Solid matrix-antibody-antigen complexes induce antigen-specific CD8+ cells that clear a persistent paramyxovirus infection

We have previously shown that the adoptive transfer of splenocytes, isolated from mice immunized by infection with the paramyxovirus simian virus 5 (SV5), enhance the speed of clearance of SV5 from the lungs of immunodeficient mice; clearance is mediated primarily through CD8+ effector cells and not by serum neutralizing antibody (D.F. Young, R.E. Randall, J.A. Hoyle, and B.E. Souberbielle, J. Virol. 64:5403-5411, 1990). In this article we demonstrate that immunization of mice with solid matrix-antibody-antigen (SMAA) complexes also induces CD8+ effector cells that are responsible for clearing persistent SV5 infections in immunodeficient mice. The demonstration that immunization with SMAA complexes (an exogenous antigen) can induce class I-restricted cytotoxic T lymphocytes (CTLs) suggests that that these cells may be responsible for virus clearance in vivo. This premise is supported indirectly by the observation that immunization with SMAA complexes was less efficient in inducing class I-restricted CTLs (as measured in vitro) than was infectious virus and that splenocytes isolated from mice immunized with SMAA complexes were also less efficient in clearing virus from lungs of immunodeficient mice than were splenocytes isolated from mice immunized by infection with virus. This was not because the SMAA complexes were generally less immunogenic than infectious virus, since mice immunized with SMAA complexes (which contained the HN protein of SV5) produced higher levels of neutralizing antibody than mice immunized with infectious virus. In the majority of experiments, fixed and killed suspensions of Staphylococcus aureus Cowan strain A were used as the solid matrix in the construction of SMAA complexes. However, in this article we present evidence that alum-antibody-antigen complexes are as immunogenic as S. aureus A-antibody-antigen complexes. These results suggest that the immunological reactivity of the solid matrix itself does not influence the intensity of the immune response to the antigens of interest in the SMAA complexes. The significance of these results for vaccine design are discussed.     

Immunisation of macaques with SIV env recombinants: Specificity of T cell and antibody responses and evaluation of protective efficacy

Macaques were immunised with lentil lectin purified recombinant SIVmac (BK28) derived gp160 (rgp160) with or without live vaccinia (vac)-env (BK28) priming, followed by a final boost with solid matrix antibody antigen (SMAA)-gp160 (J5) complexes and challenged with the SIVmac molecularly cloned virus J5M. Rgp160 and vac-env plus gp160 induced strong Ab responses against the homologous virus. Live vac-env did not enhance or prolong the antibody response, however, T cell responses were stronger. Analysis of the specificity of the immune response demonstrated that sequence variation within SIVmac viruses can affect antibody and T cell recognition. A single booster immunisation with the heterologous SIVmac J5 env recombinant protein was not sufficient to protect against the molecularly cloned virus J5M. These findings further illustrate the difficulty of generating protective immunity with immunogens based on single sequence recombinants.     

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.     

Synthetic multivalent ligands in the exploration of cell-surface interactions

Processes such as cell-cell recognition and the initiation of signal transduction often depend on the formation of multiple receptor-ligand complexes at the cell surface. Synthetic multivalent ligands are unique probes of these complex cell-surface-binding events. Multivalent ligands can be used as inhibitors of receptor-ligand interactions or as activators of signal transduction pathways. Emerging from these complementary applications is insight into how cells exploit multivalent interactions to bind with increased avidity and specificity and how cell-surface receptor organization influences signaling and the cellular responses that result.     

Broadly Protective Adenovirus-Based Multivalent Vaccines against Highly Pathogenic Avian Influenza Viruses for Pandemic Preparedness

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced.     

Repertoire and immunofocusing of CD8 T cell responses generated by HIV-1 gag-pol and expression library immunization vaccines

