Nanotechnology for the delivery of vaccines

Liposomes offer an ideal platform for the delivery of subunit vaccines, due to their versatility and flexibility, which allows for antigen as well as immunostimulatory lipids and TLR agonists to become associated with these bilayered vesicles. Liposomes have the ability to protect vaccine antigen, as well as enhance delivery to antigen presenting cells, whilst the importance of cationic surface charge for delivery of TB subunit vaccines and formation of an ‘antigen depot’ may play a key role in boosting cell-mediated immunity and Th1 immune responses. The rational design of vaccine adjuvants requires the thorough investigation into the physicochemical characteristics that dictate the function of a liposomal adjuvant. Within this thesis, physicochemical characteristics were investigated in order to show any effects on the biodistribution profiles and the ensuing immune responses of these formulations. Initially the role of liposome charge within the formulation was investigated and subsequently their efficacy as vaccine adjuvants in combination with their biodistribution was measured to allow the role of formulation in vaccine function to be considered. These results showed that cationic surface charge, in combination with high loading of H56 vaccine antigen through electrostatic binding, was crucial in the promotion of the ‘depot-effect’ at the injection site which increases the initiation of Th1 cell-mediated immune responses that are required to offer protection against tuberculosis. To further investigate this, different methods of liposome production were also investigated where antigen incorporation within the vesicles as well as surface adsorption were adopted. Using the dehydration-rehydration (DRV) method (where liposomes are freeze-dried in the presence of antigen to promote antigen encapsulation) and the double emulsion (DE) method, a range of liposomes entrapping antigen were formulated. Variation in the liposome preparation method can lead to antigen entrapment within the delivery system which has been shown to be greater for DRV-formulated liposomes compared to their DE-counterparts. This resulted in no significant effect on the vaccine biodistribution profile, as well as not significantly altering the efficacy of cationic liposomal adjuvants. To further enhance the efficacy of these systems, the addition of TLR agonists either at the vesicle surface as well as within the delivery system has been displayed through variation in the preparation method. Anionic liposomal adjuvants have been formulated, which displayed rapid drainage from the injection site to the draining lymph nodes and displayed a reduction in measured Th1 immune responses. However, variation in the preparation method can alter the immune response profile for anionic liposomal adjuvants with a bias in immune response to Th2 responses being noted. Through the use of high shear mixing and stepwise incorporation, the efficient loading of TLR agonist within liposomes has been shown. However, interestingly the conjugation between lipid and non-electrostatically bound TLR agonist, followed by insertion into the bilayer of DDA/TDB resulted in localised agonist retention at the injection site and further stimulation of the Th1 immune response at the SOI, spleen and draining lymphatics as well as enhanced antibody titres.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris): Morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n = 190) and manatees (n = 56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immunocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included T$\rm\sb{h}$, T$\rm\sb{c/s}$, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating T$\rm\sb{h}$ cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris) : morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n=180) and manatees (n=56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immuocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included Th, Tes, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating Th cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Impacto de la haploinsuficiencia de CD3 en la expresión y función del complejo TCR/CD3 en humanos y ratones

