Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Single-domain antibodies as intracellular inhibitors of a bacterial toxin

Los camélidos poseen anticuerpos que carecen de cadena liviana y de una fracción de la cadena pesada. El sitio de unión al antígeno está formado por el dominio variable de la cadena pesada, denominado VHH. Su largo CDR3 resulta especialmente apto para la unión a epitopes cóncavos, como los sitios activos enzimáticos. Salmonella tyhimurium es una bacteria intracelular patógena que posee una ADP‐ribosiltransferasa denominada SpvB, esencial para la virulencia. SpvB induce la depolimerización de los filamentos de actina, llevando a la muerte celular. En este trabajo de Tesis, mostramos la generación de una biblioteca de expresión en fagos, a partir de linfocitos de llamas inmunizadas con el dominio catalítico de SpvB, para obtener VHHs que reconocen a SpvB e inhiben su actividad enzimática. Los clones de VHHs anti‐SpvB se secuenciaron y clasificaron de acuerdo al CDR3. Fueron seleccionados 5 clones, los cuales se expresaron y purificaron como proteína recombinante. Mediante dos ensayos independientes, mostramos que estos VHHs inhiben a SpvB. Uno de los ensayos nos permitió cuantificar la relación molar VHH:SpvB para lograr un 100% de inhibición identificando así a VHH5, que inhibe a SpvB en relación equimolar. Se transfectaron macrófagos con VHH5 y se infectaron con Salmonella typhimurium. El análisis de las células por microscopía de fluorescencia mostró claramente que VHH5 expresado en el interior celular, tiene la capacidad de proteger su citoesqueleto. Por otro lado, mediante la translocación de SpvB al citoplasma, realizamos un ensayo en células transfectadas con VHH5 para discriminar el efecto de SpvB de otros factores de virulencia. Los resultados mostraron que la expresión intracelular de VHH5 protege el citoesqueleto del efecto citopático causado por SpvB. Estos resultados constituyen una prueba de concepto sobre el uso de anticuerpos de dominio único de llama para la inhibición de enzimas intracelulares.Camelids produce unusual antibodies composed only of heavy chains. The antigen combining site of these antibodies is formed solely by the heavy‐chain variable domain (VHH). Their CDR3s form long finger‐like extensions that can protrude into cavities on antigens, e.g. the active site crevice of enzymes. Salmonella tyhimurium are pathogenic intracellular bacteria that express an ADP‐ ribosyltransferase called SpvB, which is essential for its virulence. SpvB induces the depolymerization of actin filaments leading to cell death. In the present work, we show the generation of an expression phage library from SpvB‐immunized llama lymphocytes to obtain VHHs that inhibit SpvB activity. SpvB‐recognizing VHHs clones were sequenced and classified according to their CDR3. Five independent clones were isolated, and the VHH proteins codified by them were expressed and purified. All VHHs were able to inhibit SpvB in two different assays. We quantitatively analyzed the inhibition of SpvB activity by fluorescence using pyrene‐labelled actin as substrate. VHH5 completely inhibited SpvB activity in an equimolar ratio. Furthermore, VHH5 was subcloned in a vector for eukaryotic expression. As such, eukaryotic cells were transfected and cells expressing VHH5 were infected with Salmonella typhimurium to test their effect on virulence. Fluorescence microscopy analyses clearly showed that VHH5, when expressed as an intrabody, effectively protected cells from wild type SpvB‐ expressing strains of Salmonella. Transfected cells were also protected against the cytotoxic activity of a translocation competent chimeric SpvB‐C2 toxin. These results constitute a proof of principle of the use of single domain antibodies for the specific intracellular inhibition of normal or pathogenic enzymatic functions.Fil:Alzogaray, Vanina A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Polymeric Display of Proteins through High Affinity Leucine Zipper Peptide Adaptors

The polymeric display of proteins is a method that could be used to increase the immunogenicity of antigens and to enhance the interaction strength of binding domains for their target ligands through an avidity effect. However, the coupling of proteins to oligomeric scaffolds is challenging. The chemical conjugation and recombinant fusion techniques have limitations that prevent their general use. In this work we describe a simple and effective method for coupling proteins to the decameric structure of Brucella abortus Lumazine Synthase based on the use of a pair of high affinity heterodimeric coiled coil peptides complementary fused to the scaffold and the target protein. Results obtained with a series of proteins demonstrate the capability of this approach to generate polyvalent particles. Furthermore, we show that the method is able to increase the immunogenicity of antigens and produce polyfunctional particles with promising biomedical and nanotechnological applications

