Contact dependent suppression of CD4 T cell activation and proliferation by B cells activated through IgD cross-linking.

Although the co-stimulatory interaction between B and T cells is well defined, recent evidence suggests that B cells also have a regulatory role. Here, we show that B cells activated using anti-IgD conjugated to dextran (α-δ-dex) directly inhibit TCR-induced CD4 T cell activation, proliferation and cytokine production. This effect was observed in CD4 T cells activated both with and without CD28 co-stimulation. T cell viability was unaffected, and the T cell suppressive effect was mediated by contact with IgD activated purified B cells and not by IL-10 or other soluble factors. This is the first evidence of IgD activated B cells mediating inhibition of activation and proliferation of CD4 T cells in humans. This article is protected by copyright. All rights reserved.

Conjugation of lymphoma idiotype to CD40 antibody enhances lymphoma vaccine immunogenicity and antitumor effects in mice.

Personalized immunotherapy of lymphoma based on tumor idiotype (Id) has shown anti-idiotype humoral immune responses in 40%-50% and cellular immune responses in 50%-75% of follicular lymphoma patients, indicating that this therapy can be clinically successful. We have developed a novel vaccine against lymphoma consisting of an anti-CD40 Ab (ADX40) chemically conjugated to the tumor idiotype A20 and tested it in a murine lymphoma model. BALB/c mice were immunized with 2 doses of immunogen alone or in conjunction with additional adjuvants before tumor challenge. ADX40-Id vaccination resulted in significantly retarded tumor growth and reduced mouse morbidity. Moreover, similar mouse survival was obtained with 2 injections of ADX40-Id as with 8 injections using the standard therapy of keyhole limpet hemocyanin Id + GM-CSF. Co-administration of ADX40-Id with 3-O-deacyl-4'-monophosphoryl lipid A further significantly enhanced vaccine efficacy, resulting in an increased overall survival. Anti-Id-specific Abs were detected at elevated levels after ADX40-Id immunization; however, in vivo depletion of CD4 and/or CD8 T cells before challenge showed that CD8 effector T cells were the major mediators of tumor protection. The results of the present study show that the ADX40-Id conjugate vaccine is a potential candidate as a stand-alone vaccine or in combination with currently licensed adjuvants for lymphoma immunotherapy.

An examination of acute physiological and perceptual responses following blood flow restriction exercise using a traditional research device or novel, automated system.

Objective. To compare the acute physiological and perceptual responses to blood flow restriction (BFR) exercise using a traditional research device or novel, automated system.Methods. Forty-four resistance trained individuals performed four sets of unilateral elbow flexion exercise (30% one-repetition maximum) to volitional failure using two distinct restrictive devices [SmartCuffs PRO BFR Model (SMARTCUFF), Hokanson E20 Rapid Inflation device (HOKANSON)] and with two levels of BFR [40% limb occlusion pressure (LOP), 80% LOP]. Blood pressure (BP), muscle thickness (MT), and isometric strength (ISO) were assessed prior to and following exercise. Perceptual responses [ratings of perceived exertion (RPE), discomfort] were assessed prior to exercise and following each exercise set.Main results. Data are displayed as means (SD). Immediately following exercise with 40% LOP, there were no statistical differences between devices for BP, MT, and ISO. However, only following Set 1 of exercise, RPE was greater with SMARTCUFF compared to HOKANSON (p< 0.05). In addition, only following Set 2 of exercise, discomfort was greater with HOKANSON compared to SMARTCUFF (p< 0.001). Immediately following exercise with 80% LOP, there were no statistical differences between devices for BP, MT, and ISO. However, only following Set 4 of exercise, RPE was greater with HOKANSON compared to SMARTCUFF (p< 0.05). In addition, following all exercise sets, discomfort was greater with HOKANSON compared to SMARTCUFF (p< 0.001). For repetitions completed with 40% LOP there were no statistical differences between SMARTCUFF and HOKANSON across any exercise sets. For repetitions completed with 80% LOP there were no statistical differences between SMARTCUFF and HOKANSON across Set 1 of exercise (p= 0.34), however, for Sets 2-4 of exercise, significantly greater number of repetitions were completed during SMARTCUFF than HOKANSON.Significance. The present study provides valuable insight into the efficacy of a novel, automated BFR system (SMARTCUFF) eliciting comparable acute physiological responses to BFR exercise and in some cases favorable perceptual responses when compared to a traditional research device (HOKANSON).