TCR/CD3 (αβTCRand γδTCR) complexes are members of a family of modular biosensors that are responsible for driving T-cell development, activation, and effector functions. They inform essential checkpoint decisions by relaying key information from their ligand-binding modules (TCR chains) to their signaling modules (CD3γε, CD3δε and CD3ζζ) and onto the intracellular signaling apparatus. Their actions shape the T-cell repertoire, as well as T-cell-mediated immunity; yet, the mechanisms that underlie their activity are still to be precisely determined. As with any molecular machine, understanding how they function depends critically on dissecting how their parts fit and work together. T-cell receptor immunodeficiencies (TCRID) are low-prevalence autosomal recessive diseases characterized by impaired surface TCR expression, frequently associated with peripheral blood T lymphocytopenia, severe combined ID (SCID) and (or) autoimmune symptoms, but not associated with B or natural killer (NK) lymphocytopenia. Several CD3, CD247, and TCR deficiencies have been described which can be classified as complete or partial according to the absence or presence of residual levels of the affected protein. Although rare and sometimes based on single cases, TCRID offer rich information about the underpinnings of human TCR structure and function, which in turn impact our understanding of T-cell development and function... NOTA 520 8 Las inmunodeficiencias humanas del receptor de antígeno de los linfocitos T (TCR) son enfermedades autosómicas recesivas con baja prevalencia, caracterizadas por un defecto de expresión del TCR asociado a una linfopenia T selectiva. La ausencia congénita de componentes del TCR tiene un impacto diferencial en el desarrollo y función de los linfocitos T, que depende de la cadena del TCR afectada y de la especie, siendo en algunos casos diferente en los pacientes humanos en comparación con los modelos en ratones. Las inmunodeficiencias del TCR han sido ampliamente estudiadas, en particular las de las sub-unidades CD3 del complejo receptor. En contraste, hasta ahora es muy poca la atención que se le ha prestado a los potenciales efectos de la haploinsuficiencia de componentes del TCR en el desarrollo, fenotipo y función de los linfocitos T. Esto es debido en gran parte al hecho de que los individuos haploinsuficientes (portadores de mutaciones nulas en heterocigosis) no presentan alteraciones obvias de la inmunidad mediada por células T o signos de enfermedad. En este trabajo se analiza por primera vez, en humanos y modelos de ratones, el impacto de la haploinsuficiencia de CD3 y CD3 en la expresión y funcionalidad del TCR, y su potencial relevancia en el desarrollo y función de los linfocitos T. Los resultados indican que la haploinsuficiencia de CD3 y, en menor medida, la de CD3causa una disminución en la expresión del TCR en la superficie de las células T y T, y provoca alteraciones claras en el desarrollo y función de los linfocitos T en ambas especies...

Étude de la reconstitution de l’immunité spécifique au cytomégalovirus et au virus de la varicelle suite à la transplantation de sang de cordon ombilical

La transplantation de sang de cordon ombilical (TSCO) constitue un traitement de choix pour une multitude de pathologies hématologiques malignes et non malignes chez l’enfant et dans certains cas l’adulte. La TSCO est associée à certaines complications, dont une reconstitution immunitaire plus lente et une incidence élevée d’infections opportunistes, notamment celles reliées au cytomégalovirus (CMV) et au virus varicella-zoster (VZV). Dans le cadre de ce travail, nous nous sommes intéressés dans un premier temps à la caractérisation de la reconstitution immunitaire spécifique au CMV et au VZV. Nos résultats ont démontré que la reconstitution de l’immunité cellulaire ne requiert ni un statut séropositif pré-transplantation ni le développement de la maladie. De plus, des reconstitutions spontanées ont été détectées chez certains patients séronégatifs vis-à-vis du CMV ou du VZV. Outre le fait qu’elle se manifeste surtout à partir de 6 mois post-transplantation, ladite reconstitution mérite le qualificatif de « protectrice » en termes de réactivations virales et du développement de signes cliniques lorsqu’une fréquence de 150 cellules produisant l’IFN-γ/million est dépassée. Toutefois, moins de 5% des patients développent une réponse T anti-VZV et anti-CMV au cours 100 premiers jours suivant la TSCO. Il est donc possible que les lymphocytes CD8+ T provenant du SCO, comparativement à leurs homologues provenant de la moelle osseuse (MO), présentent un défaut de fonctionnalité, communément appelé « épuisement clonal ». La caractérisation du répertoire de récepteurs inhibiteurs exprimés par les cellules T CD8+ suivant la TSCO ou la transplantation de moelle osseuse (TMO) a révélé une augmentation significative de la fréquence des cellules exprimant PD-1 tôt suivant la transplantation. Cette population, caractérisée majoritairement par un phénotype effecteur-mémoire (EM), démontre une perte significative de la capacité proliférative et exprime moins d'IFN-γ, d'IL-2, de TNF-α et de CD107a. Une meilleure caractérisation de la reconstitution immunitaire après TSCO permettrait, d'une part de sélectionner des biomarqueurs en vue d’une meilleure gestion des patients à risques de développer des infections virales et/ou de rechuter, et d'autre part d'améliorer leur pronostic.

