Resistance to foot-and-mouth disease virus mediated by trans-acting cellular products.

Upon serial passage of BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) C-S8c1, cells with increased resistance to the virus were selected (J. C. de la Torre, E. Martinez-Salas, J. Diez, and E. Domingo, J. Virol. 63:59-63, 1989). Two highly resistant cell clones, 74A11 and 74D12, were transformed to puromycin resistance (Purr) and were fused to BHK-21 cells transformed to neomycin resistance (Neor). The hybrid Neor Purr cells showed the specific resistance to FMDV C-S8c1 characteristic of clones 74A11 and 74D12. The results suggest that resistance to FMDV C-S8c1 is mediated by trans-acting cellular products. The possibility of engineering constitutive resistance to FMDV is discussed.     

Hepatitis C virus infection of human T lymphocytes is mediated by CD5

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease. Although infection of hepatocytes is mainly responsible for manifestations of hepatitis C, the virus also invades the immune system by a yet-to-be-identified mechanism. Using human T cell lines and primary T lymphocytes as targets and patient-derived HCV as inocula, we aimed to identify how HCV gains entry into these cells. HCV replication was determined by detection of the HCV RNA replicative (negative) strand and viral proteins, while specific antibodies, knocking down gene expression and making otherwise-resistant cells prone to HCV, were employed to identify a receptor molecule determining T lymphocyte permissiveness to HCV infection. The results revealed that T cell susceptibility to HCV requires CD5, a lymphocyte-specific glycoprotein belonging to the scavenger receptor cysteine-rich family. Blocking of T cell CD5 with antibody or silencing with specific short hairpin RNA (shRNA) decreased cell susceptibility to HCV, while increasing CD5 expression by mitogen stimulation had the opposite effect. Moreover, transfection of naturally CD5-deficient HEK-293 fibroblasts with CD5 facilitated infection of these otherwise HCV-resistant cells. In contrast to T cells, hepatocytes do not express CD5. The data revealed that CD5 is a molecule important for HCV entry into human T lymphocytes. This finding provides direct insight into the mechanism of HCV lymphotropism and defines a target for potential interventions against HCV propagating in this extrahepatic compartment.     

Typing foot-and-mouth disease virus by fluorescent antibody technique.

Typing of foot-and-mouth disease (FMD) virus was performed by the direct fluorescent antibody (FA) technique. Type-specific FA was prepared from the following two sorts of procedures: (1) FA against live virus (FA-live) was prepared from hyperimmune serum taken from guinea pigs having received live FMD virus. Then it was adsorbed with concentrated heterotype antigen. (2) FA against inactivated virus (FA-Inact) was prepared from antiserum taken from guinea pigs immunized with purified FMD virus inactivated with acetylethyleneimine. Seventeen strains of FMD virus (seven strains of type A, seven strains of type O, and three strains of thpe C) were used. Type-specific FMD virus antigen was detected distinctly from the monolayer of BHK cells infected with each type of virus and fixed in acetone, in spite of negative results obtained from the cells fixed in methyl alcohol. All the 17 strains were typed successfully by the implementation of these two FA methods.     

Reduced Fas ligand-expressing splenic CD5+ B lymphocytes in severe collagen-induced arthritis

Introduction  

The objective was to study immune regulation in a mouse model of rheumatoid arthritis that exhibits considerable heterogeneity of disease activity.     

Rapid response to Con A by CD4+CD45R- rat memory lymphocytes as compared to CD4+CD45R+ lymphocytes

Functionally distinct subpopulations within the CD4+ subset of T lymphocytes have been described in man, rat, and mouse. In the rat different functions have been assigned to CD45R+ and CD45R- T helper cells. The CD45R+ in contrast to the CD45R- T helper cells have been reported to produce IL-2 and to proliferate well in response both to Con A and in MLR. In the present investigation the kinetics of the response to Con A by the CD45R+ and CD45R- rat T helper subsets have been analyzed. We confirm a strong proliferative response to Con A by CD4+CD45R+ rat T lymphocytes and also that they are the best IL-2 producers. We further demonstrate that CD4+CD45R- cells also produce IL-2, although in order to appreciate this production quantitatively by assays of the culture supernatants it was necessary to block IL-2 absorption by IL-2 receptor (IL-2R) antibodies. This blockage was of importance also in comparisons of the two subsets, since they showed different kinetics of IL-2R appearance. It is demonstrated that the CD4+CD45R- cells respond more rapidly to Con A than the CD4+CD45R+ cells as reflected by phenotypic conversion, IL-2 production, and proliferation. The fast response of the CD4+CD45R- T subset shown in the present study of rat cells and analogous studies of human cells suggests that the memory compartment of T cells besides other characteristics also has the capacity for a more rapid response than naive lymphocytes.     

