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Rotavirus Infection Activates Dendritic Cells from Peyer's Patches in Adult Mice

ABSTRACT This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyers patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIMwt). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF-α), and beta interferon (IFN-β), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response.

Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo

Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection.

Cell biology of T cell activation and differentiation.

T cells are major components of the adaptive immune system. They can differentiate into two different populations of effector cells-type one and type two-and may also become tolerant. T cells respond to immune challenges by interacting with antigen-presenting cells of the innate immune system. These latter cells can identify the nature of any immune challenge and initiate adaptive immune responses. Dendritic cells are the most important antigen-presenting cells in the body. The T cell recognizes both peptides associated with MHC molecules on the antigen-presenting cells and also other molecules in a complex structure known as an immunological synapse. The nature of the antigen, the cytokine environment, and other molecules on the dendritic cell surface instruct the T cells as to the response required. A better understanding of the biology of T cell responses offers the prospect of more effective therapeutic interventions.

The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice.

Viral protein assemblies have shown to be superior immunogens used in commercial vaccines. However, little is known about the effect of protein assembly structure in immunogenicity and the protection conferred by a vaccine. In this work, rotavirus VP6, a polymorphic protein that assembles into nanotubes, icosahedra (dlRLP) or trimers was used to compare the immune response elicited by three different assemblies. VP6 is the most antigenic and abundant rotavirus structural protein. It has been demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it a vaccine candidate. Groups of mice were immunized with either nanotubes, dlRLP or trimers and the humoral response (IgG and IgA titers) was measured. Immunized mice were challenged with EDIM rotavirus and protection against rotavirus infection, measured as viral shedding, was evaluated. Immunization with nanotubes resulted in the highest IgG titers, followed by immunization with dlRLP. While immunization with one dose of nanotubes was sufficient to reduce viral shedding by 70%, two doses of dlRLP or trimers were required to obtain a similar protection. The results show that the type of assembly of VP6 results in different humoral responses and protection efficacies against challenge with live virus. This information is important for the design of recombinant vaccines in general.

Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

BackgroundGag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB) when fused to p24.ResultsA fusion between ricin toxin B subunit and p24 HIV (RTB/p24) was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin.ConclusionIn this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when conjugated to an HIV structural protein. This is the first report in which a eukaryotic toxin produced in E. coli is employed as an adjuvant to elicit immune responses to p24 HIV core antigen.

Heterologous Prime-Boost Oral Immunization with GK-1 Peptide from Taenia crassiceps Cysticerci Induces Protective Immunity

ABSTRACT Oral immunization is a goal in vaccine development, particularly for pathogens that enter the host through the mucosal system. This study was designed to explore the immunogenic properties of the Taenia crassiceps protective peptide GK-1 administered orally. Mice were orally immunized with the synthetic GK-1 peptide in its linear form with or without the Brucella lumazine synthase (BLS) protein adjuvant or as a chimera recombinantly bound to BLS (BLS-GK-1). Mice were boosted twice with GK-1 only at 15-day intervals. A significant rate of protection of 64.7% was achieved in GK-1-immunized mice, and that rate significantly increased to 91.8 and 96% when mice were primed with GK-1 coadministered with BLS as an adjuvant and BLS as a carrier, respectively. Specific antibodies and T cell activation and proliferation accompanied the protection induced, revealing the potent immunogenicity of GK-1. Through immunohistochemical studies, GK-1 was detected in T and B cell zones of the Peyers patches (PP) and mesenteric lymph nodes. In the latter, abundant proliferating cells were detected by 5′-bromo-2′-deoxyuridine incorporation. No proliferation was detected in PP. Altogether, these results portray the potent immunogenic properties of GK-1 administered orally and reinforce the usefulness of BLS as an adjuvant and adequate vaccine delivery system for oral vaccines.