Several gene-based vaccine approaches are being tested to drive multivalent cellular immune responses to control HIV-1 viral variants. To compare the utility of these approaches, HLA-A*0201 transgenic mice were genetically immunized with plasmids encoding wild-type (wt) gag-pol, codon-optimized (CO) gag-pol, and an expression library immunization (ELI) vaccine genetically re-engineered to express non-CO fragments of gag and pol fused to ubiquitin for proteasome targeting. Equimolar delivery of each vaccine into HLA-A*0201 transgenic mice generated CD8 T cell responses, with the ELI vaccine producing up to 10-fold higher responses than the wt or CO gag-pol plasmids against cognate and mutant epitopes. All three vaccines generated multivalent CD8 responses against varying numbers of epitopes after priming. However, when the animals were immunized again, the wt and CO gag-pol vaccines boosted only the responses against a subset of epitopes and attenuated the responses against all other Ags including epitopes from clade and drug-resistant viral variants. In contrast, the ELI vaccine boosted CD8 responses against all of the gag-pol Ags and against mutant epitopes from clade and drug-resistant variants. These data suggest that HIV-1 vaccines expressing structurally intact gag and pol proteins drive immunofocused CD8 responses that reduce the repertoire of T cell responses. In contrast, the genetically re-engineered ELI vaccine appears to better maintain the multivalent CD8 responses that may be required to control HIV-1 viral variants.     

The complexity of complexes in signal transduction

Many activities of cells are controlled by cell-surface receptors, which in response to ligands, trigger intracellular signaling reactions that elicit cellular responses. A hallmark of these signaling reactions is the reversible nucleation of multicomponent complexes, which typically begin to assemble when ligand-receptor binding allows an enzyme, often a kinase, to create docking sites for signaling molecules through chemical modifications, such as tyrosine phosphorylation. One function of such docking sites is the co-localization of enzymes with their substrates, which can enhance both enzyme activity and specificity. The directed assembly of complexes can also influence the sensitivity of cellular responses to ligand-receptor binding kinetics and determine whether a cellular response is up- or downregulated in response to a ligand stimulus. The full functional implications of ligand-stimulated complex formation are difficult to discern intuitively. Complex formation is governed by conditional interactions among multivalent signaling molecules and influenced by quantitative properties of both the components in a system and the system itself. Even a simple list of the complexes that can potentially form in response to a ligand stimulus is problematic because of the number of ways signaling molecules can be modified and combined. Here, we review the role of multicomponent complexes in signal transduction and advocate the use of mathematical models that incorporate detail at the level of molecular domains to study this important aspect of cellular signaling.     

Multi-gene vaccination against malaria: A multistage, multi-immune response approach

An ideal malaria vaccine will induce immune responses against each stage of the Plasmodium spp life cycle. During its complicated life cycle, the parasite exists extracellularly in the host's bloodstream, within cells that express major histocompatibility complex (MHC) molecules (hepatocytes), within cells that do not express MHC molecules (erythrocytes) and within the mosquito vector. Different arms of the immune system are required to attack the parasite at the different stages. Therefore, a multistage vaccine must be a multi-immune response vaccine. In addition, given the unique antigenicities of the different stages of the life cycle, implicit in this definition is that the vaccine be multivalent. Here, Denise Doolan and Stephen Hoffman present the rationale for developing a multistage, multivalent, multi-immune response malaria vaccine and explain why, among currently available technologies, DNA vaccines may offer the best prospect for success.     

The role of T cell antagonism and original antigenic sin in genetic immunization

To counter highly mutable pathogens like HIV-1, a number of vaccines are being developed to deliver multiple mutant forms of viral Ags to provoke multivalent antiviral CTLs. However, it is uncertain whether such multiple mutant epitope vaccines will generate the diverse CTL responses desired or will instead create immune interference. To characterize the role of immune interference by mutant epitopes in this process, we have tested a "worst case" scenario in which the immunodominant epitope of OVA (SIINFEKL) and its in vitro TCR antagonist (SIINFEDL) have been used to genetically immunize C57BL/6 mice. We demonstrate here that sequential delivery of these mutant epitopes provokes original antigenic sin in CD8 T cells as demonstrated by attenuation of CTLs, intracellular IFN-gamma production, and MHC I peptide-tetramer staining. By contrast, simultaneous exposure of the immune system to this agonist/antagonist pair not only fails to generate T cell antagonism in vivo, but also avoids original antigenic sin. These observations suggest that simultaneous immunization with vaccines containing mutant epitopes, even T cell antagonists, can indeed generate a diverse array of T cell responses and that at least some immune interference can be avoided by delivering mutant Ags to the immune system simultaneously.     

Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model

The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4(+) Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.     

