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.

Mucosal and systemic immune responses induced by a single time vaccination strategy in mice.

Vaccination is considered by the World Health Organization as the most cost-effective strategy for controlling infectious diseases. In spite of great successes with vaccines, many infectious diseases are still leading killers, because of the inadequate coverage of many vaccines. Several factors have been responsible: number of doses, high vaccine reactogenicity, vaccine costs, vaccination policy, among others. Contradictorily, few vaccines are of single dose and even less of mucosal administration. However, more common infections occur via mucosa, where secretory immunoglobulin A plays an essential role. As an alternative, we proposed a novel protocol of vaccination called Single Time Vaccination Strategy (SinTimVaS) by immunizing 2 priming doses at the same time: one by mucosal route and the other by parenteral route. Here, the mucosal and systemic responses induced by Finlay adjuvants (AF Proteoliposome 1 and AF Cochleate 1) implementing SinTimVaS in BALB/c mice were evaluated. One intranasal dose of AF Cochleate 1 and an intramuscular dose of AF Proteoliposome 1 adsorbed onto aluminum hydroxide, with bovine serum albumin or tetanus toxoid as model antigens, administrated at the same time, induced potent specific mucosal and systemic immune responses. Also, we demonstrated that SinTimVaS using other mucosal routes like oral and sublingual, in combination with the subcutaneous route elicits immune responses. SinTimVaS, as a new immunization strategy, could increase vaccination coverage and reduce time-cost vaccines campaigns, adding the benefits of immune response in mucosa.

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines.

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10(7) M(-1). The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.

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.

Four monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, C, Y and W135: its application in identity tests.

Murine hybridoma monoclonal antibodies (MAbs) were produced against the capsular polysaccharide (CPs) of serogroups A, C, W135 and Y meningococci (MenA, MenC, MenW, MenY) in order to develop immunological reagents for the identification of meningococcal polysaccharides. Each serogroup-specific MAb reacted with the CPs from its homologous serogroup only and did not react with CPs from the other three serogroups. The affinity constant (Ka) of the four MAbs measured by non-competitive ELISA was 6.62 × 10(9), 2.76 × 10(9), 1.48 × 10(9) and 3.8 × 10(9) M(-1) for MenA, MenC, MenW and MenY MAbs respectively. The application of these MAbs for identity tests was demonstrated by their abilities to correctly identify the CPs from serogroups A, C, W135 and Y in meningococcal CPs-based vaccines through ELISA. The MAbs obtained in this work are a very valuable set of tools for study meningococcal polysaccharides vaccines.

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 and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1.

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.

VA-MENGOC-BC Vaccination Induces Serum and Mucosal Anti Neisseria gonorrhoeae Immune Responses and Reduces the Incidence of Gonorrhea.

BACKGROUND: Overall, there are over 30 different sexually transmitted infections with Neisseria gonorrhoeae being the third most frequent with a reported 78 million cases per year. Gonococcal infection causes genital inflammation, which can be a risk factor for others sexually transmitted infections, particularly human immunodeficiency virus. Gonorrhea is a treatable disease, but recently an increase in antibiotic resistance has been of concern. There are currently no vaccines available. However, parenteral vaccination with anti N. meningitidis serogroup B vaccine has been reported to decrease the incidence of gonococcal burden in New Zealand and in Cuba despite the fact that parenteral vaccination is not deemed to induce mucosal IgA. Here we explore possible mechanisms of protection against gonococcal infection through parenteral meningococcal B vaccination. METHODS: Ninety-two serum, saliva and oropharyngeal swabs samples of young adults (healthy and Neisseria carriers) of the internal higher school were obtained. They have been vaccinated with VA-MENGOC-BC (MBV) during their infancy and boosted with a third dose during this study. Serum and saliva samples were analyzed by ELISA and Western blot to measured IgG and IgA antibodies against N. meningitidis and N. gonorrhoeae antigens. N. meningitidis carriers were determined by standard microbiologic test. In addition, we reviewed epidemiologic data for N. meningitidis and N. gonorrhoeae infections in Cuba. RESULTS: Epidemiologic data show the influence of MBV over gonorrhea incidence suggesting to be dependent of sexual arrival age of vaccines but not over syphilis. Laboratorial data permit the detection of 70 and 22 noncarriers and carriers of N. meningitidis, respectively. Serum anti-MBV antigens (PL) responses were boosted by a third dose and were independent of carriage stages, but saliva anti-PL IgA responses were only present and were significant induced in carriers subjects. Carriers boosted with a third dose of MBV induced similar antigonococcal and -PL saliva IgA and serum IgG responses; meanwhile, serum antigonococcal IgG was significantly lower. In saliva, at least 2 gonococcal antigens were identified by Western blot. Finally, gonococcal-specific mucosal IgA antibody responses, in addition to the serum IgG antibodies, might contributed to the reduction of the incidence of N. gonorrhoeae. We hypothesize that this might have contributed to the observed reductions of the incidence of N. gonorrhoeae. CONCLUSION: These results suggest a mechanism for the influence of a Proteoliposome-based meningococcal BC vaccine on gonococcal incidence.

