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Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab') 2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab') 2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab') 2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab') 2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab') 2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.

The insulin-like growth factor type 1 receptor (IGF-1R) is important in tumorigenesis, and its overexpression occurs in numerous tumor tissues. To date, therapeutic approaches based on mAbs and tyrosine kinase inhibitors targeting IGF-1R have only shown clinical benefit in specific patient populations. We report a unique IGF-1R-targeted antibody-drug conjugate (ADC), W0101, designed to deliver a highly potent cytotoxic auristatin derivative selectively to IGF-1R overexpressing tumor cells. The mAb (hz208F2-4) used to prepare the ADC was selected for its specific binding properties to IGF-1R compared with the insulin receptor, and for its internalization properties. Conjugation of a novel auristatin derivative drug linker to hz208F2-4 did not alter its binding and internalization properties. W0101 induced receptor-dependent cell cytotoxicity in vitro when applied to various cell lines overexpressing IGF-1R, but it did not affect normal cells. Efficacy studies were conducted in several mouse models expressing different levels of IGF-1R to determine the sensitivity of the tumors to W0101. W0101 induced potent tumor regression in certain mouse models. Interestingly, the potency of W0101 correlated with the expression level of IGF-1R evaluated by IHC. In an MCF-7 breast cancer model with high-level IGF-1R expression, a single injection of W0101 3 mg/kg led to strong inhibition of tumor growth. W0101 provides a potential new therapeutic option for patients overexpressing IGF-1R. A first-in-human trial of W0101 is currently ongoing to address clinical safety., (©2019 American Association for Cancer Research.)

The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.

c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers., (© 2016 UICC.)

The type IV C-X-C-motif chemokine receptor (CXCR4) is expressed in a large variety of human cancers, including hematologic malignancies, and this receptor and its ligand, stromal cell-derived factor-1 (SDF-1), play a crucial role in cancer progression. We generated a humanized immunoglobulin G1 mAb, hz515H7, which binds human CXCR4, efficiently competes for SDF-1 binding, and induces a conformational change in CXCR4 homodimers. Furthermore, it inhibits both CXCR4 receptor-mediated G-protein activation and β-arrestin-2 recruitment following CXCR4 activation. The binding of the hz515H7 antibody to CXCR4 inhibits the SDF-1-induced signaling pathway, resulting in reduced phosphorylation of downstream effectors, such as Akt, Erk1/2, p38, and GSK3β. Hz515H7 also strongly inhibits cell migration and proliferation and, while preserving normal blood cells, induces both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against neoplastic cells. In mouse xenograft models, hz515H7 displays antitumor activities with multiple hematologic tumor cell lines, with its Fc-mediated effector functions proving essential in this context. Furthermore, hz515H7 binds to primary tumor cells from acute myeloid leukemia and multiple myeloma patients. Collectively, our results demonstrate two major mechanisms of action, making hz515H7 unique in this regard. Its potential as a best-in-class molecule is currently under investigation in a phase I clinical trial. Mol Cancer Ther; 15(8); 1890-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA )-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms,electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glycoprofiling and demonstration of the absence of additional conjugation was easily achieved. As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.

To identify new potential targets in oncology, functional approaches were developed using tumor cells as immunogens to select monoclonal antibodies targeting membrane receptors involved in cell proliferation. For that purpose cancer cells were injected into mice and resulting hybridomas were screened for their ability to inhibit cell proliferation in vitro. Based on this functional approach coupled to proteomic analysis, a monoclonal antibody specifically recognizing the human junctional adhesion molecule-A (JAM-A) was defined. Interestingly, compared to both normal and tumor tissues, we observed that JAM-A was mainly overexpressed on breast, lung and kidney tumor tissues. In vivo experiments demonstrated that injections of anti-JAM-A antibody resulted in a significant tumor growth inhibition of xenograft human tumors. Treatment with monoclonal antibody induced a decrease of the Ki67 expression and downregulated JAM-A levels. All together, our results show for the first time that JAM-A can interfere with tumor proliferation and suggest that JAM-A is a potential novel target in oncology. The results also demonstrate that a functional approach coupled to a robust proteomic analysis can be successful to identify new antibody target molecules that lead to promising new antibody-based therapies against cancers., (Copyright © 2012 UICC.)