A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation

Germinación de Flaveria bidentis bajo diferentes ambientes térmicos y lumínicos

Ponencia presentada en las VIII Jornadas Integradas de Investigación, Extensión y Enseñanza. Córdoba, 20 de noviembre de 2019Flaveria bidentis (L.) Kuntze es una especie nativa perteneciente a la familia de las Asteraceae conocida por ser fuente de flavonoides con alto grado de sulfatación, estructuras muy poco frecuentes en la naturaleza, y con gran potencial como antitrombótico. En estas especies de interés medicinal es fundamental la generación de conocimientos agronómicos que faciliten su cultivo, tales como sus requerimientos térmicos para la germinación, y si su semilla presenta fotoblastismo. El objetivo de este trabajo fue estudiar la germinación de F. bidentis bajo diferentes ambientes térmicos y lumínicos. Se realizaron dos ensayos con semillas cosechadas de plantas de crecimiento espontáneo en el Valle de Punilla, Córdoba, año 2017. Se sembraron 4-6 repeticiones de 30 semillas en placas de Petri, y se colocaron a tres niveles de temperatura (T): 20, 25 y 30ºC; y dos niveles de fotoperiodo (F): 12 h luz, y 24 h oscuridad. Al cabo de 13-16 días, en cada combinación de tratamiento se calculó el poder germinativo (PG), el PG acumulado a mitad de conteo (PGt/2), el índice de germinación relativa a la luz (GRL; Milberg et al., 2007), y la tasa de germinación (TG; El-Keblawy and Al-Rawai 2005). El valor máximo de PG obtenido fue de 36 %, indicando una baja viabilidad, probable de esperar al cosechar semillas de plantas de crecimiento espontáneo, con maduración heterogénea. Se suma además el deterioro provocado por el tiempo transcurrido desde 2017. El GRL fue de 0.53, lo cual clasifica a la especie como indiferente al fotoperiodo. Pese a ello, el efecto de F fue significativo para el PG (no para PGt/2 ni TG), lo cual indica que la luz favorecería a la germinación, aunque F.bidentis no presente requerimientos absolutos. El efecto de la T sobre PG, PGt/2 y TG también fue significativo. La T de 25°C fue la que mostró el mayor PG tanto al final como a mitad de tiempo ensayado. TG también fue mayor a 25°C indicando una mayor velocidad de germinación en esta condición. Estos resultados sirven de base para continuar con el estudio ecofosiologico de la especie a los fines de su domesticacion y cultivo.Fil: Manero, Miranda Nicole. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Andrés, Natanael. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Alzogaray, Maximiliano Miguel. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina.Fil: Agnese, A. M. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina.Fil: Cabrera, J. L. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina.Fil: Davidenco, Vanina. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias; Argentina

IFN-γ induced by BLS.

<p>A: C57BL/10J or C57BL/10ScNJ were immunized with BLS in the right hind footpad. At 48 h total RNA from draining lymph nodes was obtained. The expression of IFN-γ was determined by RT-PCR. Data are expressed as the fold increase of mRNA in draining versus non-draining lymph nodes (n = 6). B: C57BL/10J or C57BL/10ScNJ mice were immunized with BLS or PBS. Splenocytes were re-stimulated with BLS <i>in vitro</i>. IFN-γ in the supernatants were measured by ELISA. ND: Not detectable. Bars represent means+SDs (n = 6). *p<0.05 compared to control. Data of two independent experiments have been pooled.</p

Early activation of specific cells induced by BLS-OVA_257–264.

<p>C57BL/6J mice received CFSE-labeled OT-I CD8+ cells and were then immunized s.c. with BLS-OVA_257–264 (BLS-OVAp), OVA in IFA, OVAp, OVAp in IFA, BLS or PBS (control). At 20 h, draining (inguinal) lymph nodes were removed, and CD69 fluorescence was analyzed by FACS. A: Histograms show CD69 expression in CFSE+ cells. B: Percentage of CFSE+ cells expressing CD69. Bars represent the fold increase of the mean % + SD (n = 9). **p<0.001 and *p<0.05 compared to control. Data of two independent experiments have been pooled.</p

Changes induced by BLS-OVA_257–264 in the phenotype of CD8+-specific cells.

<p>C57BL/6J mice received CFSE-labeled OT-I CD8+ cells and were then immunized s.c. with BLS-OVAp, OVA in IFA, OVAp, OVAp in IFA, BLS or PBS (control). After 5 days, inguinal lymph nodes were removed, and the expression of CD44 and CD127 was analyzed by FACS. A: Dot plots show CD44 and CFSE expression in lymph node cells. B: Bars represent the percentage+SD of CFSE+ cells expressing high levels of CD44+ (n = 10); *p<0.001 compared to control. C: Bars represent the MFI+SD of CD127 in CD44^high CFSE+ cells (n = 10); *p<0.05 compared to control. Pooled data of three experiments are shown.</p

Specific cytotoxicity induced by BLS-OVA_257–264.

<p>A: Representative overlayed histograms of CFSE^high and CFSE^low populations within CFSE+ cells from inguinal lymph nodes of C57BL/6J mice immunized with BLS-OVA_257–264 (BLS-OVAp) in PBS, BLS-OVAp in AlOH or PBS (control). B: Bars represent the mean percentage+SD of specific cytotoxicity in lymph nodes of mice immunized with BLS-OVAp in AlOH, OVA in AlOH, BLS-OVAp in PBS, OVA in PBS, OVAp in PBS, BLS in PBS or with PBS (n = 12). *p<0.05 compared to control. Data from two independent experiments have been pooled (6 mice per group).</p