Stable, soluble, model immune complexes made with a versatile multivalent affinity-labeling antigen

We describe here the synthesis of a family of multivalent affinity-labeling antigens based on the soluble carbohydrate polymer Ficoll. Ficoll was derivatized successively with chloroacetate, ethylenediamine, and glutaric anhydride and finally esterified with 2,4-dinitrophenol. Prior to esterification, the polymer could also be derivatized with tyramine to allow trace iodination and with the monosaccharides galactose or mannose. The numbers of substituent groups could be controlled at several points in the synthesis. The resulting multiple dinitrophenyl esters on a Ficoll or glycosylated Ficoll polymer specifically cross-linked anti-dinitrophenyl antibodies to form covalently cross-linked antigen-antibody complexes. The glycosylated Ficolls were particularly made for studies of the influence of antigen structure on the behavior of immune complexes. The intermediates in the synthesis are suitable for other derivatizations as well. These model immune complexes are stable and soluble, they can be separated by size, and they overcome some of the limitations on the study of complexes imposed by previous techniques of preparing them.     

Intra-Golgi formation of IgM-glycosaminoglycan complexes promotes Ig deposition

Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. However, it is not known whether intracellular Ag-Ab interactions also contribute to the formation of extracellular immune complexes. In this study, we report that certain murine B cell hybridomas accumulate intracellular IgM and release large, spherical IgM complexes. The complexes (termed "spherons") reach 2 μm in diameter, detach from the cell surface, and settle out of solution. The spherons contain IgM multimers that incorporate the J chain and resist degradation by endoglycosidase H, arguing for IgM passage through the Golgi. Treatment of cells with inhibitors of proteoglycan synthesis, or incubation of spherons with chondroitinase ABC, degrades spherons, indicating that spheron formation and growth depend on interactions between IgM and glycosaminoglycans. This inference is supported by direct binding of IgM to heparin and hyaluronic acid. We conclude that, as a consequence of IgM binding to glycosaminoglycans, multivalent IgM-glycan complexes form in transit of IgM to the cell surface. Intra-Golgi formation of immune complexes could represent a new pathogenic mechanism for immune complex deposition disorders.     

A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation

Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.     

Polynucleotide vaccines: potential for inducing immunity in animals.

Polynucleotide immunization has been described as the Third Revolution in Vaccinology. Early studies suggest the potential benefits of this form of immunization including: long-lived immunity, a broad-spectrum of immune responses (both cell mediated immunity, and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using a murine model, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to mucosal surfaces have been attempted and are described. Expansion of studies in various species, using natural models, should be extremely helpful in demonstrating the universality of this approach to immunization and more importantly, accurately identify parameters that are critical for the development of protective immunity.     

B cell responses to a peptide epitope. VIII. Immune complex-mediated regulation of memory B cell generation within germinal centers.

Using an in vivo reconstitution assay, we examine here the role of immune complexes in both formation of germinal centers (GC) and processes that occur subsequently within. The presence of Ag, as immune complexes, was found not to constitute a limiting requirement for the initiation of GC formation. No detrimental effect either on numbers or sizes of the resulting GC was observed when Ag-containing immune complexes were omitted during reconstitution. Thus, both recruitment and proliferation of Ag-activated B cells within GC appear not to be limited by Ag concentrations. In contrast, the presence of immune complexes was observed to be obligatory for the generation of Ag-specific memory B cells. This optimally required immune complexes to be constituted by IgG-class Abs with epitope specificities that were homologous to those of the GC B cells. The GC reaction was also found to be characterized by an enhancement of Ab specificity for the homologous epitope. Although some improvement in specificity was noted in recall responses from immune complex-deficient GC, the presence of appropriate immune complexes served to further optimize the outcome. Here again, isotype and epitope-specificity of the Ab constituent in immune complexes proved to be important.     

Strategies for designing vaccines eliciting Th1 responses in humans

There is currently a major interest in designing vaccines capable of eliciting strong cellular immune responses. The induction of cytotoxic and Th1 helper cellular responses is for example highly desirable for vaccines targeting either chronic infectious diseases or cancers (therapeutic vaccines). Similarly, Th1 vaccines would be useful in redirecting inappropriate antigen-specific immune responses in patients with autoimmune diseases and allergies. Importantly, emerging technologies and a better understanding of the physiology of immune responses offer new avenues to rationally design such vaccines. Approaches based on the identification and selection of immunogens containing T cell epitopes can be used, together with epitope-enhancement strategies, to increase binding to MHC, or to improve recognition by T cell receptor complexes. Optimized immunogens can subsequently be presented to the immune system with appropriate vectors allowing to target professional antigen-presenting cells, such as dendritic cells. Such antigen presentation platforms can be used alone or in association, as part of mixed immunization regimens (heterologous prime-boosts), in order to elicit broad immune responses. The rational design of Th1 adjuvants can also benefit from our better understanding of the nature of proinflammatory signals leading to the initiation of both innate and adaptive immune effector mechanisms. Candidate Th1 vaccines (or components such as vectors or adjuvants) will have to be tested in exploratory clinical studies, implying a need for new assays and methods allowing to assess in a qualitative and quantitative manner low-frequency T cell responses in humans.     