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130-230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130-230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

CD151 is a plasma membrane protein belonging to the tetraspanin superfamily which is expressed on normal cells such as endothelial cells and platelets and frequently overexpressed on cancer cells. It is known to be functionally linked to cancer metastasis. In humans, increased expression of CD151 is indicative of a poor prognosis in different cancer types. Whereas its mechanism of action remains obscure, CD151 was shown to regulate cell motility and adhesion through association with laminin-binding integrins such as α3β1 or α6β4. Several anti-CD151 mAbs (monoclonal antibodies) have been shown to display anti-metastatic activity in vivo. Inhibition of metastasis was not attributed to any effect of these mAbs on tumour cell growth, but was essentially attributed to inhibition of cell motility. We have generated anti-CD151 mAbs which can inhibit the tumoral growth in different xenograft cancer models. As expected, these mAbs were also able to inhibit metastasis in orthotopic cancer models. These data suggest that CD151 could function at multiple cancer stages, including not only metastasis cascade steps, but also earlier steps of primary tumour growth, thus reinforcing the interest of this innovative target in oncology. mAbs targeting CD151 may be of significant interest for cancer biotherapy.

Monoclonal antibodies (mAbs) and derivatives are currently the fastest growing class of therapeutic molecules. More than 30 G-type immunoglobulins (IgG) and related agents have been approved over the past 25 years mainly for cancers and inflammatory diseases. In oncology, mAbs are often combined with cytotoxic drugs to enhance their therapeutic efficacy. Alternatively, small anti-neoplastic molecules can be chemically conjugated to mAbs, used both as carriers (increased half-life) and as targeting agents (selectivity). Potential benefits of antibody-drug conjugates (ADCs), strategies, and development challenges are discussed in this review. Several examples of ADCs are presented with emphasis on three major molecules currently in late clinical development as well as next generation thio-mAbs conjugates with improved therapeutic index.

Monoclonal antibodies constitute a growing class of therapeutic agents. They are classically used in combination with chemotherapeutic drugs for cancer treatment. The concept of coupling a cytotoxic agent to an antibody can be viewed as a means to confer a selectivity for tumoral cells to highly cytotoxic drugs which cannot be used in human, or a higher power to antibodies which have a low anti-tumoral activity on their own. Gemtuzumab ozogamicin is the only drug-armed antibody available on the market, for the treatment of acute myeloid leukaemia. Other immunoconjugates are currently under clinical development. The most used cytotoxic agents derive from calicheamicin, maytansin and auristatin, compounds which are 100 to 1 000 fold more toxic than the classical chemotherapeutic drugs. Today, we know that the efficacy of an immunoconjugate depends not only on the coupled cytotoxic agent, but also on the selected target, the coupling method and the linker.

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells. Many alternative production systems and improved constructs are also being actively investigated. IgGs glycans represent only an average of around 3% of the total mass of the molecule. Despite this low percentage, particular glycoforms are involved in essential immune effector functions. On the other hand, glycoforms that are not commonly biosynthesized in human may be allergenic, immunogenic and accelerate the plasmatic clearance of the linked antibody. These glyco-variants have to be identified, controlled and limited for therapeutic uses. Glycosylation depends on multiple factors like production system, selected clonal population, manufacturing process and may be genetically or chemically engineered. The present account reviews the glycosylation patterns observed for the current approved therapeutic antibodies produced in mammalian cell lines, details classical and state-of-the-art analytical methods used for the characterization of glycoforms and discusses the expected benefits of manipulating the carbohydrate components of antibodies by bio- or chemical engineering as well as the expected advantages of alternative biotechnological production systems developed for new generation of therapeutic antibodies and Fc-fusion proteins.

Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.

The proteasome is a proteolytic complex that constitutes the main pathway for degradation of intracellular proteins in eukaryotic cells. It regulates many physiological processes and its dysfunction can lead to several pathologies like cancer. To study the 20S proteasome structure/activity relationship in cells that derive from human biopsy samples, we optimized an immuno-purification protocol for the analysis of samples containing a small number of cells using magnetic beads. This scaled-down protocol was used to purify the cytoplasmic 20S proteasome of adjacent normal and tumor colorectal cells arising from tissue samples of several patients. Proteomic analyses based on two-dimensional gel electrophoresis (2DE) and mass spectrometry showed that the subunit composition of 20S proteasomes from these normal and tumor cells were not significantly different. The proteasome activity was also assessed in the cytoplasmic extracts and was similar or higher in tumor colorectal than in the corresponding normal cells. The scaled-down 20S proteasome purification protocol developed here can be applied to any human clinical tissue samples and is compatible with further proteomic analyses.

Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies. They are also valuable analytical reference compounds for the structural characterisation by liquid chromatography and mass spectrometry of recombinant proteins used as biopharmaceuticals. As for other therapeutic applications, formulation, solubilisation, batch consistency and stability, issues have to be addressed to allow the pre-clinical and clinical development of this class of compounds as immunotherapeutic drugs. In the present review, three case studies dealing with (i) the design and the characterisation of Respiratory Syntycial Virus subunit vaccines, (ii) peptide-based melanoma vaccines, and (iii) therapeutic monoclonal antibodies, all investigated in clinical trials, are reported and discussed.

