The Role of Antibodies in the Treatment of SARS-CoV-2 Virus Infection, and Evaluating Their Contribution to Antibody-Dependent Enhancement of Infection.

Antibodies play a crucial role in the immune response, in fighting off pathogens as well as helping create strong immunological memory. Antibody-dependent enhancement (ADE) occurs when non-neutralising antibodies recognise and bind to a pathogen, but are unable to prevent infection, and is widely known and is reported as occurring in infection caused by several viruses. This narrative review explores the ADE phenomenon, its occurrence in viral infections and evaluates its role in infection by SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19). As of yet, there is no clear evidence of ADE in SARS-CoV-2, though this area is still subject to further study.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa.

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi.

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.

Pilot scale production of the vaccine adjuvant Proteoliposome derived Cochleates (AFCo1) from Neisseria meningitidis serogroup B.

The use of new adjuvants in vaccine formulations is a subject of current research. Only few parenteral adjuvants have been licensed. We have developed a mucosal and parenteral adjuvant known as AFCo1 (Adjuvant Finlay Cochleate 1, derived from proteoliposomes of N. meningitidis B) using a dialysis procedure to produce them on lab scale. The immunogenicity of the AFCo1 produced by dialysis has been already evaluated, but it was necessary to demonstrate the feasibility of a larger-scale manufacturing process. Therefore, we used a crossflow diafiltration system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The aim of this work was to produce AFCo1 on pilot scale, while conserving the adjuvant properties. The proteoliposomes (raw material) were resuspended in a buffer containing sodium deoxycholate and were transformed into AFCo1 under the action of a calcium forming buffer. The detergent was removed from the protein solution by diafiltration to a constant volume. In this CFS, we used a hollow fiber cartridge from Amicon (polysulfona cartridge of 10 kDa porosity, 1mm channel diameter of fiber and 0.45 m² area of filtration), allowing production of a batch of up to 20 L. AFCo1 were successfully produced by tangential filtration to pilot scale. The batch passed preliminary stability tests. Nasal immunization of BALB/c mice, induced specific saliva IgA and serum IgG. The induction of Th1 responses were demonstrated by the induction of IgG2a, IFNγ and not IL-5. The adjuvant action over Neisseria (self) antigens and with co-administered (heterologous) antigens such as ovalbumin and a synthetic peptide from haemolytic Streptococcus B was also demonstrated.

Mucosal and systemic immune responses following mucosal immunisation of tetanus toxoid entrapped in lipid nanoparticles prepared by microwave reactor.

In this study, the use of a microwave reactor, which allowed high input of energy into a pressurised system in a short period of time, was investigated for preparation of lipid nanoparticles (LNPs). The aim was to optimise the formulation process by reducing manufacturing time. Two types of LNPs were prepared; non-ionic surfactant vesicles (NISV) and bilosomes (modified NISV incorporating bile salts), with a model antigen (tetanus toxoid, TT) and the immune response induced after mucosal (nasal and oral, respectively) administration was assessed. The TT loaded LNPs were characterised in terms of particle size, size distribution, morphology, and entrapment efficiency. Immunisation was evaluated by lethal challenge with tetanus toxin in an animal model. The efficiency of vaccination was evaluated by measuring the anti-TT IgG antibody levels in the vaccinated animals. Bilosomes formed by this method showed an immunogen entrapment efficiency of ∼30% which was significantly (p < 0.05) higher than entrapment efficiency in the NISV. The percentage of animals that survived when challenged with tetanus toxin correlated with the level of IgG determined in the serum of mice immunised with LNPs by the mucosal route. Moreover, there were significant (p < 0.05) differences between orally and nasally immunised groups. Animal groups immunised bilosomes via the oral route showed the highest level of IgG (1.2 ± 0.13) compared to the positive control, LN + Xn, and no immunised group. Similarly, groups immunised via the nasal route showed significantly (p < 0.0001) higher titres compared with the control group. Mucosal TT was capable of inducing systemic specific IgG anti-TT responses that were higher than the parenteral vaccine.

AFCo1 as nasal adjuvant of capsular polysaccharide from Neisseria meningitidis serogroup C induces systemic and mucosal immune responses.

Increasing emphasis is being placed on the mucosal administration of vaccines in order to stimulate mucosal as well as systemic responses. Findings from our group suggest that proteoliposome-derived cochleate (AFCo1) acts as a potent mucosal adjuvant. As an alternative to chemical conjugation, the current study aimed to determine the benefit of using AFCo1 to improve the mucosal and systemic immune responses to capsular polysaccharide of Neisseria meningitidis serogroup C (PsC), a model of a thymus-independent (TI) antigen. Therefore, intranasal (i.n.) immunization of 3 doses 1 week apart with AFCo1 plus PsC in mice was conducted. Highly specific anti-PsC IgA responses and an anti-PsC IgG response were obtained. The subclass pattern induced against PsC was similar to that induced with the meningococcal vaccine. In summary, AFCo1 as nasal adjuvant was demonstrated to be capable of eliciting mucosal and systemic specific responses against a TI antigen.

