Anti-Pseudomonas aeruginosa serotype O11 LPS immunoglobulin M monoclonal antibody panobacumab (KBPA101) confers protection in a murine model of acute lung infection.

OBJECTIVES: To investigate the in vivo efficacy in a murine pulmonary infection model of panobacumab (KBPA101), a human IgM monoclonal antibody directed against the O-polysaccharide moiety of Pseudomonas aeruginosa serotype O11, and to describe the anti-inflammatory effects in the lung as a consequence of the treatment. METHODS: We established an experimental murine model of acute pneumonia by intranasal administration of P. aeruginosa serotype O11. Mice were treated, after infection, with a single intravenous injection of panobacumab and panobacumab lung bioavailability was assessed. Inflammatory parameters such as pro-inflammatory cytokine production and neutrophil recruitment in broncho-alveolar lavage fluid (BALF) were measured and bacterial load in the lung was analysed. RESULTS: Panobacumab plays a significant role in addition to the host innate immune response, leading to improved control of pulmonary infection. The IgM antibody is able to reach the broncho-alveolar space and reduce the pulmonary bacterial load as well as lung inflammation in a dose-dependent manner. Furthermore, panobacumab treatment leads to enhanced neutrophil recruitment in BALF while reducing the host-derived production of pro-inflammatory mediators and lung injury. CONCLUSIONS: These data provide evidence that panobacumab, an IgM-based immunotherapeutic, is highly efficacious in controlling acute lung infection by enhancing the natural innate immune response.

Human dendritic cells are activated by chimeric human papillomavirus type-16 virus-like particles and induce epitope-specific human T cell responses in vitro.

Human papillomavirus (HPV)-derived chimeric virus-like particles (VLPs) are the leading candidate vaccine for the treatment or prevention of cervical cancer in humans. Dendritic cells (DCs) are the most potent inducers of immune responses and here we show for the first time evidence for binding of chimeric HPV-16 VLPs to human peripheral blood-derived DCS: Incubation of immature human DCs with VLPs for 48 h induced a significant up-regulation of the CD80 and CD83 molecules as well as secretion of IL-12. Confocal microscopy analysis revealed that cell surface-bound chimeric VLPs were taken up by DCS: Moreover, DCs loaded with chimeric HPV-16 L1L2-E7 VLPs induced an HLA-*0201-restricted human T cell response in vitro specific for E7-derived peptides. These results clearly demonstrate that immature human DCs are fully activated by chimeric HPV-16 VLPs and subsequently are capable of inducing endogenously processed epitope-specific human T cell responses in vitro. Overall, these findings could explain the high immunogenicity and efficiency of VLPs as vaccines.

Human T-cell responses to HLA-A-restricted high binding affinity peptides of human papillomavirus type 18 proteins E6 and E7.

Human Papillomaviruses (HPVs) are sexually transmitted pathogens, which are implicated in the etiology of cervical cancer. The early proteins E6 and E7 of HPV have transforming capacity and interfere with the cell cycle control of infected host cells and are essential for the maintenance of the transformed state. Identification of MHC class I-restricted, immunogenic peptides derived from either the E6 or the E7 protein is essential for the design of vaccines as well as the monitoring of clinical trials and immunotherapeutic approaches for the treatment of HPV-18-induced carcinomas. We have determined the binding affinities for all possible 9-mer peptides spanning the entire E6 and E7 amino acid sequence for the HLA-A*0101, HLA-A*0201, HLA-A*0302, HLA-A*1102, and HLA-A*2402101 molecules by a competition assay with reference peptides, thereby establishing the binding peptides as potential cytotoxic T-cell epitopes. From the HLA-A*0201 binding peptides, we selected five E6-derived and one E7-derived peptide with high affinities for HLA-A*0201. These six peptides were tested for their immunogenicity by in vitro immunization assays with purified human CD8+ T cells. We identified three HPV-18 E6-derived peptides (ELTEVFEFA, KTVLELTEV, and KLPDLCTEL) and the E7-derived peptide TLQDIVLHL to be highly immunogenic. Overall, these results will help to design vaccines for the prevention or treatment of HPV-18-induced cervical cancer.

Cervical cancer vaccines: emerging concepts and developments.

Certain human cancers are linked to infection by oncogenic viruses that are able to cause transformation of the normal host cell into a cancerous cell. Human papillomavirus (HPV) DNA and expression of viral transforming proteins are found in virtually all cervical cancer cells, indicating an important role of this virus in the pathogenesis of the disease. Evidence exists that the immune response to cancer cells can play a major role in determining the outcome of disease. The fact that HPV is a necessary cause for cervical cancer provides a clear opportunity to develop a therapeutic vaccine against the virus to treat patients with cervical cancer at its early and late stages. Development of a prophylactic vaccine for HPV would also reduce the incidence of cervical neoplasias by preventing virus infection. Various candidate HPV vaccines are being developed and tested in animal models and/or in human clinical trials. These HPV vaccines, both preventive and therapeutic, are the subjects of this review.

Molecular characterization of melanoma-derived antigens.