An improved high-performance liquid chromatographic (HPLC) method for the separation of zwitterionic detergents is described. It is based on a reversed-phase liquid chromatography with evaporative light-scattering detection (ELSD). The method was shown to be highly specific, allowing the separation of three detergents of the alkyl sulfobetaine family: 3-(N-dodecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB12), 3-(N-tetradecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB14) and 3-(N-hexadecyl-N,N-dimethyl-ammonio)-propane-1-sulfonate (SB16). It was further used to develop a quantitation method for SB14, which was validated for linearity, precision, robustness, limits of detection and quantitation, specificity and accuracy. Linearity was found in the range of 50-500 microg/ml with a correlation coefficient of 0.9938+/-0.0029. The mean value of slope and intercept were 1.567+/-0.06 and 0.1541+/-0.0271, respectively. The limits of detection (LOD) and quantitation (LOQ) were 2 and 10 microg/ml, respectively. The validated method was used to determine the concentration of SB14 in different biological samples, specially in bulks of a recombinant membrane protein, the Klebsiella pneumoniae outer membrane protein A, which is produced at the pilot scale for human clinical studies.

Immunisation of BALB/c mice with a vaccine containing Vi polysaccharide conjugated to the Klebsiella pneumoniae outer membrane 40 kDa protein (rP40), in combination with Escherichia coli heat-labile toxin adjuvant (LT), elicited anti-Vi IgG antibodies after administration using different routes. Testing of the immune serum in opsonisation assays demonstrated the specific enhancement of Vi-positive bacterial uptake by cultured murine bone marrow derived macrophages. Intra-peritoneal challenge of mice immunised with the Vi-based vaccine elicited a degree of protection against virulent Vi+ Salmonella enterica serovar typhimurium (S. typhimurium). In contrast, Vi vaccination did not confer protection against oral challenge with virulent Vi-positive S. typhimurium or S. dublin.

7H2HM is a new humanized recombinant monoclonal antibody (MAb) directed against insulin-like growth factor-1 receptor and produced in CHO cells. Homogeneity of intact antibody, reduced light and heavy chains, Fab and Fc fragments were investigated by analytical methods based on mass (SDS-PAGE, SEC), charge (IEF, C-IEX) and hydrophobicity differences (RP-HPLC, HIC) and compared side-by-side with A2CHM, produced in NS0 cells. Primary structures and disulfide bridge pairing were analyzed by microsequencing (Edman degradation), mass spectrometry (MALDI-TOF, ES-TOF) and peptide mapping after enzymatic digestion (Trypsin, endoprotease Lys-C, papain). The light chains demonstrated the expected sequences. The heavy chains yielded post-translational modifications previously reported for other recombinant humanized or human IgG1 such as N-terminal pyroglutamic acid, C-terminal lysine clipping and N-glycosylation for asparagine 297. More surprisingly, two-thirds of the 7H2HM heavy chains were shown to contain an additional 24-amino-acid sequence, corresponding to the translation of an intron located between the variable and the constant domains. Taken together these data suggest that 7H2HM is a mixture of three families of antibodies corresponding (i) to the expected structure (17%; 14,9297 Da; 1330 amino acids), (ii) a variant with a translated intron in one heavy chains (33%; 15,2878 Da; 1354 amino acids) and (iii) a variant with translated introns in two heavy chains (50%; 15,4459 Da; 1378 amino acids), respectively. RP-HPLC is not a commonly used chromatographic method to assess purity of monoclonal antibodies but unlike to SEC and SDS-PAGE, was able to show and to quantify the family of structures present in 7H2HM, which were also identified by peptide mapping, mass spectrometry and microsequencing.

Outer membrane protein A (OmpA) is a conserved major component of the outer membrane of Enterobacteriaceae. Here, we report that OmpA from Klebsiella pneumoniae (KpOmpA) activates macrophages and dendritic cells (DCs) in a TLR2-dependent way. However, TLR2 does not account for binding of KpOmpA to innate immune cells. KpOmpA binds the scavenger receptors (SRs) LOX-1 and SREC-I, but not other members of the same family. LOX-1 colocalizes and cooperates with TLR2 in triggering cellular responses. The TLR2-activated functional program includes production of the long pentraxin PTX3, a soluble pattern recognition receptor involved in resistance against diverse pathogens. PTX3, in turn, binds KpOmpA but does not affect recognition of this microbial moiety by cellular receptors. KpOmpA-elicited in vivo inflammation is abrogated in TLR2(-/-) mice and significantly reduced in PTX3(-/-) mice. Thus, SR-mediated KpOmpA recognition and TLR2-dependent cellular activation set in motion a nonredundant PTX3-mediated humoral amplification loop of innate immunity.