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.

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.

Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroups A, C, W and Y in multivalent vaccines.

Neisseria meningitidis is a Gram negative bacterium that has been classified in 13 serogroups according to the biochemical composition of the capsular polysaccharide (CP). However, invasive infections are most frequently caused by six of these serogroups: A, B, C, W, X and Y (MenA, MenB, MenC, MenW, MenX, MenY). Individual CP quantitation in multivalent meningococcal CP-based vaccines is required for quality control testing of these products. In this regard, four sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the quantitation of CP. The quantitation and detection limits of the four ELISAs were below 1ng/mL. The assays showed good reproducibility and repeatability as calculated for each point of the standard curve (CV<15%). In addition, five multivalent meningococcal CP-based vaccines were evaluated and the proposed ELISAs showed that these vaccines were found into the accepted range (±30%) of CP content. These assays are suitable for screening multiple plain or conjugated meningococcal CP-based vaccines and could be useful for monitoring lot-to-lot consistency and stability analysis.

The golden era of vaccine adjuvants.

A 5-day workshop on adjuvant for vaccines was held in Trinidad, Cuba, on the 16-21 May 2010. Organized by the Latin American Association for Immunology and the Cuban Society for Immunology, the workshop attracted more than 60 scientists from different parts of the world. The meeting summarizes current knowledge regarding vaccine adjuvants, including delivery systems and parasitic vaccines. Main discussion topics were the discovery of new adjuvant formulations, action mechanisms for adjuvants, adjuvants for a different route of immunization, focused on mucosal vaccines, and nano- and micro-particles as a delivery system. This article highlights the most important issues presented.

Intranasal immunization with a proteoliposome-derived cochleate containing recombinant gD protein confers protective immunity against genital herpes in mice.

The purpose of this study was to investigate the potential of intranasal (IN) immunization with Neisseria meningitides B proteoliposome (AFPL1) and AFPL1-derived cochleate (AFCo1), containing glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) for induction of protective immunity against genital herpes infection in mice. We could show that IN immunization with both AFPL1 and AFCo1 containing gD induced gD-specific IgG antibody and lymphoproliferative responses. However, IFN-gamma response could only be detected in CD4(+) splenic cells and genital lymph node cells of the AFCo1gD immunized mice upon recall antigen stimulation in vitro. Importantly, IN immunization with AFCo1gD could elicit a complete protection against an otherwise lethal vaginal challenge with HSV-2, while the AFPL1gD immunized mice were only partially protected. Further, we could show that the IFN-gamma response and protective immunity observed after IN immunization with AFCo1gD are mediated via the adaptor molecule myeloid differentiation factor 88. These data may have implications for the development of a mucosal vaccine against genital herpes.

New meningococcal C polysaccharide-tetanus toxoid conjugate Physico-chemical and immunological characterization.