Mucosal immunization using proteoliposome and cochleate structures from Neisseria meningitidis serogroup B induce mucosal and systemic responses.

Most pathogens either invade the body or establish infection in mucosal tissues and represent an enormous challenge for vaccine development by the absence of good mucosal adjuvants. A proteoliposome-derived adjuvant from Neisseria meningitidis serogroup B (AFPL1, Adjuvant Finlay Proteoliposome 1) and its derived cochleate form (Co, AFCo1) contain multiple pathogen-associated molecular patterns as immunopotentiators, and can also serve as delivery systems to elicit a Th1-type immune response. The present studies demonstrate the ability of AFPL1and AFCo1 to induce mucosal and systemic immune responses by different mucosal immunizations routes and significant adjuvant activity for antibody responses of both structures: a microparticle and a nanoparticle with a heterologous antigen. Therefore, we used female mice immunized by intragastric, intravaginal, intranasal or intramuscular routes with both structures alone or incorporated with ovalbumin (OVA). High levels of specific IgG antibody were detected in all sera and in vaginal washes, but specific IgA antibody in external secretions was only detected in mucosally immunized mice. Furthermore, antigen specific IgG1 and IgG2a isotypes were all induced. AFPL1 and AFCo1 are capable of inducing IFN-gamma responses, and chemokine secretions, like MIP-1alpha and MIP-1beta. However, AFCo1 is a better alternative to induce immune responses at mucosal level. Even when we use a heterologous antigen, the AFCo1 response was better than with AFPL1 in inducing mucosal and systemic immune responses. These results support the use of AFCo1 as a potent Th1 inducing adjuvant particularly suitable for mucosal immunization.

Intranasal administration of proteoliposome-derived cochleates from Vibrio cholerae O1 induce mucosal and systemic immune responses in mice.

Conservative estimates place the death toll from cholera at more than 100,000 persons each year. A particulate mucosal vaccine strategy combining antigens and immune stimulator molecules from Vibrio cholerae to overcome this problem is described. Proteoliposomes extracted from V. cholerae O1 were transformed into cochleates (AFCo2, Adjuvant Finlay cochleate 2) through a calcium inducible rotary dialysis method. Light microscopy was carried out and tubules of 16.25+/-4.57 microm in length were observed. Western blots were performed to verify the immunochemical properties of the main AFCo2 incorporated antigens, revealing full recognition of the outer membrane protein U (OmpU), lipopolysaccharide (LPS), and mannose-sensitive hemagglutinin (MSHA) antigens. AFCo2 were administered by the intranasal route using a two or three dose schedule and the immune response against V. cholerae antigens was assessed. Three AFCo2 doses were required to induce significant (p<0.05), antigen specific IgA in saliva (1.34+/-0.135) and feces (0.60+/-0.089). While, two or three doses of AFCo2 or proteoliposomes induce similar specific IgG and vibriocidal activity responses in sera. These results show for the first time that AFCo2 can be obtained from V. cholerae O1 proteoliposomes and have the potential to protect against the pathogen when administered intranasally.

Monoclonal Antibodies Against the Capsular Polysaccharides A, C, Y, W, and X of Neisseria meningitidis: A Platform for the Quality Control of Meningococcal Vaccines.

Vaccination has reduced morbidity and mortality of many diseases that previously caused devastating epidemics and deaths globally. Vaccines as a biological product may contain microorganisms or their derivatives. This aspect together with the fact that they are administered to healthy individuals (mainly children) means that approximately 70% of vaccines development time is dedicated to quality control. Monoclonal antibodies (MAbs) have become essential analytical tools for application in ELISAs, Western and Dot blotting, immunoprecipitation, and flow cytometric assays that ensure the quality control of vaccines. The aim of this work is to present a review of the methods used to obtain a platform of MAbs against Neisseria meningitidis polysaccharide antigens to use as an analytical tool for quality control of anti-meningococcal polysaccharide (Ps) vaccines. The MAbs obtained are used in five sandwich ELISAs developed for Ps quantification. The assays showed good reproducibility and repeatability, with quantitation and detection limits below 1 ng/mL. Dot Blot, as the Identity test of the Ps vaccine, was carried out to positively identify licensed and experimental vaccines. All assays described are suitable for the screening of multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability.