Maternal antibodies (MatAbs) may protect the offspring against infections but may also interfere with their immune responses to vaccination. We have previously shown that maternal immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus protein (Pnc-TT) protected the offspring against infections caused by three important pediatric serotypes. To study the influence of MatAb on the immune response to Pnc-TT early in life, adult female mice were immunized twice with Pnc-TT of serotype 1 (Pnc1-TT), and their offspring received Pnc1-TT subcutaneously three times at 3-week intervals starting at 1 week (neonatal) or 3 weeks (infant) of age. High levels of PPS-1-specific MatAb (>3 log) in offspring of Pnc1-TT-immunized dams completely inhibited their anti-PPS-1 response elicited by Pnc1-TT. In contrast, low or moderate ( approximately 1 to 2 log) levels of MatAb did not interfere with and even enhanced the immune response of the offspring, and a booster response to a second Pnc1-TT dose was observed. Carrier-specific MatAbs had little effect on the response of offspring to the conjugate. All Pnc1-TT-immunized offspring were protected against pneumococcal bacteremia and had reduced lung infection. These results demonstrate that in the presence of MatAb, Pnc1-TT may elicit a protective PPS-1-specific antibody response and prime for PPS-1-specific memory in young offspring. Importantly, low or moderate levels of PPS-1-specific MatAb not only provided protection against pneumococcal infections but also enhanced the immune response elicited by Pnc1-TT in neonatal and infant mice. This murine model will be used to develop novel strategies combining maternal and neonatal immunization to protect against infections caused by encapsulated bacteria in early life.

Interaction of insulin-like growth factor receptor I (IGF-IR) with its ligands has been reported to induce cell proliferation, transformation and blockade of cell apoptotic functions. IGF-IR is overexpressed on numerous tumor cell types and its blockade could be of importance for anti-cancer therapy. We have generated a humanized anti-IGF-IR antibody h7C10 that blocks in vitro IGF-I and IGF-II-induced cell proliferation of MCF-7 breast cancer cells. Analysis of the IGF-I transduction cascade demonstrated that the humanized anti-IGF-IR antibody and its murine parental form block IGF-I-induced tyrosine phosphorylation, both its beta-chain and IRS-1 tyrosine phosphorylation. This presumably leads to cell cycle arrest and, consequently, growth inhibition. Treatment of nude mice bearing either human breast cancer cells (MCF-7) or non small lung cancer cells (A549) with h7C10, or its murine parental form 7C10, inhibited significantly tumor growth. An almost complete inhibition of A549 tumor growth was observed when mice were treated with the anti-IGF-IR antibody combined with either a chemotherapeutic agent, Vinorelbine or an anti-epidermal growth factor receptor (EGFR) antibody, 225. Combined therapy prolonged significantly the life span of mice in an orthotopic in vivo model of A549; the combination of the anti-IGF-IR antibody with an anti-EGFR antibody was superior to the Vinorelbine combination. The present results indicate that the humanized anti-IGF-IR antibody h7C10 has a great potential for cancer therapy when combined with either a chemotherapeutic agent or an antibody that targets other growth factor receptors, such as the epidermal growth factor receptor.

The immune system of the newborn is immature, and therefore it is difficult to induce protective immunity by vaccination in the neonatal period. Immunization of mothers during pregnancy against infections caused by encapsulated bacteria could thus be particularly attractive, as infants do not respond to polysaccharide (PS) antigens. Transmission of maternal vaccine-specific antibodies and protection of offspring against pneumococcal bacteremia and/or lung infection were studied in a neonatal murine model of pneumococcal immunization and infections. Adult female mice were immunized with native pneumococcal PS (PPS) of serotypes 1, 6B, and 19F or PPS conjugated to tetanus protein (Pnc-TT), and PPS-specific antibodies were measured in sera of mothers and their offspring. Effective transmission of maternal antibodies was observed, as PPS-specific immunoglobulin G levels in 3-week-old offspring of immunized mothers were 37 to 322% of maternal titers, and a significant correlation between maternal and offspring antibody levels was observed. The PPS-specific antibodies persisted for several weeks but slowly decreased over time. Offspring of Pnc-TT-immunized mothers were protected against pneumococcal infections with homologous serotypes, whereas PPS immunization of mothers did not protect their offspring, in agreement with the low titer of maternal PPS specific antibodies. When adult female mice were immunized with a meningococcal serogroup C conjugate vaccine (MenC-CRM), antibody response and transmission were similar to those observed for pneumococcal antibodies. Importantly, bactericidal activity was demonstrated in offspring of MenC-CRM-immunized mothers. These results demonstrate that this murine model of pneumococcal immunization and infections is suitable to study maternal immunization strategies for protection of offspring against encapsulated bacteria.

