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 (α-δ-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.The meningococcal serogroup C conjugate (MCC) vaccine has markedly reduced the incidence of serogroup C meningococcal disease (MD) where it has been used (21), but the initial campaign did not protect all vaccinated people. By 2004, 53 vaccine failures (defined as a case of serogroup C MD occurring more than 10 days after vaccination with MCC vaccine) had been identified in the United Kingdom (1).Acute- and convalescent-phase sera from these patients suggested there had been some initial protection induced by MCC vaccine that was boosted by disease, but the serum bactericidal antibody (SBA) titer at the time of subsequent contact with meningococci was not protective, and the memory response was too slow to confer protection (1). Most vaccine failures were infants. Since the majority of infants have a rapid wane of SBA titer (20), a booster dose of MCC vaccine at 1 year has now been added to the infant vaccine schedule in the United Kingdom. However, some vaccine failures were vaccinated in adolescence, a group that received a single dose of vaccine but in whom mature immune responses should occur (2).Capsular polysaccharide (PS) is a T-cell-independent (TI-2) antigen. Polysaccharide-protein conjugate vaccines comprise the appropriate capsular polysaccharide covalently attached to a carrier protein, such as tetanus toxoid (TT), diphtheria toxoid (DT), or cross-reacting material (CRM) mutant toxin. Their superior efficacy compared to plain polysaccharide vaccines is due to the presentation of carrier epitopes by polysaccharide-specific B cells to carrier-specific T cells, followed by the provision of T-cell help to the PS-specific B cells. This T-cell help enhances B-cell proliferation, class switching, and memory B-cell formation (10). The levels of memory B cells induced correlate with both the initial antibody response and the persistence of the antibody response (3). The induction of a strong immune response against the polysaccharide component of the MCC vaccine therefore requires effective B-cell-T-cell cross talk in both directions.As the immunological response to this vaccine is complex, it is possible that relative inefficiency of one or more components of the response might underlie variation in immunogenicity between individuals, the extreme manifestation of which is serogroup C disease in adolescents despite vaccination. Therefore, we tested the hypothesis that adolescent vaccine failures have an unidentified intrinsic defect in T- or B-cell function, or in cross talk between these cells, expressed as reduced activation and proliferation in response to appropriate stimuli.     

CD40 antibody as an adjuvant induces enhanced T cell responses

Monoclonal antibodies against CD40, conjugated to antigen, act as potent immunological adjuvants for primary antibody responses. We show here that CD40mAbs can also act as strong adjuvants for memory antibody responses, and for T cell responses as measured by ex vivo T cell proliferation to antigen, and delayed type hypersensitivity. Interferon gamma secretion in response to antigen is also enhanced. Finally, the adjuvant effect of CD40mAbs for secondary antibody responses is transferred with T cells rather than B cells. CD40mAb apparently have potent adjuvant effects on both Th1-like cells, and on T cells able to promote B cell antibody production. It is possible that the adjuvant effects of CD40 are mediated at least in part, indirectly, through enhanced antigen presentation by specific B cells, to T cells.     

Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses

Uveal melanoma is a rare malignancy with a poor prognosis despite current therapeutic intervention. The current investigation focuses on the immunogenicity of uveal melanoma cells genetically modified with recombinant adenovirus encoding CD80 (AdCD80) in contrast to their parental counterpart. We demonstrate that costimulation provided by uveal melanoma cells improved immune responses in vitro as determined by mixed lymphocyte tumour cell cultures and cytotoxic T-cell assays using lymphocytes from healthy donors and uveal melanoma patients. Flow cytometry revealed T-cell stimulation by activated CD4+ and CD8+ T cells. Additionally, autologous lymphocytes proliferated in response to CD80-expressing primary uveal melanomas, indicating that this patient group is suitable for immunotherapy. Moreover, this study utilized AdCD80 modified and parental apoptotic tumour cells, loaded onto immature dendritic cells, as a source of tumour antigen. The ability of live or apoptotic tumour cells to stimulate lymphocyte proliferation and activation was determined. Apoptotic uveal melanoma cells expressing CD80 were efficient at inducing an immune response and served as a potent immunogen. The use of apoptotic uveal melanoma cells in combination with expression of costimulatory molecules could prove a novel adjuvant therapy for the treatment of this disease.     