Establishment of an interleukin 2-dependent T cell line derived from a patient with severe aplastic anemia, which inhibits in vitro hematopoiesis

Circulating mononuclear cells from a patient developing severe aplastic anemia during the course of non-A, non-B hepatitis were found to be virtually entirely composed of in vivo activated suppressor T cells (Ia+T8+). These cells were used to establish a new permanent cell line, termed SMAA, by using phytohemagglutinin, Ebstein-Barr virus-transformed irradiated B cells, allogeneic irradiated peripheral blood mononuclear cells, and recombinant interleukin 2 to investigate the relationship of aplastic anemia-derived circulating T cells to bone marrow failure. SMAA cells, now in continuous culture for more than 9 mo, were shown to inhibit proliferation of purified myeloid progenitors and their differentiation into early and late appearing neutrophil and eosinophil colonies by 90%, whereas monocyte colonies were much less affected. Similarly, growth of erythroid colonies and bursts was almost completely inhibited, as was anti-mu-induced B cell proliferation and lectin-induced T cell proliferation. This inhibition of hematopoiesis was mediated by the release of a soluble factor that was sensitive to acid (pH 2), heat (56 degrees C), and trypsin. Monoclonal and polyclonal antibodies to interferon-gamma could abrogate the inhibitory effects of SMAA supernatant, but more than 10(4) neutralizing U/ml had to be added. The effects of SMAA could be duplicated by adding 10(4) U/ml of purified recombinant interferon-gamma to colony and proliferation assays. The concentration of interferon-gamma in SMAA supernatant was estimated to be greater than 3 X 10(3) National Institutes of Health reference U/ml by immunoradiometric assay. These results demonstrate that some patients with aplastic anemia have circulating T cells that are capable of prolonged in vitro secretion of interferon-gamma causing severe inhibition of in vitro hematopoiesis, and these cells can be expanded into permanent lines for studies on their regulatory properties.     

Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4⁺ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund’s Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated.     

Vitellogenin, a multivalent sensor and an antimicrobial effector

Vitellogenin (Vg), the precursor of yolk proteins, was traditionally regarded as the energy reserve for nourishment of the developing embryos. However, its roles to extend beyond the nutrient function. Here we discuss recent developments in the understanding of Vg. Accumulating data have demonstrated that Vg fulfils important roles in innate immune responses. It acts as a multivalent pattern recognition receptor capable of binding to lipopolysaccharide, lipoteichoic acid, peptidolycan, glucan and virons. It is also a bactericidal molecule capable of damaging bacterial cell walls. Moreover, it is an acute phase protein with bacterial-binding and inhibiting activities, and possibly functions in the immune responses of host in vivo. Further understanding of Vg and its derived yolk proteins should provide new insights into the mechanisms of host defense, and reveal if they can be used as alternative strategies promoting the immunity of cultured fish as well as developing embryos.     

Feedback regulation of immune responses by immune complexes; possible involvement of a suppressive lymphokine by FcR gamma-bearing B cell

Inoculations of antigen-antibody complexes (immune complexes) with the intact Fc portion generates suppressor cells in vivo by binding to FcR gamma on B cells via Fc portions. The cell type responsible for the suppression appears to be B cells bearing FcR gamma. Neither T cells nor macrophages participate in both the inductive and effective phases of this type of regulation. The suppression caused by splenic B cells, previously stimulated with immune complexes in vivo, is mediated by humoral factor(s) released from them. The suppressive factor(s) have H-2 gene product(s) coded by the right-hand side of the H-2 gene complex, but not for FcR gamma themselves or immunoglobulins. It has shared component(s) with suppressive B cell factor (SBF) released from FcR gamma + B cells stimulated with immune complexes in vitro, and it resembles SBF in its mode of action. These findings indicate that immune complexes, the final products of antibody responses, control the immune responses by stimulating surface FcR gamma on B cells. It is of interest that this type of regulation functions in vivo.