The polysaccharides (Ps) are thymus-independent 2 (TI-2) antigens and poor immunogens in infants and young children; as a result of this delayed response to Ps antigens during ontogeny, infants and young children are highly susceptible to infections caused by encapsulated bacteria. Meningococcal group C polysaccharide (PsC)-proteins conjugate vaccines have been reported to induce significant serum IgG antibodies and immunologic memory in infants resulting in very effective vaccines. We describe here the obtainment, by a new method, of a neoglycoconjugate intended to immunize against Neisseria meningitidis serogroup C, its characterization by physico-chemical methods, including (1)H NMR and fluorescence spectroscopy methods, as well as the characterization of the immune response induced in mice by such conjugate. Amine groups generated by basic hydrolysis in the PsC were successfully conjugated to carboxyl groups of tetanus toxoid (TT), using carbodiimide-mediated coupling. The specific anti-Ps IgG and anti-Ps IgG subclasses (IgG1 and IgG2a) were measured by ELISA methods, the bactericidal activity in sera and the cytokines response (IFNgamma or IL5) in spleen cell of mice immunized with conjugated and native Ps were evaluated. The (1)H NMR spectra and the result obtained by the fluorescence spectroscopy method showed that the PsC and TT maintained structural identity after conjugation process. Conjugated PsC elicited an increase of anti-PsC IgG responses, anti-PsC IgG subclass (IgG1, IgG2a), an eight-fold increase in bactericidal activity in sera of mice immunized with conjugate compared with native PsC, was also observed. Higher titres of IFNgamma were observed in mice immunized with conjugated Ps. These results indicated that, the PsC and TT maintained its chemical and antigenic structure after the conjugation process. A change in the immunological pattern of responses of PsC, from TI-2 to a thymus-dependent (TD) pattern, was also demonstrated.

La gD2 coadministrada con el AFCo1 por vía intranasal induce inmunidad protectora contra virus de herpes simple tipo 2 en ratones / gD2 coadministered with AFCo1 by intranasal route induces protective immunity against Virus Herpes Simplex type 2 in mice

La infección por virus herpes simple tipo 2 (VHS-2) continúa siendo un problema de salud mundial. Esta infección es transmitida sexualmente y es la principal causa de úlceras genitales. La prevención de esta enfermedad requiere de la utilización de vacunas mucosales, pues las vacunas parenterales no han sido exitosas. Por otra parte, no existen adyuvantes mucosales, por lo que el desarrollo de estos es esencial para la estrategia de estas vacunas. La administración intranasal (IN) de la glicoproteína D del VHS-2 (gD2), coadministrada con el cocleato (AFCo1+gD2) sería igualmente efectiva con la gD2 incluida (AFCo1-gD2). Se inocularon ratones hembras C57BL/6 por la vía IN con gD2, contenida dentro del cocleato, coadministrada con el cocleato o gD2 sola. Se determinaron los niveles de IgG anti gD2 en suero y lavado vaginal, así como las subclases de IgG anti gD2 por ELISA. Se determinó la respuesta linfoproliferativa en células de bazo, el perfil de citoquinas Th1/Th2, los signos de la enfermedad y la protección frente al reto viral. Se observaron altos títulos de IgG e IgG2c anti gD2 en el suero de los animales inoculados con la gD2 y el AFCo1 como adyuvante. No se observaron diferencias significativas (p>0,05) entre los grupos que recibieron AFCo1+gD2 y los que recibieron AFCo1-gD2. Se observó un perfil de citoquinas tipo Th1 y un 100 por ciento de sobrevida en los grupos que recibieron el AFCo1 como adyuvante de la gD2, mientras que en el grupo que recibió la gD2 sola no se observó protección. Estos resultados indican que la gD2 puede ser utilizada coadministrada con AFCo1 por vía IN como un potencial candidato vacunal contra VHS-2(AU)