Human cellular immune response to the saliva of Phlebotomus papatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes

Background

The saliva of sand flies strongly enhances the infectivity of Leishmania in mice. Additionally, pre-exposure to saliva can protect mice from disease progression probably through the induction of a cellular immune response.

Methodology/Principal Findings

We analysed the cellular immune response against the saliva of Phlebotomus papatasi in humans and defined the phenotypic characteristics and cytokine production pattern of specific lymphocytes by flow cytometry. Additionally, proliferation and IFN-γ production of activated cells were analysed in magnetically separated CD4+ and CD8+ T cells. A proliferative response of peripheral blood mononuclear cells against the saliva of Phlebotomus papatasi was demonstrated in nearly 30% of naturally exposed individuals. Salivary extracts did not induce any secretion of IFN-γ but triggered the production of IL-10 primarily by CD8+ lymphocytes. In magnetically separated lymphocytes, the saliva induced the proliferation of both CD4+ and CD8+ T cells which was further enhanced after IL-10 blockage. Interestingly, when activated CD4+ lymphocytes were separated from CD8+ cells, they produced high amounts of IFN-γ.

Conclusion

Herein, we demonstrated that the overall effect of Phlebotomus papatasi saliva was dominated by the activation of IL-10-producing CD8+ cells suggesting a possible detrimental effect of pre-exposure to saliva on human leishmaniasis outcome. However, the activation of Th1 lymphocytes by the saliva provides the rationale to better define the nature of the salivary antigens that could be used for vaccine development.     

Fine mapping of a foot-and-mouth disease virus epitope recognized by serotype-independent monoclonal antibody 4B2

VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-in-dependent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to ⁶ETTLLE¹¹ at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif ⁶ETTLLE¹¹ of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide ²KKTEETTLLEDR¹³ was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr⁷, Thr⁸, and Leu¹⁰ are the functional residues of the 4B2 epitope Glu⁶ and Leu⁹ are required residues, and Glu¹¹ plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.     

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.     

Type I IFN signaling on both B and CD4 T cells is required for protective antibody response to adenovirus

Recombinant adenoviruses have been used as vehicles for gene therapy as well as vaccination against infectious diseases and cancer. Efficient activation of host B cell response to adenoviral vectors that leads to the generation of protective, neutralizing Ab, represents a major barrier for gene therapy, but an attractive feature for vaccine development. What regulate(s) potent B cell response to adenoviral vectors remains incompletely defined. In this study, we showed that type I IFNs induced upon adenoviral infection are critical for multiple stages of adaptive B cell response to adenovirus including early B cell activation, germinal center formation, Ig isotype switching as well as plasma cell differentiation. We further demonstrated that although type I IFN signaling on dendritic cells was important for the production of virus-specific IgM, the generation of protective neutralizing Ab critically depended on type I IFN signaling on both CD4 T and B cells. The results may suggest potential strategies for improving adenovirus-mediated gene therapy in vivo and/or the design of effective vaccines for cancer and infectious diseases.     

Tolerance to cyclosporin A-induced autologous graft-versus-host disease is mediated by a CD4+CD25+ subset of recent thymic emigrants

Our previous studies revealed that both the autoeffector and immunoregulatory T cells in cyclosporin A (CSA)-induced autologous graft-vs-host disease are recent thymic emigrants (RTEs). The autoeffector cells appear in and are released from the thymus during the first week of CSA treatment, whereas the immunoregulatory thymocytes appear during the second week but are not released until several days after cessation of CSA treatment. In the present study, the antigenic phenotypes of these functional T cell subsets were determined by immunomagnetic separation and flow immunocytometric analysis. During CSA wk 1, the autoeffector T cells in both the thymus and lymph node (LN) expressed a CD4+8+ double-positive (DP) phenotype, after which those in the LN became CD8 single positive (SP). Timed thymectomy experiments confirmed that the CD8-SP autoeffector T cells in LN originated from these DP RTEs. During CSA wk 2, the immunoregulatory thymocytes also displayed a DP phenotype. However, they were not exported to the periphery until several days after CSA treatment had been interrupted and they had acquired a CD4-SP phenotype. In LN, these immunoregulatory RTEs expressed the CD25+ marker characteristic of anergic/suppressor T cells. Cell separation and mixing experiments demonstrated that the autoeffector T cells persist in LN after cessation of CSA treatment, but their activity is not detectable in the presence of recently exported CD4+ T cells. Hence, the results indicate that tolerance to CSA-induced autologous graft-vs-host disease is actively mediated by CD25+CD4+ RTEs that suppress the function of CD8 autoeffector T cells.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