In the last decade, many antigens expressed by tumors and recognized by the immune system have been identified. Melanoma was among the first tumors found to express such tumor-associated antigens, and, therefore, melanoma is currently one of the best and extensively studied tumors for which new techniques have been introduced to optimize the characterization of tumor antigens. In this chapter, we discuss the techniques used for identification of melanoma-expressed antigens recognized by cytotoxic T-lymphocytes (CTLs). In more detail, we describe in Subheading 3. the reverse immunology method.

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

Oral anti-IgE immunization with epitope-displaying phage.

An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope-displaying M13 bacteriophages as carriers for an experimental oral anti-IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti-idiotypic Fab of the non-anaphylactogenic monoclonal anti-IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII-expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti-IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti-phage but no anti-IgE antibodies. After feeding a single dose of mimotope-displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope-displaying bacteriophages can be used to induce an epitope-specific antibody response via the oral route.

Molecular mimicry of the unidentified antigen of myeloma antibody IgE-ND.

Antigen-specific human IgE is in short supply. Thus, we sought to determine the yet unknown specificity of a widely available human IgE, namely the myeloma cell line U266-derived IgE-ND. For this purpose highly specific peptides able to mimic the putative antigen recognized by IgE-ND were isolated from phage-display random peptide libraries. Interestingly, we found linear sequence homologies of the IgE-ND-binding peptides with self antigens and a xenoantigen from Thiobacillus ferrooxidans. However, none of these antigens was recognized by IgE-ND. Nevertheless, our approach may be applied to identify antigen specificities of myeloma antibodies. Importantly, the mimotopes were anaphylactogenic in a histamine release assay using human basophils sensitized with IgE-ND. Thus, our mimotopes represent functional albeit synthetic antigens and may be used to study human antigen-specific IgE responses.

Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles.

It has been postulated that upon binding to a cell surface receptor, papilloma virus-like particles (VLPs) gain entry into the cytosol of infected cells and the capsid proteins L1 and L2 can be processed in the MHC class I presentation pathway. Vaccination of mice with human papilloma virus-like particles consisting of capsid proteins L1 and L2 induced a CD8-mediated and perforin dependent protective immune response against a tumor challenge with human papilloma virus transformed tumor cells, which express only minute amounts of L1 protein. Here we show that HPV16 capsid proteins stimulate a MHC class I restricted CTL response with human peripheral blood lymphocytes (PBL) in vitro. The vigorous response was specific for VLP-infected target cells and was MHC class I restricted. Moreover we show the presence of at least one HLA-A*0201 restricted CTL epitope within the HPV-16 capsid proteins by using a VLP-'infected' HLA-A*0201 transfected human cell line as target cells. These results demonstrated that VLPs can induce a HPV16 capsid protein-specific immune response in humans, allowing the monitoring of immune responses induced by vaccines based on chimeric VLPs carrying additional immunogenic peptides or proteins in therapeutical applications in human patients.

Papillomavirus virus-like particles as anticancer vaccines.

Papillomavirus virus-like particles (VLPs) are empty, non-replicative, non-infectious particles that retain conformationally correct epitopes for the generation of antibody responses to the viral capsid proteins. Chimeric human papillomavirus (HPV) virus-like particles incorporating non-structural virus proteins offer an exciting approach for combined prophylactic and therapeutic vaccines against HPV-induced lesions. Both HPV VLPs and chimeric VLPs can induce potent humoral and cellular immune responses when injected into mice, leading to the generation of virus-neutralizing antibodies, priming of CD8+ T-cells and activation of cytotoxic T-cell effector functions. This review summarizes recent advances in the production of chimeric VLPs, the immune response elicited by VLPs and chimeric VLPs, and their ability to generate strong protective and therapeutic antitumor immune responses.

Cellular immunity and immunotherapy against deoxyribonucleic acid virus-induced tumors.

An important role in the immune defense against deoxyribonucleic acid virus induced tumors is mediated by T-cells, as is evident from aetiological, animal model, and clinical data. In this review the most recent observations in this field are described for three prominent members of this family of viruses, namely human papillomavirus associated with human cervical cancer, human adenovirus associated with lung infections in humans and tumors in rodents, and simian virus 40 associated with rodent tumors and human mesothelioma, osteosarcoma and ependymoma.

Epitope-specific antibody response to IgE by mimotope immunization.

We have previously described a mouse monoclonal anti-human IgE antibody (BSW17) capable of recognizing receptor-bound IgE without inducing mediator release from human basophils or mast cells. Moreover, immune complexes of IgE and BSW17 are not able to bind to the IgE receptor. An initial attempt to map the precise epitope recognized by this mAb by using Fc epsilon-derived peptides of variable length was unsuccessful. However, by screening random peptide phage display libraries we isolated circular nona- and octapeptides specifically recognized by BSW17. These constrained peptides mimic at least a part of a conformational epitope and are thus called mimotopes. These mimotopes, either phage displayed or synthetically synthesized, did not react with any other anti-human IgE antibody tested, but efficiently inhibited the binding of human IgE to BSW17 only. The use of Rhodol-Green-labeled free cyclic peptide proved that these interactions were not carrier dependent. Immunization of rabbits with phage clones displaying the specific peptides on the surface induced an anti-human IgE response specific for the epitope of BSW17. Therefore, we conclude that such mimotopes or mimotope-derived peptides might be used for vaccination to induce in vivo a beneficial anti-IgE response as a novel immunotherapy.