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.     

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.     

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.     

A potent adjuvant effect of CD40 antibody attached to antigen

There is great potential for novel vaccines based on recombinant proteins and synthetic peptides. Unfortunately these antigens often lack the immunogenicity of whole, killed pathogens used in traditional vaccines. Thus there is strong interest in the identification of immunological adjuvants with low reactogenicity, but high potency, to enhance immune responses and realize the potential of these new vaccine strategies. CD40 antibodies have been shown to have adjuvant effects when administered at very high doses. These large doses are impractical and induce a cascade of cytokine release giving rise to septic shock-like symptoms, as well as splenomegaly and polyclonal antibody production. We show here that a very small amount of CD40 antibody can exhibit potent adjuvant effects when attached to soluble antigen. The lack of detectable systemic effects indicates that this method may be a powerful and practical means of enhancing the efficacy of recombinant vaccines.     

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 T-cell receptor-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 interleukin-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.     

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 (α-δ-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 α-δ-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.Meningococcal disease is more common in infants than in adults, likely due to increasing protection over time due to cumulative immunizing episodes of natural nasopharyngeal carriage of Neisseria meningitidis. Several factors are known to determine susceptibility to meningococcal disease, but chief among these is the possession of protective levels of serum bactericidal antibody (SBA) directed against the meningococcus (2). The generation of natural immunity is thought to be the result of recurrent oronasopharyngeal colonization by meningococci. Most colonizing events are transient and result in expulsion of the organism, along with natural generation of protective immunoglobulin G (IgG) antibody against the colonizing strain (23). During this natural exposure to bacteria, responses are generated against the capsular polysaccharide and against T-cell-dependent (TD) protein antigens. Antibodies against both polysaccharides and proteins can exert bactericidal activity in vitro. Carriage of Neisseria spp. is common (14). In older children and adults in the United Kingdom, the prevalence of carriage of N. meningitidis is 25 to 37% (3), and therefore the probability that an adult has never been exposed to the organism is low and cumulative immunizing exposure to the meningococcus should be significant by the time a person reaches adulthood. Nevertheless, there remains a significant disease burden in adults. Goldschneider and colleagues (5) collected serum over the course of an epidemic caused by serogroup C among adult army recruits and demonstrated that patients with meningococcal disease did not have bactericidal antibody against the cognate serogroup prior to the onset of their disease. It was also discovered that patients were relatively deficient of antibody to heterologous serogroup strains. Although this might reflect reduced prior meningococcal exposure for recruits drawn from some geographic regions of the United States with a low incidence of carriage, there remains the possibility that adults who suffer serogroup C meningococcal disease have defective humoral immunity to pathogenic meningococci in general. Although carriage of serogroup C N. meningitidis is infrequent in the general population (<0.5%), most individuals who become colonized by serogroup C do not experience disease (26), despite the fact that adults in an unvaccinated population do not have serum serogroup C bactericidal activity or high concentrations of antipolysaccharide antibody (27).The chief target for protective immunity against the meningococcus is the polysaccharide capsule. Capsular polysaccharides, as T-cell-independent type 2 (TI-2) antigens, generate relatively weak IgG responses when delivered alone, but the response becomes TD when the polysaccharide is presented with protein, for example, when it is delivered as part of a protein conjugate vaccine (22). Capsular polysaccharides contain multiple repeating epitopes and therefore have the ability to cross-link B-cell receptors and induce B-cell activation directly (16), unlike TD protein antigens. However, immune responses against protein antigens can also contribute to SBA titers, as immunization with outer membrane vesicles (24) or colonization with neisserial species or strains not sharing the same capsular antigen (12) can each induce SBA. Antibody responses against TD antigens such as proteins require T-cell help, but there is also some evidence from model systems that responses against capsular polysaccharides presented on whole organisms can also benefit from T-cell help (10).We postulated that adults who suffer meningococcal disease have a subtle deficiency of the humoral immune response to colonization by Neisseria, leading to a failure to rapidly generate protective antibody titers. We tested this hypothesis by using a polyclonal TI-2 antigen mimic and polyclonal T-cell activators to stimulate peripheral B and T lymphocytes derived from recovered meningococcal patients and controls.