Assessing the immunogenicity and toxicity of the AFPL1-conjugate nicotine vaccine using heterologous and homologous vaccination routes.

Despite the increased risks of cancers and cardiovascular related diseases, tobacco smoking continues to be prevalent in the population due largely in part to the addictive nature of nicotine. Nicotine vaccines are an attractive alternative to the current smoking cessation options but have yet to be successful enough in clinical trials to reach the market due to a lack of neutralizing antibodies and inconsistent results. Using AFPL1 derived from the Cuban meningococcal vaccine as an adjuvant, we have previously published promising results with an intranasally administered nicotine vaccine. In order to examine the immunogenicity and safety of this vaccine in mice we set up a pilot trial administering the vaccine either intranasally, intramuscularly or utilizing both routes simultaneously and evaluated immune responses and clinical symptoms throughout the duration of the vaccination protocol and post-mortem. These data further demonstrate the ability of the AFPL1 nicotine conjugate vaccine to be a safe and potential candidate for clinical use.

Repeat-Dose Toxicity Study Using the AFPL1-Conjugate Nicotine Vaccine in Male Sprague Dawley Rats.

Tobacco smoking is the cause of 20% of Canadian deaths per year. Nicotine vaccines present a promising alternative to traditional smoking cessation products, but to date, no vaccine has been able to move through all phases of clinical trials. We have previously demonstrated that the AFPL1-conjugate nicotine vaccine does not induce systemic or immunotoxicity in a mouse model and that a heterologous vaccination approach is more advantageous than the homologous routes to inducing mucosal and systemic anti-nicotine antibodies. The purpose of this study was to confirm the safety profile of the vaccine in a repeat-dose toxicity study. The heterologous vaccination strategy was again used, and Sprague Dawley rats were administered a dose five times greater than in our previous studies. Physiological conditions, food and water consumption, body temperature, injection site inflammation, relative weights of organs, histopathology, and blood chemistry and hematology were evaluated during the course of the vaccination period to determine the safety of the vaccine. The AFPL1-conjugate nicotine vaccine did not induce clinically relevant changes or induce symptoms that would be associated with toxicity, making it a promising candidate for future investigations.

The Global Meningococcal Initiative meeting on prevention of meningococcal disease worldwide: Epidemiology, surveillance, hypervirulent strains, antibiotic resistance and high-risk populations.

INTRODUCTION: The 2018 Global Meningococcal Initiative (GMI) meeting focused on evolving invasive meningococcal disease (IMD) epidemiology, surveillance, and protection strategies worldwide, with emphasis on emerging antibiotic resistance and protection of high-risk populations. The GMI is comprised of a multidisciplinary group of scientists and clinicians representing institutions from several continents. AREAS COVERED: Given that the incidence and prevalence of IMD continually varies both geographically and temporally, and surveillance systems differ worldwide, the true burden of IMD remains unknown. Genomic alterations may increase the epidemic potential of meningococcal strains. Vaccination and (to a lesser extent) antimicrobial prophylaxis are the mainstays of IMD prevention. Experiences from across the globe advocate the use of conjugate vaccines, with promising evidence growing for protein vaccines. Multivalent vaccines can broaden protection against IMD. Application of protection strategies to high-risk groups, including individuals with asplenia, complement deficiencies and human immunodeficiency virus, laboratory workers, persons receiving eculizumab, and men who have sex with men, as well as attendees at mass gatherings, may prevent outbreaks. There was, however, evidence that reduced susceptibility to antibiotics was increasing worldwide. EXPERT COMMENTARY: The current GMI global recommendations were reinforced, with several other global initiatives underway to support IMD protection and prevention.

Evaluating the immunogenicity of an intranasal vaccine against nicotine in mice using the Adjuvant Finlay Proteoliposome (AFPL1).

Tobacco smoking is recognized as a global pandemic resulting in 6 million deaths per year. Despite a variety of anti-smoking products available to aid with tobacco cessation, the majority of people who attempt to quit smoking relapse within 6 months due to the addictive nature of nicotine. An immunotherapy approach could offer a promising treatment option by inducing a potent selective antibody response against nicotine in order to block its distribution to the brain and its addictive effects in the central nervous system. Our nicotine vaccine candidate was administered intranasally using the Neisseria meningitidis serogroup B Adjuvant Finlay Proteoliposome 1 (AFPL1) as a part of the delivery system. This system was designed to generate a robust immune response by stimulating IL-1ß production through Toll-like receptor 4 (TLR4), a potent mechanism for mucosal immunity. The vaccine induced high antibody titers in mice sera in addition to inducing mucosal antibodies. The efficacy of our vaccine was demonstrated using in vivo challenge experiments with radioactive [(3)H]-nicotine, followed by an analysis of nicotine distribution in the lung, liver, blood and brain. Our results were encouraging as the nicotine concentration in the brain tissue of mice vaccinated with our candidate vaccine was four times lower than in non-vaccinated controls; suggesting that the anti-nicotine antibodies were able to block nicotine from crossing the blood brain barrier. In summary, we have developed a novel nicotine vaccine for the treatment of tobacco addiction by intranasal administration and also demonstrated that the AFPL1 can be used as a potential adjuvant for this vaccine design.