Identification of peptides for immunotherapy of cancer. It is worth the effort.

As the nature of the T cell immune response is defined by T cell receptor recognition of small protein fragments, referred to as peptides, the identification of peptides would lead us to understanding and directing the T-cell-mediated immune response. Immunogenic peptides might be used for vaccination and activation of the immune reaction against cancer- and virus-infected cells. Additionally, the knowledge of immunogenic peptides was expected to lead to blocking of allergic reactions and autoimmune diseases. Based on these assumptions, the search for immunogenic peptides was started in mice and man in the mid-1980s. After a decade of peptide identification and testing in vitro and in vivo, this may be a proper time to evaluate the results from the peptide-related work and determine the possible applications of this knowledge for the next decade. In this review we discuss the identification of peptides, their use in murine models, as well as clinical data from peptide vaccinations or therapies. Potential hazards and limitations of peptide use in immunotherapy and other possible applications for peptides or peptide motifs in immunotherapy are evaluated.

Antigen interaction and heat inactivation expose new epitopes on human IgE.

It is well established that heat-denatured IgE is no longer capable of binding to FcepsilonRI. We have found an antibody that interacts with heat-denatured IgE. Interestingly, this antibody can also be used to detect some serum IgE, but not IgE synthesized de novo in vitro. However, native IgE can be transformed into an IgE that is recognized by this antibody, if antigen is added. Our data indicate that physiological mechanisms exist that biologically inactivate IgE which might still be mistaken for 'functional' IgE by assays based on polyclonal antibodies.

Can active immunization redirect an anti-IgE immune response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.

Effect of anti-IgE antibodies on Fc epsilonRI-bound IgE.

Anti-IgE mAbs have always been used to trigger mediator release from basophils or mast cells. Now nonanaphylactogenic anti-human IgE Abs are in clinical evaluation as a therapeutic agent against atopic disease. We have found a mAb that is nonanaphylactogenic but recognizes receptor-bound IgE. Interestingly, the Ab prevents the association of IgE with its receptor if immune complexes were formed between IgE and anti-IgE mAb. This explained the phenomenon that addition of anti-IgE Ab to receptor-bound IgE resulted in a decrease of receptor-bound IgE, because IgE dissociating from the receptor was complexed, altering the thermodynamic balance of receptor-bound vs free IgE. Our data show that there are nonanaphylactogenic Abs that do not directly interfere with the receptor binding site on IgE and are capable of preventing the association of IgE with its high affinity receptor. This unique feature would render such an anti-IgE Ab a possible candidate for immunotherapy of atopy.

Anti-IgE in allergic sensitization.

Anti-IgE autoantibodies exist predominantly in the sera of patients with atopic disease. For some time such anti-IgE autoantibodies have been considered a phenomenon that may not be of clinical importance. The cloning of such anti-IgE autoantibodies has eliminated doubts of whether these antibodies exist, but it is still unclear whether such autoantibodies play a pathophysiological role. However, there are ongoing clinical trials that use humanized anti-IgE antibodies for passive immunization of atopic individuals. While this approach may not definitely clarify the role of anti-IgE autoantibodies, it will nevertheless clarify the role of IgE.

Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation.

Amino acid sequence homology between tetanus toxoid, a common vaccine and melittin, a bee venom allergen provided us with two antigen models for studying the production of cross reactive antibodies after immunisation. Analysis of the serum of an atopic patient recently boosted with tetanus toxoid revealed the presence of anti-melittin and anti-tetanus antibodies. From this donor a phage display Fab library was constructed five days after vaccination. Screening of this library either with melittin alone or with tetanus toxoid followed by melittin allowed the isolation of Fab fragments specific to melittin but also Fab fragments which cross react with melittin and tetanus toxoid. Amino acid analysis revealed diversity in the heavy and the light Ig chains of the melittin specific and of the cross reactive clones. Interestingly we found that the light chain recognised melittin whereas the heavy chain preferentially bound to tetanus toxoid suggesting that cross reactivity may be due to the different binding specificities of the individual Ig chains.

A specific feedback by anti-IgE autoantibodies on the cytokine network in allergy.

During recent years we have shown that anti-IgE antibodies can have different biological functions. Depending on their epitope specificity they can be anaphylactogenic or not, they interfere with IgE binding to its receptor or not, and they enhance or inhibit IgE synthesis. Therefore we propose a theoretical model implying that anti-IgE autoantibodies are specific feed back molecules that neutralize IgE induced by the cytokine network. In the normal individual this system would be beneficial, where as the atopic individual, due to differences in its B cell repertoire, will produce the wrong type of anti-IgE antibody. The wrong type of anti-IgE antibody may even aggravate the disease as some of these autoantibodies may induce IgE synthesis or trigger effector cells that in turn generate a Th2 like cytokine pattern.