Adult survivors of invasive pneumococcal disease exhibit defective B cell function.

Adults who have recovered from an episode of invasive pneumococcal disease demonstrate defective B cell activation in response to αδ-dex, a polyclonal polysaccharide mimic, compared with matched control subjects. The defect is not overcome by CD4(+) T cell assistance and may explain the relatively poor response to pneumococcal vaccination in survivors of invasive pneumococcal disease.

Kinetics of immune responses to nasal challenge with meningococcal polysaccharide one year after serogroup-C glycoconjugate vaccination.

BACKGROUND: Recipients of serogroup-C glycoconjugate meningococcal vaccine (MCC) exhibit waning of serum bactericidal antibody (SBA) titers, but the rate of decline and the speed of their immunological memory in response to new meningococcal nasopharyngeal colonization are unknown. METHODS: In a prospective challenge study, we measured persistence of SBA and anti-Neisseria meningitidis serogroup-C (MenC) immunoglobulin (Ig) G and IgA in adults aged 18-39, 28 days and 12 months after receiving MCC. Volunteers were then challenged intranasally with 50 µg MenC polysaccharide to mimic meningococcal colonization, and systemic and mucosal antibody responses were measured. RESULTS: All subjects had protective SBA titers (≥8) 28 days after MCC vaccination, but 12.3% and 20.2% had unprotective (<8) or low (<128) levels, respectively, after 12 months. Following rechallenge (12 months postvaccination) and measurement of antibody responses after 4, 7, and 10 days, rises in SBA titers were only observed in subjects with low (<128) or nonprotective (<8) prerechallenge SBA titers. In subjects with pre rechallenge SBA titers <8, the majority did not reach a protective SBA titer until 7 days post-rechallenge. MenC-specific IgG levels rose in both serum and saliva in correlation with SBA titers. No detectable rise in salivary IgA was observed. CONCLUSIONS: In those individuals who fail to retain protective SBA 12 months after MCC, immunological memory fails to generate protective systemic and mucosal antibodies until 7 days post intranasal challenge with cognate meningococcal polysaccharide. This is likely too slow to protect from natural meningococcal infection. MCC vaccinees rely on persistence of antibody levels rather than immunological memory for sustained protection.

CD40mAb adjuvant induces a rapid antibody response that may be beneficial in post-exposure prophylaxis.

Active vaccination can be effective as a post-exposure prophylaxis, but the rapidity of the immune response induced, relative to the incubation time of the pathogen, is critical. We show here that CD40mAb conjugated to antigen induces a more rapid specific antibody response than currently used immunological adjuvants, alum and monophosphoryl lipid A.

Adjuvanticity of anti-cD40 in vaccine development.

CD40 antibodies can be effective adjuvants for humoral and cell-mediated immune responses. As their mode of action is not via toll-like receptors, these adjuvants can avoid many of the side effects associated with other adjuvants that function through these receptors. CD40 antibodies can be effective in high doses, administered separately from antigen, or in very low doses conjugated to the antigen. In the former case, there are likely to be side effects such as splenomegaly, while in the case of conjugates, side effects may be avoided.

Correlation of group C meningococcal conjugate vaccine response with B- and T-lymphocyte activity.

Despite the success of conjugate vaccination against meningococcal group C (MenC) disease, post-vaccination, some individuals still exhibit rapid waning of initially protective bactericidal antibody levels. The mechanism of this relative loss of humoral protection remains undetermined. In this report we have investigated the relationship between T- and B-cell activation and co-stimulation and the loss of protective antibody titers. We have found that healthy volunteers who lose protective MenC antibody levels one year after receipt of glycoconjugate vaccine exhibit no detectable cellular defect in polyclonal B- or T-cell activation, proliferation or the B-memory pool. This suggests that the processes underlying the more rapid loss of antibody levels are independent of defects in either initial T- or B-cell activation.

CD154-CD40 interactions in the control of murine B cell hematopoiesis.

