Heparan sulfates targeting increases MHC class I- and MHC class II-restricted antigen presentation and CD8(+) T-cell response.

Heparan sulfates (HS) are carbohydrate moieties of HS proteoglycans (HSPGs). They often represent alternative attachment points for proteins or microorganisms targeting receptors. HSPGs, which are ubiquitously expressed, thereby participate in numerous biological processes. We previously showed that MHC class II-restricted antigen presentation is increased when antigens are coupled to HS ligands, suggesting that HSPGs might contribute to adaptive immune responses. Here, we examined if HSPG targeting influences other aspects of immune responses. We found that coupling of an HS ligand to the antigen increases antigen presentation to CD4(+) and CD8(+) T-cells after antigen targeting to membrane immunoglobulins or to MHC-II molecules. Moreover, this increased stimulating capacity correlates with an enhanced CD8(+) immune response in mice. Last, animals control more effectively the growth of Ova-expressing tumour cells when they are immunized with an Ova construct targeting HSPGs and MHC-II molecules. Our results indicate that ubiquitous molecules can influence both MHC class I- and MHC class II-restricted antigen presentation and behave as co-receptors during T-cell stimulation. Moreover, they suggest that tumour-antigens endowed with the ability to target both HSPGs and MHC-II molecules could be of value to increase CD8(+) immune response and control tumour-growth, opening new perspectives for the design of highly immunogenic protein-based vaccines.

Obtaining anti-type 1 melatonin receptor antibodies by immunization with melatonin receptor-expressing cells.

Antibodies (Abs) specific to cell-surface receptors are attractive tools for studying the physiological role of such receptors or for controlling their activity. We sought to obtain such antibodies against the type 1 receptor for melatonin (MT1). For this, we injected mice with CHO cells transfected with a plasmid encoding human MT1 (CHO-MT1-h), in the presence or absence of an adjuvant mixture containing Alum and CpG1018. As we previously observed that the immune response to a protein antigen is increased when it is coupled to a fusion protein, called ZZTat101, we also investigated if the association of ZZTat101 with CHO-MT1-h cells provides an immunogenic advantage. We measured similar levels of anti-CHO and anti-MT1-h Ab responses in animals injected with either CHO-MT1-h cells or ZZTat101/CHO-MT1-h cells, with or without adjuvant, indicating that neither the adjuvant mixture nor ZZTat101 increased the anti-cell immune response. Then, we investigated whether the antisera also recognized murine MT1 (MT1-m). Using cloned CHO cells transfected with a plasmid encoding MT1-m, we found that antisera raised against CHO-MT1-h cells also bound the mouse receptor. Altogether our studies indicate that immunizing approaches based on MT1-h-expressing CHO cells allow the production of polyclonal antibodies against MT1 receptors of different origins. This paves the way to preparation of MT1-specific monoclonal antibodies.

Acidosis increases MHC class II-restricted presentation of a protein endowed with a pH-dependent heparan sulfate-binding ability.

Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed molecules that participate in numerous biological processes. We previously showed that HSPGs expressed on the surface of APCs can serve as receptors for a hybrid protein containing an HS ligand and an Ag, which leads to more efficient stimulation of Th cells. To investigate whether such behavior is shared by proteins with inherent HS-binding ability, we looked for proteins endowed with this characteristic. We found that diphtheria toxin and its nontoxic mutant, called CRM197, can interact with HS. However, we observed that their binding ability is higher at pH 6 than at pH 7.4. Therefore, as extracellular acidosis occurs during infection by various micro-organisms, we assessed whether HS-binding capacity affects MHC class II-restricted presentation at different pHs. We first observed that pH decrease allows CRM197 binding to HSPG-expressing cells, including APCs. Then, we showed that this interaction enhances Ag uptake and presentation to Th cells. Lastly, we observed that pH decrease does not affect processing and presentation abilities of the APCs. Our findings show that acidic pH causes an HSPG-mediated uptake and an enhancement of T cell stimulation of Ags with the inherent ability to bind HSPGs pH-dependently. Furthermore, they suggest that proteins from micro-organisms with this binding characteristic might be supported more efficiently by the adaptive immune system when acidosis is triggered during infection.

Transfer of the ability of HIV-1 Tat to raise an adjuvant-free humoral immune response to unrelated antigens.

The HIV-1 Tat protein is able to raise a humoral immune response in the absence of adjuvant. Here, we investigated whether this property can be transferred to unrelated antigens. We first observed that Tat self-adjuvanticity is a T cell-dependent phenomenon in which a Th2 profile predominates. Then, we showed that the determinant governing the property is located in the region 1-57 of Tat and that fragment Tat1-57 can make two unrelated model antigens immunogenic in the absence of adjuvant. We found a Th2 pattern of immune response for both antigens, suggesting that Tat1-57 mediates this response. Next, we showed that, although less efficient than Tat1-57, the Tat37-57 fragment suffices to transfer the adjuvant property to other antigens. We also observed that preservation of cysteine 37 is absolutely required for the transfer, suggesting the role of disulphide-mediated dimerization in the transfer of the adjuvant property. Our observations suggest that for various antigens, the use of Tat37-57 or Tat1-57 or Tat22-57C(22-34)A might represent an alternative to adjuvants in humans, thereby opening up new perspectives in vaccination.

Cell surface heparan sulfate proteoglycans influence MHC class II-restricted antigen presentation.