Outer membrane proteins (OMP) are expressed in Gram-negative bacterial cell wall. OmpA from Klebsiella pneumoniae (KpOmpA) has been shown to bind and to activate selectively antigen presenting cells (APCs), eliciting protective CTL responses. In this study, we investigated whether OmpX, another member of the OMP family and structurally related to OmpA, exhibits the same immune properties. Using recombinant OmpX from Escherichia coli (EcOmpX), we report that EcOmpX binds to and is internalized by human APCs. However, EcOmpX does not activate APCs. EcOmpX acts as an efficient carrier protein as it induces a potent and Th1/Th2 mixed anti-TNP humoral response. However, adjuvant is required to generate a protective anti-tumoral immune response in mice injected with a tumor model antigen coupled to EcOmpX. Collectively, these data show that EcOmpX is recognized by innate cells but does not activate them, suggesting that EcOmpX does not provide a signal danger to APCs. In conclusion, this study provides information on the molecular mechanisms involved in the recognition and activation of innate cells by bacterial outer membrane proteins.

Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB.

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. We have observed that antigen presenting cells (APCs) recognize and are activated by the recombinant OmpA from Klebsiella pneumoniae (KpOmpA). KpOmpA triggers cytokine production by macrophages and dendritic cells (DC), induces DC maturation and signals via Toll-like receptor 2. KpOmpA also interacts with endocytic receptor(s) expressed on DC and macrophages. Tumor antigens coupled to KpOmpA are taken up by APCs and gain access to the MHC class I pathway, triggering the initiation of protective anti-tumor cytotoxic responses in the absence of CD4 T cell help and adjuvant. Thus, OmpA appears as a new type of pathogen-associated molecular pattern (PAMP) usable as a vector in anti-infectious and therapeutic anti-tumor vaccines to elicit CTLs.

Previous work has suggested that the peptide-carrier, heat-shock protein (hsp)70, could directly activate APC. Here we show that this ability is related to endotoxin contamination of the human rhsp70 produced in Escherichia coli. Hence, the ability of 1-3 microg/ml of rhsp70 to induce the maturation of human monocyte-derived DC is abrogated in the presence of the LPS-antagonist polymyxin B or when the rhsp70 contains less than 60 IU/mg endotoxin. Such a level of contamination of the rhsp70 is, however, sufficient - in the presence of soluble rCD14, the LPS co-receptor - to induce cytokine secretion from monocytes and DC, despite the presence of polymyxin B. However, when endotoxin contamination is below 10 IU/mg, rhsp70 does not induce cytokine secretion - even in the presence of soluble rCD14 - or activate p38 mitogen-activated protein kinase signaling pathways, thus showing that an "endotoxin free" hsp70 does not activate APC.

BBG2Na is a recombinant protein, composed in part of carrier protein BB and of the central conserved domain of the attachment glycoprotein G of human respiratory syncytial virus (HRSV) subgroup A. This protein is a potent vaccine candidate against HRSV. G2Na contains several contiguous B-cell epitopes, occupying sequential positions in the linear sequence of the protein. One of the epitopes contains four cysteines that are completely conserved in known strains of HRSV and form a 'cysteine noose' motif. In this study, we analysed circular dichroism (CD) spectra of BBG2Na and its B-cell epitopes. We also used NMR and molecular dynamics simulations to determine the three-dimensional structure of the cysteine noose domain. We observed significant structural differences related to the length of peptides containing the cysteine noose. These differences show good correlation with the immunogenic activity of the peptides. It is shown that a single Val(171) addition induces a pronounced structure stabilization of the cysteine noose peptide G4a (1-4/2-3) (residues 172-187), which is associated with a 100-fold increase in its antigenicity vis-à-vis a G-protein specific monoclonal antibody.

Polysaccharides (PSs) derived from Streptococcus pneumoniae include more than 90 serotypes and differ greatly in their immunogenicity. In addition, immunization with PSs does not induce high affinity antibody production and no memory B-cells are generated. Coupling PSs to carrier proteins has been reported to induce B-cell maturation and to install a B-cell memory. As an alternative carrier protein, the outer membrane protein A (OmpA) derived from Klebsiella pneumoniae has been coupled to various PSs. We evaluated the immunogenicity of two PS conjugates, using PS derived from S. pneumoniae types 14 and 19. In this report, we show that anti-PS IgG responses are generated after the conjugation of PSs to P40. In addition, the humoral response generated is able to protect mice from a bacterial challenge. Our results indicate that P40 could be included in the development of new PS conjugate vaccines.

Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A from Klebsiella pneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive or K. pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection.