Interactions between CD40 and CD154 play a very important role in control of immune responses, including the delivery of T cell help to B cells and other APCs. Thus far, there has been no role postulated for CD40-CD154 interactions in hematopoiesis. We show here that CD40 is expressed on murine pro-B cells and that its ligation enhances pro-B cell proliferation in vitro and in vivo. In addition, CD154 mRNA is present in the BM. Moreover, we show that a deficiency in CD154 expression has effects on B cell hematopoiesis. Aged, CD154-deficient mice have significantly lower levels of B hematopoietic subsets downstream of pro-B cells in the BM. In addition, B lineage cells reconstitute more slowly following BMT into CD154-deficient recipients. We hypothesize that CD154 is expressed by radio-resistant cells in the BM and plays a role in fine-tuning B cell hematopoiesis.

Functional T-cell deficiency in adolescents who experience serogroup C meningococcal disease despite receiving the meningococcal serogroup C conjugate vaccine.

Some individuals have experienced meningococcal disease despite receiving the meningococcal serogroup C conjugate (MCC) vaccine in adolescence. We sought to determine whether this is due to subclinical functional B- or T-cell immunodeficiency. Of 53 vaccine failures identified by enhanced surveillance of England and Wales from 1999 to 2004, 15 received MCC vaccine in adolescence, 9 of whom were recruited 2 to 6 years following convalescence from meningococcal disease. Their peripheral blood mononuclear cells (PBMCs) were incubated with polyclonal activators designed to mimic T-cell-independent B-cell stimulation by bacterial polysaccharides and the T-cell stimulation provided by the protein component of the conjugate vaccine. Subsequent proliferation and activation of T and B lymphocytes were measured, along with T-cell help to B cells. Compared to age-, sex-, geographically, and ethnicity-matched controls, CD4 T-cell proliferation rates in response to both anti-CD3 (T-cell receptor [TCR]) stimulation and anti-CD3 in the presence of B cells activated through anti-IgD conjugated to dextran (alpha-delta-dex) were lower in PBMCs derived from vaccine failures (P = 0.044 and P = 0.029, respectively). There was reduced CD4 cell activation of the patient cells compared to controls following stimulation by CD3 (P = 0.048). B-cell activation during incubation of PBMCs with the T-cell stimuli, anti-CD3 (P = 0.044), or anti-CD3 plus anti-CD28 (P = 0.018) was relatively impaired in patients. Anti-tetanus toxoid IgG concentrations were lower in the vaccine failure group (P = 0.0385). There was a relative defect of T-cell responsiveness to T-cell-dependent antigen stimulation in MCC vaccine failures, which was manifested in reduced T-cell help to B cells.

B-cell-T-cell activation and interaction in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a heterogeneous group of disorders, characterized by hypogammaglobulinemia and normal or low numbers of B cells, which predispose patients to recurrent infections. Peripheral blood mononuclear cells from 19 patients with CVID, and age- and sex-matched controls, were subjected to an in vitro assay of B-cell-T-cell activation and interaction, using anti-immunoglobulin (Ig)-D conjugated to dextran (alpha-delta-dex), as a polyclonal T independent type 2 antigen mimic, with and without anti-CD3/anti-CD28, as polyclonal T-cell stimuli. Stimulation of lymphocytes with either anti-CD3 or anti-CD3 plus anti-CD28 induced T-cell activation and proliferation in CVID patients who were similar to age- and sex-matched controls, but B cells of patients were significantly less activated when peripheral blood mononuclear cells were stimulated with polyclonal T-cell agonists alone. Comparison of CD86 expression in the patients with matched controls revealed that patients had low B-cell activation in response to T-cell stimuli (bystander T-cell help). In conclusion, this sample of CVID patients exhibits a defect of T-cell "help" to B cells, and/or B-cell response to T-cell help.

Evidence of a functional B-cell immunodeficiency in adults who experience serogroup C meningococcal disease.

After adolescence, the incidence of meningococcal disease decreases with age as a result of the cumulative immunizing effect of repeated nasopharyngeal colonization. Nevertheless, some adults succumb to meningococcal disease, so we hypothesized that this is due to a subtle functional immunological defect. Peripheral blood lymphocytes derived from survivors of serogroup C meningococcal disease and from age- and sex-matched controls were incubated with a polyclonal B-cell activator containing anti-immunoglobulin D (alpha-delta-dex) employed to mimic antigen-specific stimuli encountered during immune responses to bacterial polysaccharides, with and without T-cell activation (using anti-CD3/anti-CD28). Subsequent proliferation and activation of T and B lymphocytes were measured. In patients, T-cell responses to polyclonal stimuli and the delivery of T-cell help to B cells were unimpaired. Levels of B-cell proliferation in response to alpha-delta-dex stimulation alone were low in all samples but were significantly lower in patients than in controls, and these differences were more pronounced with the addition of T-cell help. The data are consistent with the presence of a subtle immunodeficiency in adults who have exhibited susceptibility to meningococcal disease. This defect is manifested as an impaired B-cell response to T-cell-independent type 2 antigens analogous to bacterial capsular polysaccharide.