Étude de la reconstitution de l’immunité spécifique au cytomégalovirus et au virus de la varicelle suite à la transplantation de sang de cordon ombilical

La transplantation de sang de cordon ombilical (TSCO) constitue un traitement de choix pour une multitude de pathologies hématologiques malignes et non malignes chez l’enfant et dans certains cas l’adulte. La TSCO est associée à certaines complications, dont une reconstitution immunitaire plus lente et une incidence élevée d’infections opportunistes, notamment celles reliées au cytomégalovirus (CMV) et au virus varicella-zoster (VZV). Dans le cadre de ce travail, nous nous sommes intéressés dans un premier temps à la caractérisation de la reconstitution immunitaire spécifique au CMV et au VZV. Nos résultats ont démontré que la reconstitution de l’immunité cellulaire ne requiert ni un statut séropositif pré-transplantation ni le développement de la maladie. De plus, des reconstitutions spontanées ont été détectées chez certains patients séronégatifs vis-à-vis du CMV ou du VZV. Outre le fait qu’elle se manifeste surtout à partir de 6 mois post-transplantation, ladite reconstitution mérite le qualificatif de « protectrice » en termes de réactivations virales et du développement de signes cliniques lorsqu’une fréquence de 150 cellules produisant l’IFN-γ/million est dépassée. Toutefois, moins de 5% des patients développent une réponse T anti-VZV et anti-CMV au cours 100 premiers jours suivant la TSCO. Il est donc possible que les lymphocytes CD8+ T provenant du SCO, comparativement à leurs homologues provenant de la moelle osseuse (MO), présentent un défaut de fonctionnalité, communément appelé « épuisement clonal ». La caractérisation du répertoire de récepteurs inhibiteurs exprimés par les cellules T CD8+ suivant la TSCO ou la transplantation de moelle osseuse (TMO) a révélé une augmentation significative de la fréquence des cellules exprimant PD-1 tôt suivant la transplantation. Cette population, caractérisée majoritairement par un phénotype effecteur-mémoire (EM), démontre une perte significative de la capacité proliférative et exprime moins d'IFN-γ, d'IL-2, de TNF-α et de CD107a. Une meilleure caractérisation de la reconstitution immunitaire après TSCO permettrait, d'une part de sélectionner des biomarqueurs en vue d’une meilleure gestion des patients à risques de développer des infections virales et/ou de rechuter, et d'autre part d'améliorer leur pronostic.

Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Developing a therapeutic anti-dendritic cell antibody to prevent graft versus host disease

Host and donor dendritic cells (DC) stimulate alloreactive donor T lymphocytes, and initiate GVHD. We have shown that polyclonal antibody to the DC surface activation marker human CD83 (anti hCD83), which depletes activated DC, can prevent human DC and T cell induced lethal xenogeneic GVHD in SCID mice without impairing T cell mediated anti-leukaemic and anti-viral (CMV and influenza) immunity (J Exp Med 2009; 206: 387). Therefore, we made and tested a polyclonal anti mouse CD83 (RAM83) antibody in murine HSCT models and developed a human mAb against hCD83 as a potential new therapeutic immunosuppressive agent.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense. © 2012 Nature America, Inc. All rights reserved.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

A Safe Vaccine (DV-STM-07) against Salmonella Infection Prevents Abortion and Confers Protective Immunity to the Pregnant and New Born Mice

Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain delta pmrG-HM-D (DV-STM-07) in female mice before the onset of pregnancy and followed the immune reaction against challenge with virulent S. Typhimurium in pregnant mice. Here we demonstrate that DV-STM-07 vaccine gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. These results suggest that our vaccine DV-STM-07 can be very useful in preventing abortion in the pregnant individuals and confer immunity to the new born. Since there are no such vaccine candidates which can be given to the new born and to the pregnant women, this vaccine holds a very bright future to combat Salmonella induced pregnancy loss.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca²⁺ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. © 2008 Elsevier Inc. All rights reserved.

Humoral immunity in brain aging and Alzheimer's disease.

Although the contribution of inflammatory processes in the etiology of late-onset Alzheimer's disease (AD) has been suspected for years, most studies were confined to the analysis of cell-mediated immunological reactions thought to represent an epiphenomenon of AD lesion development. Based on the traditional view of the "immunological privilege" of the brain, which excludes a direct access of human immunoglobulins (Ig) to the central nervous system under normal conditions, little attention has been paid to a possible role of humoral immunity in AD pathogenesis. In the first part of this review, we summarize evidences for a blood-brain barrier (BBB) dysfunction in this disorder and critically comment on earlier observations supporting the presence of anti-brain autoantibodies and immunoglobulins (Ig) in AD brains. Current concepts regarding the Ig turnover in the central nervous system and the mechanisms of glial and neuronal Fc receptors activation are also discussed. In the second part, we present new ex vivo and in vitro data suggesting that human immunoglobulins can interact with tau protein and alter both the dynamics and structural organization of microtubules. Subsequent experiments needed to test this new working hypothesis are addressed at the end of the review.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance.

Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.