The decapeptide ELA (ELAGIGILTV), a Melan-A/MART-1 antigen immunodominant peptide analogue, is an interesting melanoma vaccine candidate alone or in combination with other tumour antigens. P40, the recombinant outer membrane protein A of Klebsiella pneumoniae (kpOmpA), was recently shown to target dendritic cells and to induce peptide-specific CTLs. Here we investigated the adjuvant role of P40 mixed or chemically conjugated to ELA. This compound is an N-terminal glutamic acid-containing peptide. However, it has been reported that the amino group and the gamma-carboxylic group of glutamic acids easily condense to form pyroglutamic derivatives. Usually, to overcome this stability problem, peptides of pharmaceutical interest were developed with a pyroglutamic acid instead of N-terminal glutamic acid, without loss of pharmacological properties. Unfortunately, the pyroglutamic acid derivative (PyrELA) as well as the N-terminal acetyl capped derivative (AcELA) failed to elicit CTL activity when mixed with P40 adjuvant protein. Despite the apparent minor modifications introduced by PyrELA and AcELA, these two derivatives have probably lower affinity than ELA for the class I Major Histocompatibility Complex. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride shows a higher stability than the acetate and may be suitable for use in man., (Copyright 2001 The International Association for Biologicals.)

Human respiratory syncytial virus (hRSV) is a major pathogen responsible for bronchiolitis and severe pulmonary disease in very young children, immunodeficient patients and the elderly. BBG2Na, a recombinant chimeric protein produced in Escherichia coli, is a promising subunit vaccine candidate against this respiratory pathogen, composed of G2Na, the central domain of RSV G glycoprotein, and BB, an albumin binding domain of streptococcal protein G. BBG2Na has a basic isoelectric point (pI 9.3) and as expected, is strongly adsorbed by aluminium phosphate (AP). Surprisingly, BBG2Na is also strongly adsorbed by aluminium hydroxide (AH), which normally binds molecules with acidic isoelectric points. This behaviour was unexpected according to the well established adsorption model of Hem and co-workers. Our observations may be explained by the bipolar two-domain structure of the BBG2Na chimera which is not reflected by the global basic isoelectric point of the whole protein: the BB domain has an acidic isoelectric point (pI 5.5) and the G2Na domain a highly basic one (pI 10.0). Importantly, formulation in either aluminium salt resulted in equally high immunogenicity and protective efficacy against RSV in mice. From a physicochemical point of view, this unique property of BBG2Na makes it eminently suitable for combination to either paediatric or elderly multivalent AH- or AP-containing vaccines already in the market or in development.

Respiratory syncytial virus (RSV) is a major respiratory pathogen responsible for severe pulmonary disease. We have developed a parenterally administered vaccine, BBG2Na, which is currently in a phase III clinical trial. BBG2Na comprises residues 130--230 of RSV-A G protein (G2Na) fused to the BB carrier protein. In this study, we show that BBG2Na can be delivered by the nasal route and generates both mucosal and systemic antibody responses when co-administered with cholera toxin B or a newly described delivery system, zwittergent 3--14. We found that nasal BBG2Na administration protects against RSV challenge and does not induce lung immunopathology upon subsequent RSV challenge.

Several cytotoxic T lymphocyte peptide-based vaccines against hepatitis B, human immunodeficiency virus and melanoma were recently studied in clinical trials. One interesting melanoma vaccine candidate alone or in combination with other tumor antigens, is the decapeptide ELA. This peptide is a Melan-A/MART-1 antigen immunodominant peptide analog, with an N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxylic group of glutamic acids, as well as the amino group and gamma-carboxamide group of glutamines, condense easily to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutical interest have been developed with a pyroglutamic acid instead of N-terminal glutamine or glutamic acid, without loss of pharmacological properties. Unfortunately compared with ELA, the pyroglutamic acid derivative (PyrELA) and also the N-terminal acetyl-capped derivative (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the apparent minor modifications introduced in PyrELA and AcELA, these two derivatives probably have lower affinity than ELA for the specific class I major histocompatibility complex. Consequently, in order to conserve full activity of ELA, the formation of PyrELA must be avoided. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride salt, shows higher stability than the acetate salt and may be suitable for use in man. Similar stability data were also obtained for MAGE-3, another N-terminal glutamic acid containing CTL peptide in clinical development, leading us to suggest that all N-terminal glutamic acid and probably glutamine-containing CTL peptide epitopes may be stabilized as hydrochloride salts.

We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV). The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches. The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing. The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm). The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm. To our knowledge, this is the first sensitive HCP assay reported for a vaccine.