Sexually transmitted infections by Herpes Simplex Virus type 2 (HSV-2) are the leading cause of genital ulcers and a major public health problem worldwide. This requires the use of mucosal vaccines, because parenteral vaccines have not been successful. Presently, there are not mucosal adjuvants, for this reason the development of adjuvants is essential for mucosal vaccine strategies. The intranasal (IN) immunizations using HSV-2 glycoprotein D (gD2), coadministered with cochleate (AFCo1+gD2), would be an efficient candidate for future vaccines against HSV2, similar to the gD2 incorporated into AFCo1(AFCo1-gD2). Female C57Bl/6 mice were inoculated with AFCo1-gD2, AFCo1+gD2 or gD2 alone by IN route. The anti gD2 IgG in sera and vaginal fluids and IgG subclasses were measured by ELISA. The lymphoproliferative response in spleen cells, the Th1/Th2 cytokine profile, the protection and the signs of disease against viral challenge were measured. High titers of IgG and IgG2c subclasses were observed in sera of mice that received the gD2 and AFCo1 as adjuvant. No significant differences (p>0.005) were observed in the animals that received AFCo1+gD2 or AFCo1-gD2. a preferential Th1 cytokine profile and 100 percent of survival after challenge were observed in both groups that received the gD2 and AFCo1, while no survival was observed in the group that only received the gD2. These results showed that the gD2 can be used coadministered with AFCo1 by IN route as a potential vaccine candidate against HSV-2(AU)

Influencia de las vías de inmunización mucosales sobre la protección contra herpes simple tipo 2 con el AFCo1 como adyuvante / Influence of mucosal immunization routes on herpes simplex virus type 2 protection with AFCol as adjuvant

Las vacunas mucosales se han planteado como una estrategia prometedora para inducir protección mucosal. El virus herpes simple tipo 2 es uno de los patógenos más frecuentes en el humano transmitidos por vía sexual. Varios candidatos vacunales contra este patógeno se han evaluado, pero no han sido efectivos, por lo que aún no se cuenta con una vacuna profiláctica ni terapéutica. La gD2 es una glicoproteína recombinante y está reportada como uno de los antígenos de importancia vacunal contra este germen. Contamos con el cocleato derivado del proteoliposoma de Neisseria meningitidis serogrupo B (AFCo1) que ha mostrado capacidades adyuvantes por varias vías de inmunización. El objetivo de este trabajo fue evaluar la protección inducida en ratones por el AFCo1-gD2, administrada por diferentes vías mucosales. Se utilizaron ratones hembras C57BL6, los cuales fueron inmunizados por vía intranasal (IN), intravaginal (IVag) o intrarrectal (IR) con AFCo1-gD2 o gD2 sola. Se determinó la IgG anti gD2, la proliferación celular específica, la replicación viral en lavado vaginal, los signos de la enfermedad y la protección frente al reto viral. Se obtuvo respuestas significativas de IgG anti gD2 por todas las vías, aunque la IN mostró los valores más elevados. Se observó proliferación celular en células de animales inmunizados IN e IVag, pero no por vía IR. Se observó la mayor protección (100 por ciento) en los animales inmunizados por vía IN. Se concluye que la vía nasal es la más prometedora en la inducción de protección contra este reto viral(AU)

Mucosal vaccines are a promising strategy to induce mucosal protection. Herpes simplex virus type 2 is the commonest pathogens in the human transmitted by exposure at the genital mucosal surfaces. Many vaccine candidates against this pathogen have been evaluated; but they have not been effective, and neither a prophylactic nor a therapeutic vaccine has been yet obtained. The gD2 is a recombinant glycoprotein and it is reported as one of the antigens of importance for vaccine against this virus. There is the cochleate (AFCo1) derived from proteoliposome of Neisseria meningitidis serogroup B. This cochleate has shown potentialities as adjuvant for several immunization routes. The objective of this study was to evaluate the protection induced in mice by the AFCo1-gD2 administered by different mucosal routes. Female mice C57BL/6 were used and immunized by: intranasal (IN), intravaginal (IVag), or intrarectal (IR) routes with AFCo1-gD2 or gD2 alone. The anti-gD2 IgG and specific cellular proliferation were determined and the viral replication in vaginal wash, the signs of disease and the protection against the viral challenge, were measured too. A significant anti-gD2 IgG response was obtained by all routes, although the IN route showed the highest values. Cellular proliferation was observed in cells of animals immunized IN and IVag route; but not by IR route. In addition, a higher protection (100 percent) in the animals immunized with AFCo1-gD2 by IN route was observed. In conclusion the intranasal is the most promising route in the protection induction against this viral challenge(AU)

Preparation and evaluation of vibrio cholerae O1 EL Tor Ogawa lipopolysaccharide-tetanus toxoid conjugates.