CD43 signals induce type one lineage commitment of human CD4(+) T cells

BackgroundThe activation and effector phenotype of T cells depend on the strength of the interaction of the TcR with its cognate antigen and additional signals provided by cytokines and by co-receptors. Lymphocytes sense both the presence of an antigen and also clues from antigen-presenting cells, which dictate the requisite response. CD43 is one of the most abundant molecules on the surface of T cells; it mediates its own signalling events and cooperates with those mediated by the T cell receptor in T cell priming. We have examined the role of CD43 signals on the effector phenotype of adult CD4+ and CD8+ human T cells, both alone and in the presence of signals from the TcR.ResultsCD43 signals direct the expression of IFNγ in human T cells. In freshly isolated CD4+ T cells, CD43 signals potentiated expression of the IFNγ gene induced by TcR activation; this was not seen in CD8+ T cells. In effector cells, CD43 signals alone induced the expression of the IFNγ gene in CD4+ T cells and to a lesser extent in CD8+ cells. The combined signals from CD43 and the TcR increased the transcription of the T-bet gene in CD4+ T cells and inhibited the transcription of the GATA-3 gene in both populations of T cells, thus predisposing CD4+ T cells to commitment to the T1 lineage. In support of this, CD43 signals induced a transient membrane expression of the high-affinity chains of the receptors for IL-12 and IFNγ in CD4+ T cells. CD43 and TcR signals also cooperated with those of IL-12 in the induction of IFNγ expression. Moreover, CD43 signals induced the co-clustering of IFNγR and the TcR and cooperated with TcR and IL-12 signals, triggering a co-capping of both receptors in CD4+ populations, a phenomenon that has been associated with a T1 commitment.ConclusionOur results suggest a key role for CD43 signals in the differentiation of human CD4+ T cells into a T1 pattern.

Immunogenicity and protective efficacy of yeast extracts containing rotavirus-like particles: A potential veterinary vaccine

Rotavirus is the most common cause of severe diarrhea in many animal species of economic interest. A simple, safe and cost-effective vaccine is required for the control and prevention of rotavirus in animals. In this study, we evaluated the use of Saccharomyces cerevisiae extracts containing rotavirus-like particles (RLP) as a vaccine candidate in an adult mice model. Two doses of 1mg of yeast extract containing rotavirus proteins (between 0.3 and 3 μg) resulted in an immunological response capable of reducing the replication of rotavirus after infection. Viral shedding in all mice groups diminished in comparison with the control group when challenged with 100 50% diarrhea doses (DD50) of murine rotavirus strain EDIM. Interestingly, when immunizing intranasally protection against rotavirus infection was observed even when no increase in rotavirus-specific antibody titers was evident, suggesting that cellular responses were responsible of protection. Our results indicate that raw yeast extracts containing rotavirus proteins and RLP are a simple, cost-effective alternative for veterinary vaccines against rotavirus.

CD43 Signals Prepare Human T Cells to Receive Cytokine Differentiation Signals

T cells are increasingly used for passive immunotherapy and bone marrow transplantation. Proper ex‐vivo management of the cells is important for the desired therapeutic effects. For differentiation into effector cells of the Th1 and Th2 phenotypes, T‐cells require signals from IFNγ and IL‐4, respectively. Naïve cells have an extremely low expression of the specific receptors that recognize these cytokines, indicating that in order to differentiate, cells need to perceive other signals that will enable them to sense the cytokine milieu. CD43 has been proposed as one of the molecules that make the initial contacts with antigen presenting cells. We report here that in cord blood, adult naïve and total human T cells, CD43 signals induced the expression of both IFNγ and IL‐4 receptors, mediate their capping, increased their signaling and augmented differentiation mediated by these receptors. CD43 signals also stimulated the expression of IFNγ and in neonatal cells that of IL‐4 as well. These data demonstrate an important role for CD43 signals in T‐cell preparedness for differentiation into effector cells. J. Cell. Physiol. 229: 172–180, 2014.

Asociación entre la presencia de anticuerpos anti-Ras y anti-VPH16 E4/E7 y lesiones intraepiteliales del cérvix

OBJETIVO: Determinar si anticuerpos sericos contra E4, E7 y Ras pueden ser utilizados como marcadores de lesiones tempranas del cervix uterino asociadas al virus del papiloma humano. MATERIAL Y METODOS: Entre marzo de 1999 y abril de 2000 se realizo un estudio sero-epidemiologico de casos y controles en la clinica de displasias del Hospital General Doctor Gea Gonzalez, en la Ciudad de Mexico, en 116 muestras de suero para evaluar la presencia de anticuerpos anti-E4, E7 y Ras utilizando un ELISA de captura. Se estimaron razones de momios e intervalos de confianza de 95% RESULTADOS: Anticuerpos anti-E7 se asociaron a mujeres con lesiones NIC III, mientras que anticuerpos anti-E4 y anti-Ras fueron mas frecuentes en lesiones NIC I-II. Al evaluar el perfil de anticuerpos que presentaron las mujeres, encontramos que a) anticuerpos contra dos proteinas predicen la existencia de una lesion NIC I-II, y b) la presencia de tres anticuerpos predicen una lesion NIC III. CONCLUSIONES: La deteccion de anticuerpos sericos contra E4, E7 y Ras en combinacion con otras tecnicas de diagnostico, podrian ser de utilidad para detectar oportunamente a mujeres con lesiones tempranas asociadas al Virus del Papiloma Humano y en riesgo de desarrollar cancer.