of this study was to assess the therapeutic potential of active immunisation with GnRH-glycys-PPD in a hormone-dependent experimental model. Mammary tumours were induced in female rats using dimethylbenzanthracene (DMBA) and the effects of GnRH immunoneutralisation on tumour development were evaluated. High titres of anti-GnRH IgG correlated with a decrease in oestrogen levels and subsequent tumour suppression. A comparison of immunised and non-immunised animals showed that when GnRH-specific IgG levels were at a maximum titre (80-100 micrograms/ml), nearly 10% of the GnRH-glycys-PPD treated animals showed mammary masses, compared with all the non-treated animals at the same stage in the study. When the antibody levels fell, tumour regrowth was observed, but to a level below that observed in the non-treated animals. Following further treatment with the analogue, the tumours regressed again, showing their retention of hormone dependency. This is consistent with other endocrine manipulations in the treatment of breast cancer; the advantages of immunisation with GnRH-glycys lies in its non-toxicity and reduction in side-effects, which were mainly adjuvant-induced.

A GnRH-neutralising vaccine, with potential applications in the treatment of human sex hormone-dependent disorders, was developed by conjugating GnRH-glycys to tetanus toxoid. An evaluation of adjuvant, dose and carrier pre-sensitisation was made. Male rats immunised with the conjugate, adsorbed onto alum, showed higher anti-GnRH antibody levels and suppressed testosterone concentrations, compared with animals immunised without adjuvant. Conjugate administration in a four injection regime proved to be the most effective in disrupting fertility, as assessed by the degree of lowered testosterone levels and gonadal atrophy. Pre-sensitisation with tetanus toxoid had an initial marked effect on immunisation, observed following 2 drug doses; the pre-sensitised animals showed a lower antibody response to the conjugate than did the non-primed animals. However, as the number of drug doses increased to 4, there was no significant difference between the primed and non-primed animals.

Immunization of male Copenhagen Fischer rats with a gonadotrophin releasing hormone (GnRH) analogue, conjugated to PPD resulted in high levels of antibody being produced which disrupted gonadal function in male rats. The antibody reduced serum testosterone levels and subsequently suppressed spermatogenesis. Alternatives to Freund's adjuvant were tested, namely, aluminium hydroxide and non-ionic surfactant vesicles (NISV). The study showed that aluminium hydroxide was as effective as Freund's adjuvant and less toxic, in both BCG and non-BCG primed animals. However, NISV were completely non-toxic and most effective in conjunction with BCG priming. The data obtained showed that NISV have the potential to be used as an alternative to FCA and aluminium hydroxide-based adjuvants.

The search for suitable boron containing compounds for 10B neutron capture therapy (BNCT) is based on the principle that boron atoms must be delivered specifically to tumour cells at a concentration high enough to be effective without being toxic to normal cells. Specificity may be achieved through monoclonal antibodies. However, it has been difficult to conjugate large numbers of boron atoms to the antibody molecules without inactivating them. We have devised a strategy to do this indirectly through the use of a boronated glutamate-lysine polymer in conjunction with biotin and streptavidin.

The aim of this study was to develop an effective and nontoxic vaccine, suitable for use in humans, which was capable of effectively controlling oestrogen levels. Female Sprague-Dawley rats were immunized with a conjugated analogue of gonadotrophin releasing hormone, GnRH-glycys-PPD. This resulted in high levels of neutralizing antibody which disrupted GnRH function and consequently caused a reduction in serum oestrogen. The effect of oestrogen deprivation correlated well with ovarian failure and gonadal atrophy. An examination was made of various adjuvants in conjunction with the analogue to determine the suitability of the combinations in the formulation of an effective human vaccine. This investigation included a novel adjuvant, non-ionic surfactant vesicles (NISV); the results showed that NISV are completely nontoxic and in terms of potentiating and sustaining an immune response, compare favourably with Freund's adjuvant and alum. In addition the long term effects of immunization were investigated and the data showed that immunoneutralization of GnRH effectively suppresses fertility on a long-term basis.