Liposomal co-entrapment of CD40mAb induces enhanced IgG responses against bacterial polysaccharide and protein.

BACKGROUND: Antibody against CD40 is effective in enhancing immune responses to vaccines when chemically conjugated to the vaccine antigen. Unfortunately the requirement for chemical conjugation presents some difficulties in vaccine production and quality control which are compounded when multivalent vaccines are required. We explore here an alternative to chemical conjugation, involving the co-encapsulation of CD40 antibody and antigens in liposomal vehicles. METHODOLOGY/PRINCIPAL FINDINGS: Anti-mouse CD40 mAb or isotype control mAb were co-entrapped individually in cationic liposomal vehicles with pneumococcal polysaccharides or diphtheria and tetanus toxoids. Retention of CD40 binding activity upon liposomal entrapment was assessed by ELISA and flow cytometry. After subcutaneous immunization of BALB/c female mice, anti-polysaccharide and DT/TT responses were measured by ELISA. Simple co-encapsulation of CD40 antibody allowed for the retention of CD40 binding on the liposome surface, and also produced vaccines with enhanced imunogenicity. Antibody responses against both co-entrapped protein in the form of tetanus toxoid, and Streptococcus pneumoniae capsular polysaccharide, were enhanced by co-encapsulation with CD40 antibody. Surprisingly, liposomal encapsulation also appeared to decrease the toxicity of high doses of CD40 antibody as assessed by the degree of splenomegaly induced. CONCLUSIONS/SIGNIFICANCE: Liposomal co-encapsulation with CD40 antibody may represent a practical means of producing more immunogenic multivalent vaccines and inducing IgG responses against polysaccharides without the need for conjugation.

CD40-mediated enhancement of immune responses against three forms of influenza vaccine.

There is potential for influenza A infections to cause massive morbidity and mortality. Vaccination may be the primary defence against pandemic influenza, and potential pandemic'flu vaccines may be produced conventionally, in embryonated eggs, or as recombinant protein or synthetic peptide vaccines. However the vaccines are produced, the supply may be limiting, and it will be important to enhance the immunogenicity of the vaccines as much as possible. We have shown that conjugation to CD40 binding antibody is a very efficient way of enhancing immune responses against model antigens, but were interested in assessing the effectiveness of this system using influenza vaccines. We produced conjugates of CD40 monoclonal antibody (mAb) and isotype control with three potential influenza vaccines: a peptide-based vaccine containing T- and B-cell epitopes from virus haemagglutinin; a whole, killed virus vaccine; and a commercially produced split virus vaccine. CD40 mAb conjugates in each case were more immunogenic, but the adjuvant effect of CD40 conjugation was greatest with the split vaccine, where antibody responses were enhanced by several hundred-fold after a single immunization, and lymphocyte proliferation in response to antigen in vitro was also strongly enhanced.

Fusion of antigen to Fas-ligand in a DNA vaccine enhances immunogenicity.

DNA vaccines have considerable potential for the prophylaxis and therapy of a range of diseases, but their potential has not been realised largely due to poor immunogenicity. Fas ligand is a pro-apoptotic molecule, able to induce death of Fas expressing cells. We describe the construction of a DNA vaccine encoding a chimeric fusion between Fas ligand and a truncated version of HIV gp120 as a model antigen. The fusion DNA was used as a priming vaccine, along with boosting with recombinant gp120 protein. Priming with fusion protein DNA resulted in a powerful enhancement of immune responses to the protein boost, and, in the presence of aluminum phosphate, to a strong enhancement in T helper 2 type responses. Fas ligand delivered in a separate plasmid also had an adjuvant effect, although it was weaker than that delivered by the fusion protein.

Enhancement of immune responses to an HIV gp120 DNA vaccine by fusion to TNF alpha cDNA.