The recent identification of tumor Ags as potential vaccines has prompted the search for efficient adjuvants and delivery systems, especially in the case of peptide-based vaccination protocols. Here, we investigated the adjuvant potential of the recombinant 40-kDa outer membrane protein of Klebsiella pneumoniae (P40) for specific CTL induction. We studied the CTL response induced in HLA-A*0201/K(b) transgenic mice immunized with peptides derived from two melanoma-associated differentiation Ags, the HLA-A*0201-restricted decapeptide Melan-A(26--35) substituted at position 2 and the K(b)-restricted tyrosinase-related protein 2(181--188) T cell epitope. We found that both peptides are able to generate a specific CTL response when mixed with the protein in the absence of conventional adjuvant. This CTL response is a function of the amount of P40 used for immunization. Moreover, the CTL response generated against the tyrosinase-related protein 2(181-188) peptide in presence of P40 is associated with tumor protection in two different experimental models and is independent of the presence of CD4(+) T lymphocytes. Thus, the recombinant bacterial protein P40 functions as a potent immunological adjuvant for specific CTL induction.

Respiratory syncytial virus (RSV) is an important respiratory pathogen in man, against which no vaccine is available. However, recent evidence suggests that antibodies to the RSV F and G proteins may play an important role in disease prevention. We previously demonstrated that BBG2Na, a subunit vaccine candidate including residues 130-230 of the Long strain G protein, protects rodents against RSV challenge. Using a panel of monoclonal antibodies (MAb) and synthetic peptides, five linear B cell epitopes were identified that mapped to residues 152-163, 165-172, 171-187 (two over-lapping epitopes) and 196-204. Antibody passive transfer and peptide immunisation studies revealed that all were protective. Pepscan analyses of anti-RSV-A and BBG2Na murine polyclonal sera suggested stronger immunogenicity of some protective epitopes (protectopes) in the context of BBG2Na compared with live virus. However, all the identified murine B cell protectopes were conserved in RSV seropositive humans. Should these protectopes correspond with protection in humans, BBG2Na may constitute a very interesting vaccine candidate against RSV.

Respiratory syncytial virus is one of the major respiratory pathogens for infants and immunocompromized children. With the exception of young children, all the population has encountered RSV and is seropositive. Recent reports have demonstrated however that the virus also affects the elderly and represents a major cause of illness associated with an excess of morbidity and mortality. We have generated a recombinant RSV vaccine, BBG2Na, which is highly protective in rodents against RSV infection. The aim of this study was to evaluate the ability of the vaccine to increase anti-RSV protection in RSV-primed mice and to characterize the induced immune responses. Immunization with BBG2Na increased the anti-RSV-A serum antibody titers of RSV-primed mice with induction of both IgG1 and IgG2a antibodies attesting for a mixed Th response. Moreover, the level of the induced anti-G2Na antibodies was greater in seropositive mice. Finally, sera from RSV-primed mice displayed a higher protective efficacy after transfer into naive mice following subsequent immunization with BBG2Na than sera of mice immunized with RSV-A only. Our results demonstrate that BBG2Na is immunogenic and increases the protective efficacy of serum antibodies in RSV-primed mice; they support the possibility of performing clinical trials in the seropositive human population.

We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4(+), but not CD8(+), T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967-1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4(+) T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.

Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumonia in infants and young children. Approximately 100000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human Respiratory Syncytial Virus vaccines (hRSV), we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulfide-containing peptides, all possible oxidized isomers were synthesized using various oxidation conditions and resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthesis and analytical methods were developed for the characterization on one hand, of recombinant fusion protein BBG2Na which is currently being investigated in advanced clinical phases as a very promising vaccine candidate, and on the other hand, for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidation and analysis conditions, should be useful for disulfide pairing studies of other peptides and proteins having the same 'xCxxCxxxxxCxxxCx' framework, such as G proteins of non-human RSV strains, developed by other groups as veterinary vaccine candidates for example.

Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumoniae in infants and young children. Approximately 100,000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human RSV vaccines, we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulphide bridges containing peptides, all possible oxidizing isomers were synthesized using various oxidising conditions, resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP-HPLC, FZCE and ES-MS after purification by preparative RP-HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC-MS analysis and peptide microsequencing. These synthetic and analytical methods were developed for the characterization of recombinant fusion protein BBG2Na which is currently investigated in clinical phase II and seems to be as a very promising vaccine candidate, and for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidising and analysis conditions, should be useful for disulphide pairing studies of other peptides and proteins having the same "xCxxCxxxxxCxxxCx" framework, such as G proteins of non-human RSV strains, developed for example as veterinary vaccine candidates.

We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1'). After immunization of mice with the rP40-G1' conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155-166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.

