Vaxcine™ Oral Formulations
Antibody-mediated protection against MERS-CoV in the murine model
Murine antisera with neutralising activity for the coronavirus causative of Middle East respiratory syndrome (MERS) were induced by immunisation of Balb/c mice with the receptor binding domain (RBD) of the viral Spike protein. The murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the MERS coronavirus (MERS-CoV). To test the neutralising capacity of these antisera in vivo, susceptibility to MERS-CoV was induced in naive recipient Balb/c mice by the administration of an adenovirus vector expressing the human DPP4 receptor (Ad5-hDPP4) for MERS-CoV, prior to the passive transfer of the RBD-specific murine antisera to the transduced mice. Subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the MERS-CoV resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. The murine antisera used were derived from mice which had been primed subcutaneously with a recombinant fusion of RBD with a human IgG Fc tag (RBD-Fc), adsorbed to calcium
phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. The data gained indicate that this dual-route vaccination with novel formulations of the RBDFc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of MERS-CoV.
New RRC. Clinical & Experimental Immunology (2019) Review Article doi: 10.1111/cei.13352
Formulation technologies for oral vaccines
Many options now exist for constructing oral vaccines, which, in experimental systems, have shown themselves to be able to generate highly effective immunity against infectious diseases. Their suitability for implementation in clinical practice, however, for prevention of outbreaks particularly in LMIC, is not always guaranteed, because of factors such as cost, logistics, and cultural and environmental conditions. This brief overview provides a summary of the various approaches which can be adopted, and evaluates them from a pharmaceutical point of view, taking into account potential regulatory issues, expense, manufacturing complexity etc., all of which can determine whether a vaccine approach will be successful in the late stages of development. Attention is also drawn to problems arising from inadequate diet, which impacts on success in stimulating effective immunity, and identifies use of lipid-based carriers as a way to counteract the problem of nutritional deficiencies in vaccination campaigns.
B.D. Moore, R.R.C. New, W. Butcher, R. Mahood, J. Steward, M. Bayliss, C. MacLeod, M. Bogus, E.D. Williamson. Vaccine 36 (2018) 5210–5217
Dual Route Vaccination for Plague with Emergency Use Applications
Here, we report a dual-route vaccination approach for plague, able to induce a rapid response involving systemic and mucosal immunity, whilst also providing ease of use in those resource-poor settings most vulnerable to disease outbreaks. This novel vaccine (VypVaxDuo) comprises the recombinant F1 and V proteins in free association. VypVaxDuo has been designed for administration via a sub-cutaneous priming dose followed by a single oral booster dose and has been demonstrated to induce early onset immunity 14 days after the primary immunisation; full protective efficacy against live organism challenge was achieved in Balb/c mice exposed to 2 x 104 median lethal doses of Yersinia pestis Co92, by the sub-cutaneous route at 25 days after the oral booster immunisation. This dual-route vaccination effectively induced serum IgG and serum and faecal IgA, specific for F1 and V, which constitute two key virulence factors in Y. pestis, and is therefore suitable for further development to prevent bubonic plague and for evaluation in models of pneumonic plague. This is an essential requirement for control of disease outbreaks in areas of the world endemic for plague and is supported further by the observed exceptional stability of the primary vaccine formulation in vialled form under thermostressed conditions (40 ⁰C for 29 weeks, and 40 ⁰C with 75% relative humidity for 6 weeks), meaning no cold chain for storage or distribution is needed.
In clinical use, the injected priming dose would be administered on simple rehydration of the dry powder by means of a dual barrel syringe, with the subsequent single booster dose being provided in an enteric-coated capsule suitable for oral self-administration.
Gabrielle R. Andrade, Roger R.C. New, Osvaldo A. Sant’Anna, Neil A. Williams, Rosely C. B. Alves, Daniel C. Pimenta, Hugo V. Barros, Bruna S. Melo, Letícia B. Rocha, Roxane M. F. Piazza, Lucia Mendonça-Previato and Marta O. Domingos. Human Vaccines & Immunotherapeutics (2014), 10 pp2864-2874
A Universal Polysaccharide Conjugated Vaccine against O111 E. coli
E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in VaxcineTM, an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in VaxcineTM generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.