The lipopolysaccharide (LPS) of Vibrio cholerae is considered one of the most important antigens from the point of view of immunogenicity in these bacteria. We have undertaken detoxification of this LPS by basic hydrolysis and the resultant amine groups were used for their conjugation to tetanus toxoid as carrier protein using carbodiimide-mediated coupling. The resulting conjugates were inoculated in Balb/c mice for immunogenicity studies. The anti-LPS IgG and vibriocidal antibodies were measured in serum. The antigenicity of this conjugated was evaluated by ELISA, with serums of humans vaccinated with a strain genetically modified. The conjugated elicited: high titers of IgG anti-LPS, high titers of vibriocidal antibodies and there was recognition of LPS by antibodies from cholerae immunised human serum. These results show that the conjugated LPS obtained by us, could be evaluated like a potential vaccine for human use.

Adjuvant properties of lipopolysaccharide from Neisseria meningitidis serogroup B detoxified and conjugated with tetanus toxoid.

We evaluated the adjuvant properties and toxicity of purified Neisseria meningitidis serogroup B lipopolysaccharide (LPS) conjugated with tetanus toxoid (TT) using a new method of conjugation to obtain amine groups in the polysaccharide structure. The endotoxic activity of treated LPS was reduced 2400 times as determined by Limulus amoebocyte assay and no mortality was observed in Balb/c mice inoculated with detoxified LPS versus 100% mortality in native LPS inoculated mice. The conjugated LPS-TT elicited in mice higher anti-TT IgG2a and IgG1 than unconjugated TT. In addition, high levels of anti-LPS IgG and IgG subclasses were detected in sera. These results evidence the adjuvant activity of detoxified LPS and may suggest that the conjugation to TT changes the LPS immune response from thymus-independent to thymus-dependent.

Mucosal immune responses to meningococcal C polysaccharide-protein conjugate in mice.

Mucosal delivery of vaccines represents an attractive approach because this is a region of first contact point for inhaled antigens. We have obtained a meningococcal group C polysaccharide-tetanus toxoid conjugate (MGCP-TT) and evaluated it for intranasal route in mice. The conjugate was obtained by a developed method in our laboratory. The specific IgA in saliva and specific IgA and IgG in serum were measured by ELISA methods and bactericidal antibodies in sera against a meningococcal group C strain were measured. The conjugated elicited a significant increase in anti-MGCP salivary IgA and serum IgG and bactericidal antibodies concentrations, while specific serum IgA was not observed. These results indicated that after conjugation, there was a change in the responses for MGCP from thymus-independent to thymus-dependent and that it was effective by intranasal route.

New method for obtaining conjugated vaccines.

We describe a new method to obtain conjugates against Neisseria meningitidis serogroups A, B, C, Vibrio cholera, and Salmonella typhi and their immunogenicity in Balb/c mice. The saccharides were activated by basic hydrolysis with the generation of amine groups in the saccharidic chain, and these groups were linked to carboxyl groups of tetanus toxoid by via carbodiimida-mediated reaction. The resultant conjugates were administered to mice for the immunogenicity studies. The pirogenicity of LPS was measured by LAL assay. The anti-saccharide IgG, IgG1, and IgG2a antibodies were evaluated. A significant decrease in the pirogenicity of LPS after basic hydrolysis treatment was observed. The conjugates elicited higher titers of anti-polysaccharides or anti-LPS IgG, IgG1, and IgG2a in conjugates than in unconjugated saccharides. The results indicate that we have a new method for obtaining conjugated vaccines and we have demonstrated that after conjugation there was a change in the responses for all saccharides, from thymus-independent to thymus-dependent responses.