Bacterial outer membrane vesicles and vaccine applications.

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.

Optimizing efficacy of mucosal vaccines.

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 derived from Vibrio cholerae O1 proteoliposomes: the impact of structure transformation on mucosal immunisation.

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.

Delivery systems: a vaccine strategy for overcoming mucosal tolerance?

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.

A proteoliposome based formulation administered by the nasal route produces vibriocidal antibodies against El Tor Ogawa Vibrio cholerae O1 in BALB/c mice.

A vaccine candidate against the enteric pathogen Vibrio cholerae was developed based on a proteoliposome (PL) formulation using a wild type strain C7258, V. cholerae O1, El Tor Ogawa as part of strategy to develop a combined formulation against enteric diseases preventable by the stimulation of the mucosal immune system. A detergent extraction method was applied to obtain the PL. Scanning electron microscopy and molecular exclusion chromatography showed the presence of two PL populations. Photon correlation spectroscopy studies were then carried out to evaluate the size (169.27+/-3.85nm), polydispersity (0.410) and zeta potential (-23.28+/-1.21mV) of the PL. SDS-PAGE and Western blot analysis revealed the presence of lipopolysaccharide (LPS), mannose-sensitive haemagglutinin (MSHA) and a range of outer membrane proteins, including OmpU. BALB/c mice were immunized intranasally with two doses of PL containing 25mug of LPS each 28 days apart. The mice showed high anti-LPS IgG titres (3.36+/-0.235) and vibriocidal antibodies (3.70+/-0.23) after two weeks from last dose. These results show for the first time that PL can be obtained from V. cholerae O1 and when administer by intranasal route has the potential to protect against this pathogen.

Estudio in vitro de liberación de proteínas de AFCo1 y sus implicaciones en la inmunización mucosal / In vitro study of protein release from AFCo1 and implications in mucosal immunisation

El cocleato adyuvante Finlay (AFCo1) 1 es derivado de un proteopolisoma de Neisseria meningitidis serogrupo B. La transformación de los proteoliposomas en AFCo1 potencia la respuesta inmune de los antígenos de Neisseria cuando se administra por vía intranasal o intragástrica. Se ha demostrado, sin embargo, que la vía intranasal es más efectiva. Los objetivos de este trabajo fueron evaluar in vitro la liberación de proteínas del AFCo1 en líquido nasal o gástrico simulado, usando para ello la prueba de microdisolución y apoyar los resultados obtenidos cuando se administró AFCo1 por vía intranasal o intragástrica en ratones BALB/c. Los resultados demostraron que la dilución de AFCo1 en líquido gástrico o nasal simulado afecta la distribución de los antígenos de proteína de Neisseria , ya que estos se liberaron de las estructuras de cocleatos más rápido cuando se utilizó líquido nasal. Se concluyó que las condiciones que simulan el entorno gástrico afectan la distribución de los antígenos de proteínas de AFCo1 y este resultado puede explicar parcialmente porqué la administración intranasal es más efectiva in vivo que la inmunización intragástrica(AU)

Adjuvant Finlay Cochleate 1 (AFCo1) is a Proteoliposome-derived cochleate obtained from Neisseria meningitidisserogroup B. Transformation of proteoliposomes into AFCo1 potentiates the immune response on Neisseriaantigens when it is administered by intranasal or intragastric (i.g) routes. However, the i.n route has beendemonstrated to be more effective. The aim of this work is to evaluate in vitro the protein release from AFCo1, insimulated gastric fluid (SGF) or simulated nasal fluid (SNF) using a microdissolution test and to provide support for the results found when AFCo1 was administered by i.g or i.n routes in BALB/c mice. Results showed that dilution of AFCo1 in simulated gastric fluid affects the delivery of Neisseria protein antigens because they were released from cochleate structures faster than when simulated nasal fluid was used. In conclusion, conditions simulating gastric environment affect the delivery of protein antigens from AFCo1 and this result could partially explain whyin administration is more effective in vivo than in immunisation(AU)