DNA vaccines have considerable potential for disease prophylaxis and therapy, but are generally poorly immunogenic. A number of means of enhancing immunogenicity have been assessed, including the co-expression of cytokines, the use of heterologous prime-boost regimes, and the addition of more conventional adjuvants. In this study we have assessed the effects on gp120 DNA immunogenicity of in-frame fusion of tumor necrosis factor alpha DNA to DNA encoding a large fragment of HIV gp120. The studies were performed using a DNA prime, protein boost regime and a heterologous boosting protein. Fusion of TNFalpha DNA enhanced Th1 related immune responses against both the priming and the boosting gp120. In-frame fusion of interferon gamma-encoding DNA at the 5' end of the chimeric molecule, to create a tripartite fusion, had no additional effect on immunogenicity.

Co-stimulatory agonists as immunological adjuvants.

The considerable advances made in the fields of molecular biology, genomics, proteomics and protein engineering have led to the identification of a vast range of potential vaccine antigens for a host of man's most serious diseases. However, experience informs us that vaccines based on recombinant proteins and synthetic peptides lack the immunogenicity of the whole, killed pathogens used in traditional vaccines and, as such, clinical use of these immunogens remains negligible. In order to fully realize the potential benefits of recombinant antigen-based vaccines there is a pressing need to identify powerful adjuvants which can safely enhance these weak responses with a minimum of undesirable side effects. Adjuvant research represents a vibrant and fast moving field and recent developments suggest the goal of generating effective, safe and affordable ways of enhancing immune responses appears to be almost within our grasp. The purpose of this article is to review recent advances in adjuvant development using approaches that directly exploit the immune system's own co-stimulatory pathways to exert their function; with a particular emphasis on CD40 and CD28 based therapies.

Antibodies against cell surface antigens as very potent immunological adjuvants.

We describe here two very potent adjuvant systems which are thought to work directly on antigen specific lymphocytes, thus by-passing the normal route for adjuvants, which is to activate antigen presenting cells (APCs) inducing release of inflammatory cytokines with resultant side effects of local and systemic reactogenicity. CD40 and CD28 based adjuvants are extremely potent and should avoid the inflammatory side effects induced by most adjuvants.

CD40 antibody as a potent immunological adjuvant: CD40 antibody provides the CD40 signal to B cells, but does not substitute for T cell help in responses to TD antigens.

Agonistic antibodies against CD40 have great potential as immunological adjuvants. We have shown that CD40mAbs induce strong antibody responses against conjugated antigen, and that this enhancement of responses extends to any sequence physically associated to the CD40 binding moiety, including the antibody's own Fc region. Thus, the CD40mAb acts as a model immunogen, containing both antigenic (i.e. Fc portion) and CD40 binding motifs (i.e. CD40 binding moiety). Using this system we examine here whether CD40mAb is able to directly mimic T cell help to B cells. CD40mAbs have no adjuvant effect in CD4 depleted mice, and thus, do not mimic T cell help. Simultaneous administration of recombinant IL-4 was unable to restore the adjuvant action of anti-CD40 in T cell depleted mice. However, CD40mAbs are effective adjuvants in CD154-/- mice, indicating that the antibodies are able to provide the CD40 stimulus to B cells which is naturally lacking in these mice. Identification of the additional stimuli required to fully mimic T cell help may be advantageous in vaccination of immunosuppressed patients.

An interferon gamma-gp120 fusion delivered as a DNA vaccine induces enhanced priming.

Nucleic acid vaccination has many potential advantages over traditional methods, but suffers from the fact that DNA vaccines tend to be relatively poorly immunogenic. Attempts to enhance DNA vaccine immunogenicity have included the addition of cytokine-encoding plasmids into the formulation, as well as the use of heterologous prime-boost regimes and the addition of conventional adjuvants, such as alum. We have previously shown that interferon gamma fusions have enhanced immunogenicity as recombinant protein vaccines. We have assessed here the immunogenicity of an interferon gamma-gp120 fusion delivered as a DNA vaccine, in the context of a prime-boost strategy and in the presence of absence of aluminium phosphate. Fusion of gp120 DNA to interferon gamma-encoding DNA resulted in strongly enhanced priming, especially of Th1 responses, including IgG2a responses to a protein boost.