Alves, R.C.B., New R.R.C., Andrade, G.R., Mendonça, R.M.Z., Sant’Anna, O.A., Mancini, D.A.P., Domingos, M.O. (2011) Memorias do Instituto Oswaldo Cruz
Vaxcine™: An oil-based adjuvant for influenza vaccine
Vaccination is the method of choice for the prevention of influenza infection. However, the quantity of the antigen available, especially in the case of pandemics, often fails to meet the global demand. However, improved adjuvants can overcome this problem. Preliminary results obtained in this study revealed that one year after a single subcutaneous immunisation with influenza A H3N2 virus in an oil-based carrier, Vaxcine™, outbreed mice produced a high immunoglobulin G response that lasted for up to one year and exhibited less variation in titre compared with the response of the control group treated with alum. The haemagglutination-inhibition titres induced by Vaxcine™ were also higher than those generated by alum. These data indicate that Vaxcine™ is a good adjuvant candidate for seasonal influenza vaccines.
Maurílio F. Santos, Roger R. C. New, Gabrielle R. Andrade, Christiane Y. Ozaki, Osvaldo A. Sant’Anna, Lucia Mendonc¸a-Previato, Luis R. Trabulsi, and Marta O. Domingos Clinical and Vaccine Immunology (2010) 7 pp1772–80
Lipopolysaccharide as an Antigen Target for the Formulation of a Universal Vaccine against Escherichia coli O111 Strains
A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.
Mookkan Prabakaran, Selvaraj Madhan, Nayana Prabhu, Grace Yuhong Geng, Roger New and Jimmy Kwang. (2010) Antiviral Research 86:2 pp 180-187.
Reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against H5N1 infection in mice.
Induction of mucosal immunity through oral immunization is an effective way to control influenza infection. In this study, baculovirus displaying influenza hemagglutinin was encapsulated within a reverse micelle structure of phosphatidylcholine and delivered into the gastrointestinal tract of mice to study its efficacy as an oral vaccine against cross-clade H5N1 infection. Mice vaccinated with encapsulated baculovirus displayingHA(En-BacHA) showed significantly enhanced HAspecific serum IgG and mucosal IgA antibodies, and higher hemagglutination inhibition (HI) titers, when compared to its non-encapsulated form (BacHA). Estimation of serum neutralizing antibodies also indicated that En-BacHA formulation was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, clade 4.0 and clade 8.0). Further, mice vaccinated with En-BacHA alone were able to confer 100% protection against 5MLD50 of HPAI heterologous H5N1 strain (clade 1). Inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant in the vaccine formulation did not show any significant effect in both systemic and mucosal immune responses. Oral delivery of encapsulated recombinant H5 HA expressed on baculovirus surface is an effective way to prime the immune system against H5N1 infection in mice and will have no biosafety concerns associated with their production or administration.
A New Oil-Based Antigen Delivery Formulation for both Oral and Parenteral Vaccination
The ability of an oil-based carrier vehicle to act as an antigen delivery system via the oral and/or parenteral routes was investigated. The formulation consists of hydrophilic macromolecules (antigens) solubilised in oil phase, in the absence of water, by virtue of being wrapped in a sheath of phospholipid amphiphile. Results obtained demonstrate that the level of mucosal IgA antibodies detected in the stools of mice immunised orally with cholera toxin B fragment (CTB) or E. coli heat-labile toxin (LT) in oil is much higher than the level of IgA produced by mice immunised with CTB or LT alone. In addition, mice immunised orally with Y. pestis antigens (F1 and V) and CTB as immunostimulant in oil produce a significantly increased (p<0.02) systemic IgG response against both antigens (F1 and V) than mice orally immunised with F1, V and CTB without oil. Six out of ten mice immunised with F1 and V antigens in oil survived an aerosol challenge of 100 LD50 doses of virulent Y. pestis. Furthermore, animals immunised sub-cutaneously with the HIV antigen (HGP-30) in oil induced much stronger humoral and cellular responses against the antigen than mice immunised with the antigen alone. Taken together, these findings indicate that oil can be used successfully as an antigen delivery system in vaccine formulations without the necessity of an aqueous phase or an emulsification process. This greatly enhances the stability and ease of production of a formulation manufactured for commercial use.
New RRC & Kirby CJ in Advanced Drug Delivery Reviews 25 pp59-69 (1997)
Solubilisation of hydrophilic drugs in oily formulations
Association of peptides with oil carriers can improve the delivery of these molecules by affording protection against intestinal proteases, improving stability in the presence of gut surfactants and altering cell permeability and lymphatic secretion. Intestinal delivery of calcitonin using an oil-based formulation is illustrated in the pig, and a new technology (“Macrosol”) is described in which hydrophilic molecules are solubilised directly in oils in the absence of an aqueous phase. Presentation of macromolecules in this form can change the way in which they interact with cells, and give rise to new possibilities for drug delivery via the intestine.