The relative immunopathogenic potential of a recombinant fusion protein incorporating residues 130-230 of respiratory syncytial virus (RSV-A) G protein (BBG2Na), formalin-inactivated RSV-A (FI-RSV), and phosphate-buffered saline (PBS) was investigated in mice after immunization and RSV challenge. FI-RSV priming resulted in massive infiltration of B cells and activated CD4(+) and CD8(+) T lymphocytes in mediastinal lymph nodes (MLN) and lungs, where eosinophilia and elevated IFN-gamma, IL-2, -4, -5, -10, and -13 mRNA transcripts were also detected. PBS-primed mice showed only elevated pulmonary IL-2 and IFN-gamma mRNAs, while an activated CD8(+) T cell peak was detected in MLN and lungs. Cell infiltration also occurred in MLN of BBG2Na-immunized mice. However, there was no evidence of T cell, B cell, or granulocyte infiltration or activation in lungs, while transient transcription of Th1-type cytokine genes was evident. The absence of pulmonary infiltration is unlikely due to insufficient viral antigen. Thus, this recombinant fusion RSV G fragment does not prime for adverse pulmonary immunopathologic responses., (Copyright 1999 Academic Press.)

BBG2Na is a protein comprising residues 130-230 of the respiratory syncytial virus subgroup A (RSV-A) G protein (G2Na) fused to the albumin-binding domain of streptococcal G protein (BB). BBG2Na was cloned, expressed in Escherichia coli and renaturated. In rodent models, this subunit RSV vaccine adjuvanted in Alhydrogel induced specific antibodies and conferred protection to RSV infection. Comparison of the antibody production in a BALB/c mouse model revealed that BBG2Na induced a stronger and earlier G2Na antibody response than G2Na alone, without altering the IgG subclass distribution. To address the role of the BB part, we explored its carrier properties and showed that it is a Th dependent antigen, generating a more potent G2Na-specific B cell memory response and able to generate Th cells that provide help for G2Na antibody production.

Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37,061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties.

By employing a novel biotin- and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.

The polypeptide of aa 130-230 of the G protein (G2Na) of respiratory syncytial virus (RSV) was fused to BB, the albumin-binding region of streptococcal G protein, producing BBG2Na, which induced protective immune responses in rodent models. Evaluation of the immune response in mice immunized with BBG2Na in the adjuvant alhydrogel revealed high amounts of interleukin (IL)-5 and some IL-4 in splenocytes restimulated in vitro. This is compatible with a Th2 response. The activation of the Th2 pathway in such mice was further supported by the detection of IL-5 and G2Na-specific IgE in vivo. Of interest, in contrast to immunization with formalin-inactivated RSV, immunization of mice with BBG2Na followed by intranasal RSV challenge did not lead to increased production of IL-5- or G2Na-specific IgE. However, IgG1- and IgG2a-specific antibodies were boosted. These results demonstrate that the Th2 pathway is not enhanced by RSV challenge in BBG2Na-immunized mice.

We have previously reported the substrate specificity of the rat liver cytosolic alpha-D-mannosidase [Haeuw, J. F., Strecker, G., Wieruszeski, J. M., Montreuil, J. & Michalski, J.-C. (1991) Eur. J. Biochem. 202, 1257-1268]. Here, we report the characterization and the purification of this alpha-D-mannosidase and the presence of two soluble forms of alpha-D-mannosidases from rat liver. The cytosolic alpha-D-mannosidase was purified nearly 660-fold with 2.66% recovery to a state approaching homogeneity using: (a) (NH4)2SO4 precipitation; (b) concanavalin-A-Sepharose chromatography; (c) affinity chromatography on a cobalt-chelating Sepharose column; (d) ion-exchange (DEAE-trisacryl M) column chromatography; (e) molecular-size chromatography (Sephacryl S 200). The enzyme was eluted from the final column at an apparent molecular mass of 113 kDa. SDS/PAGE analysis yielded a major protein band at 108 kDa. Moreover, the purification allowed to distinguish two mannosidase activities with different kinetic properties. The first cytosolic activity retained on the cobalt-chelating column was optimally active at neutral pH, was activated by Co2+, was strongly inhibited by swainsonine (Ki = 3.7 microM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (Km = 0.072 mM). Man9GlcNAc was hydrolysed by the purified enzyme down to a Man5GlcNAc structure, i.e. Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4) GlcNA c, which represents the Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The second activity not retained on the cobalt-chelating column was optimally active at neutral pH, was inhibited by swainsonine (Ki = 28.4 microM) but not by deoxymannojirimycin and was active with p-nitrophenyl alpha-D-mannoside (Km = 0.633 mM). Man9GlcNAc was broken by this enzymic activity down to Man8GlcNAc and Man7GlcNAc structures. Similitaries with endoplasmic reticulum alpha-D-mannosidase exist and this enzyme could be the cytosolic form of the endoplasmic reticulum alpha-D-mannosidase.