Heparan sulfate proteoglycans (HSPGs) are glycoproteins ubiquitously distributed on the cell surface and in the extracellular matrix. Their heparan sulfate moieties often represent alternative attachment points for extracellular proteins that target specific receptors. Thus, HSPGs modulate ligand-receptor encounters and participate in numerous biological processes. In this study, we examined whether HSPGs can also influence MHC class II-restricted Ag presentation. We selected a heparan sulfate ligand derived from the HIV-1 Tat protein and coupled it to a model protein Ag. We showed that coupling of the Tat fragment makes the Ag capable of binding cells, including APCs, and increases its ability to stimulate specific T cells up to 180-fold. The boosting effect depends on Ag processing; it vanished in the presence of an excess of heparin or free Tat fragment, indicating that HSPGs can behave as receptors involved in MHC class II processing and presentation. Furthermore, with FcγRII-bearing APCs, immune complexes containing the coupled Ag stimulated T cells up to 700-fold more efficiently than Ag-containing immune complexes. This effect vanished in the presence of heparin and is not found with FcγRII(-) APCs, indicating that HSPGs can also behave as coreceptors during FcγRIIR-mediated Ag presentation. These results indicate that ubiquitous receptors, such as HSPGs, can influence MHC class II-restricted Ag presentation and suggest that proteins will be supported more efficiently by the immune system if they have the inherent capacity to bind heparan sulfate.

Cynomolgus macaques immunized with two HIV-1 Tat stabilized proteins raise strong and long-lasting immune responses with a pattern of Th1/Th2 response differing from that in mice.

The transcriptional transactivator (Tat) of HIV-1 is regarded as an attractive target for the development of an AIDS vaccine. However, investigations have suggested that Tat is poorly immunogenic and prompted us to develop a strategy to increase its ability to raise an immune response. The strategy is based on stabilization of Tat using sulfated sugars, previously proven to boost its humoral immunogenic potency, in mice. We have now examined the pattern of immune response raised by two Tat-stabilized proteins previously mixed with Alum, in mice and macaques. We found that BALB/c and DBA/2 mice raise a Th2 immune response that contrasts with the mixed Th1/Th2 response observed in cynomolgus macaques indicating that the profile of response found in mice cannot be extrapolated to macaques. We thoroughly analyzed the macaque anti-Tat immune response and observed that the anti-Tat antibodies appear after only one immunization and that their titers remain elevated up to 8 weeks after the last injection. We found a similar behavior for the cellular immune response. Furthermore, we observed that approximately 50% of the IFN-gamma-secreting Tat-specific T-cells are CD8 lymphocytes. Last, we observed that the macaque sera neutralize the transactivating activity of Tat. This analysis shows that the two Tat-stabilized preparations raise a potent and long-lasting humoral and cellular immune response in cynomolgus macaque and opens an avenue to investigate whether a strong anti-Tat immune response can protect non-human primates against a viral challenge.

Increasing the humoral immunogenic properties of the HIV-1 Tat protein using a ligand-stabilizing strategy.

Tat is regarded as an attractive target for the development of an AIDS vaccine. However, works suggest that Tat is a poorly immunogenic protein and therefore we attempted to increase its immunogenic potency. As we observed that Tat is highly sensitive to enzymatic degradation in vitro we tried to make it less susceptible to proteolysis using ligands. We complexed Tat101 with various sulfated sugars and observed that some of these ligands made the protein more resistant to proteolysis and more immunogenic. In a more thorough study, we observed that a low-molecular-weight heparin fragment, called Hep6000, altered both the cell-binding capacity and transactivating activity of Tat101, suggesting that this sulfated polysaccharide can make the protein less toxic. Sera raised against Tat101 and Tat101/Hep6000 similarly bound mainly to the N-terminal region of the protein, indicating that formation of the complex does not alter the B-cell immunodominant region. Anti-Tat101/Hep6000 antisera neutralized the transactivating activity of Tat101 more efficiently than anti-Tat101 antisera. Altogether, these results indicate that stabilization of Tat101 using sulfated sugars increases its immunogenicity and might be of value in increasing its vaccine efficacy.

HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers.

Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region. We also show that cysteine-mediated oligomerization is a key molecular event of the adjuvant-free antibody response. In particular, a Tat dimer formed by the oxidation of two cysteine residues, at position 34 only, raises an adjuvant-free antibody response that is comparable with that observed with the wild-type protein. Unlike the parent protein, the Tat dimer has no transactivating activity and remains homogeneous for several weeks in solution. This construct might be of value for the design of an adjuvant-free Tat-based vaccine. Furthermore, we suggest that the specific autoadjuvanticity determinant of Tat could be used to provide other proteins with adjuvant-free immunogenicity.

Antigen stability controls antigen presentation.

We investigated whether protein stability controls antigen presentation using a four disulfide-containing snake toxin and three derivatives carrying one or two mutations (L1A, L1A/H4Y, and H4Y). These mutations were anticipated to increase (H4Y) or decrease (L1A) the antigen non-covalent stabilizing interactions, H4Y being naturally and frequently observed in neurotoxins. The chemically synthesized derivatives shared similar three-dimensional structure, biological activity, and T epitope pattern. However, they displayed differential thermal unfolding capacities, ranging from 65 to 98 degrees C. Using these differentially stable derivatives, we demonstrated that antigen stability controls antigen proteolysis, antigen processing in antigen-presenting cells, T cell stimulation, and kinetics of expression of T cell determinants. Therefore, non-covalent interactions that control the unfolding capacity of an antigen are key parameters in the efficacy of antigen presentation. By affecting the stabilizing interaction network of proteins, some natural mutations may modulate the subsequent T-cell stimulation and might help microorganisms to escape the immune response.