Drug Discovery and Aptamer Technologies
Roger New, Michal Bogus, Gurpal S. Bansal, Malgorzata Dryjska, Katarzyna Zajkowska and Michael Burnet. Molecules 2017, 22, 1613
Efficacy of Bioactive Cyclic Peptides in Rheumatoid Arthritis: Translation from In Vitro to In Vivo Models
Using a novel drug discovery technology reported in previous issues of this journal cyclic peptides have been created which are able to down-regulate secretion of inflammatory cytokines, in vitro, by stimulated cells of the macrophage cell line J774. The cytokines in question, TNF-alpha and IL-6, are strongly implicated in etiology of diseases such as rheumatoid arthritis. Studies are reported here using the CAIA animal model for rheumatoid arthritis, which show that the peptides identified are indeed able to impact on inflammation of joints, induced in vivo. The results suggest that these peptides are effective at a dose which could be viable in man, and at which no adverse side effects are evident in the short term.
Roger New, Gurpal S. Bansal, Malgorzata Dryjska, Michal Bogus, Patricia Green, Marc Feldmann and Fionula Brennan. Molecules 2014, 19, 21529-21540
Design and Optimisation of Bioactive Cyclic Peptides: Generation of a Down-Regulator of TNF Secretion
Although strong binding interactions between protein receptor and ligand do not require the participation of a large number of amino acids in either site, short peptide chains are generally poor at recreating the types of protein-protein interactions which take place during cell recognition and signalling process, probably because their flexible backbones prevent the side chains from forming sufficiently rigid and stable epitopes, which can take part in binding with the desired strength and specificity. In a recently-reported study, it was shown that a proto-epitope containing F, R and S amino acids has the ability to down-regulate TNF secretion by macrophages. This paper extends these findings, putting those amino acids into a short cyclic peptide scaffold, and determining the optimal configuration required to overcome the problems of conformational instability, and give rise to molecules which have potential as therapeutic agents in human disease, such as rheumatoid arthritis.
Roger New, Gurpal S. Bansal, Michael Bogus, Kasia Zajkowska, Steffen Rickelt & Istvan Toth. Molecules 2013, 18, 3427-3441
Use of Mixed Micelles for Presentation of Building Blocks in a New Combinatorial Discovery Methodology: Proof-of-Concept Studies
We describe a new method of combinatorial screening in which building blocks, instead of being linked together chemically, are placed on the surface of nanoparticles. Two- or three-dimensional structures form on the surface of these particles through the close approach of different building blocks, with sufficient flexibility to be able to adapt and interact with putative binding sites in biological systems. The particles assemble without the need for formation of chemical bonds, so libraries comprised of many structures can be prepared rapidly, with large quantities of material available for testing. Screening methods can include solid and solution-phase binding assays, or tissue culture models, for example looking for structures which can change the behaviour of cells in a disease-modifying manner.
Zyta M. Ziora, Norbert Wimmer, Roger New, Mariusz Skwarczynski and Istvan Toth International Journal of Organic Chemistry, 2012, 2, 75-81
Lipopeptides for the Fragment-Based Pharmaceutics Design
This paper describes the synthesis of peptide fragments for use in a new type of combinatorial discovery technology, in which the building blocks are brought together by non-covalent interactions, rather than direct chemical bonding. The building blocks of interest—in this case different amino acids—are converted to amphiphiles by attachment to lipid tails. The amphiphiles, when mixed together in aqueous phase, are designed so that they aggregate spontaneously to form micelles. The building blocks form the headgroups of each of the amphiphiles, and these headgroups cover the surface of the micelle in a dynamic close-packed fluid mosaic array. These building blocks come together so closely that two-or three-dimensional structures are created on the surface of the micelles, and these can be screened in biological assays to find out which combination of building blocks is able to elicit a biological response. Lipopeptides consisting of two residues of lipoamino acid and other amino acids moieties have been designed, synthesized, characterized and the ability of these constructs to form supra-molecular assemblies is demonstrated.
Zyta M. Ziora, Norbert Wimmer, Roger New, Mariusz Skwarczynski and Istvan Toth Carbohydrate Research 346 (2011) 1439–1444
Synthesis of glycolipopeptidic building blocks for carbohydrate receptor discovery
A class of glycolipopeptides for use as building blocks for a new type of dynamic combinatorial library is reported. The members of the library consist of a variable carbohydrate moiety, coded amino acids, and lipoamino acids in order to convert them into amphiphiles. Glycolipopeptidic amphiphiles interact through non-covalent bonding when mixed together in aqueous phase and form micelles in dynamic close-packed fluid mosaic arrays. The head groups of amphiphiles are exposed on the micelle surface, providing entities which could be screened in biological assays to find the most potent combination of building blocks in order to identify new bioactive carbohydrate ligands.