Selective lymphocyte depletion during the early stage of the immune response to foot-and-mouth disease virus infection in swine

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious vesicular disease of cloven-hoofed animals. In the present study we use FMDV serotype C infection of swine to determine, by analytical techniques, the direct ex vivo visualization of virus-infected immune cells during the first 17 days of infection. We report, for the first time, that FMDV C-S8c1 can infect T and B cells at short periods of time postinoculation, corresponding with the peak of the viremia. There is a significant lymphopenia that involves CD3(+) CD4(-) CD8(+/-), CD3(+) CD4(-) CD8(+)Tc, and CD3(+) CD4(+) CD8(+) memory Th but not CD3(+) CD4(+) CD8(-) na?ve Th lymphocytes. In addition, a profound depletion of the vast majority of peripheral T cells in lymph nodes and spleen is observed. This selective depletion of T cells is not due mainly to in situ death via apoptosis as visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. Thus, early infection of T cells by FMDV may be the main cause of the observed T-cell depletion. Importantly, this lack of T cells is reflected in a reduced response to mitogen activation, which in many cases is totally eliminated. These data suggest a mechanism by which the virus causes a transient immunosuppression, subvert the immune systems, and spreads. These results have important implications for our understanding of early events in the development of a robust immune response against FMDV.     

Modulation of the CD8+-T-cell response by CD4+ CD25+ regulatory T cells in patients with hepatitis B virus infection

CD4+ CD25+ regulatory T cells have been shown to maintain peripheral tolerance against self and foreign antigens. In this study we analyzed the effect of circulating CD4+ CD25+ T cells on CD8+-T-cell responses of patients with chronic and resolved hepatitis B virus (HBV) infection. We demonstrated that circulating CD4+ CD25+ T cells modulate the function and expansion of HBV-specific CD8+ cells ex vivo in all patients, regardless of whether they have chronic or resolved HBV infection. The possible role of CD4+ CD25+ T cells in the pathogenesis of chronic HBV infection is not supported by these data. However, these results might have implications for optimizing future immunotherapeutic approaches to HBV treatment.     

Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.     

Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures.

Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus. Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells. The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced. For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response. The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures. We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus.     

Fine specificity of the in vitro antibody response to influenza virus by human blood lymphocytes

The fine specificity of anti-influenza antibody produced in vitro by human PBM stimulated with different strains of influenza virus was examined by competition binding in solid phase enzyme immunoassay. Most of the antibody produced in vitro is directed to strain-specific or cross-reactive determinants on the hemagglutinin molecule. The extent of cross-reactivity is dependent on the strain of virus used to stimulate PBM as well as the individual tested and presumably on his previous exposure to influenza viruses. PBM from some individuals produced antibody that bound to the stimulating strain of influenza virus but not to other strains of the same subtype. In other individuals, antibody was produced in vitro that cross-reacted with all viruses in the same subtype (e.g., H3N2; A/X31, A/X47, and A/Texas) but did not bind to other (H2N1 or H1N1) subtypes, and in a few individuals, extensive cross-reaction between subtypes was seen. The presence of antibody to hemagglutinin in these culture supernatants was confirmed by competition binding to highly purified hemagglutinin. This in vitro culture system allows the immunologic memory of individuals to a wide range of stimulating virus strains to be examined simultaneously in terms of specificity of the antibody response by human PBM to influenza virus after natural infection or immunization.     

The epithelial integrin alphavbeta6 is a receptor for foot-and-mouth disease virus

Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin alphavbeta3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, alphavbeta6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin beta6 subunit and expression of alphavbeta6 at the cell surface. Integrin alphavbeta6 is the major site for virus attachment on the beta6-transfected cells, and binding to alphavbeta6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the beta6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes alphavbeta6. These studies establish a role for alphavbeta6 as a cellular receptor for FMDV.