Glycosylation of Therapeutic Proteins

During their development and administration, protein-based drugs routinely display suboptimal therapeutic efficacies due to their poor physicochemical and pharmacological properties. These innate liabilities have driven the development of molecular strategies to improve the therapeutic behavior of protein drugs. Among the currently developed approaches, glycoengineering is one of the most promising, because it has been shown to simultaneously afford improvements in most of the parameters necessary for optimization of in vivo efficacy while allowing for targeting to the desired site of action. These include increased in vitro and in vivo molecular stability (due to reduced oxidation, cross-linking, pH-, chemical-, heating-, and freezing-induced unfolding/denaturation, precipitation, kinetic inactivation, and aggregation), as well as modulated pharmacodynamic responses (due to altered potencies from diminished in vitro enzymatic activities and altered receptor binding affinities) and improved pharmacokinetic profiles (due to altered absorption and distribution behaviors, longer circulation lifetimes, and decreased clearance rates). This article provides an account of the effects that glycosylation has on the therapeutic efficacy of protein drugs and describes the current understanding of the mechanisms by which glycosylation leads to such effects.     

Immunogenicity of Peanut Proteins Containing Poly(Anhydride) Nanoparticles

In the last decade, peanut allergy has increased substantially. Significant differences in the prevalence among different countries are attributed to the type of thermal processing. In spite of the high prevalence and the severe reaction induced by peanuts, there is no immunotherapy available. The aim of this work was to evaluate the potential application of poly(anhydride) nanoparticles (NPs) as immunoadjuvants for peanut oral immunotherapy. NPs loaded with raw or roasted peanut proteins were prepared by a solvent displacement method and dried by either lyophilization or spray-drying. After physicochemical characterization, their adjuvant capacity was evaluated after oral immunization of C57BL/6 mice. All nanoparticle formulations induced a balanced T_H1 and T_H2 antibody response, accompanied by low specific IgE induction. In addition, oral immunization with spray-dried NPs loaded with peanut proteins was associated with a significant decrease in splenic T_H2 cytokines (interleukin 4 [IL-4], IL-5, and IL-6) and enhancement of both T_H1 (gamma interferon [IFN-γ]) and regulatory (IL-10) cytokines. In conclusion, oral immunization with poly(anhydride) NPs, particularly spray-dried formulations, led to a pro-T_H1 immune response.     

Immunogenicity to Biologics: Mechanisms,Prediction and Reduction

Currently, there is a significant rise in the development and clinical use of a unique class of pharmaceuticals termed as Biopharmaceuticals or Biologics, in the management of a range of disease conditions with, remarkable therapeutic benefits. However, there is an equally growing concern regarding development of adverse effects like immunogenicity in the form of anti-drug antibodies (ADA) production and hypersensitivity. Immunogenicity to biologics represents a significant hurdle in the continuing therapy of patients in a number of disease settings. Efforts focussed on the identification of factors that contribute towards the onset of immunogenic response to biologics have led to reductions in the incidence of immunogenicity. An in-depth understanding of the cellular and molecular mechanism underpinning immunogenic responses will likely improve the safety profile of biologics. This review addresses the mechanistic basis of ADA generation to biologics, with emphasis on the role of antigen processing and presentation in this process. The article also addresses the potential contribution of complement system in augmenting or modulating this response. Identifying specific factors that influences processing and presentation of biologic-derived antigens in different genotype and disease background may offer additional options for intervention in the immunogenic process and consequently, the management of immunogenicity to biologics.     

Ensuring the Biologic Safety of Plasma-Derived Therapeutic Proteins

Human plasma-derived proteins, such as immunoglobulins, coagulation factors, alpha1-antitrypsin, fibrin sealants, and albumin, are widely used as therapeutics for many serious and life-threatening medical conditions. The human origin of these proteins ensures excellent efficacy and compatibility but may also introduce the risk of unintentional disease transmission. Historically, only viruses, particularly hepatitis and HIV, have posed serious threats to the safety of these therapeutics. Fortunately, between 1970 and 1990, the molecular biology of each of the major viruses was elucidated. These advances led to the development and implementation of effective donor screening tests, mainly based on immunoassays and nucleic acid testing, which resulted in a significant reduction of disease transmission risk. In addition, viral inactivation and removal steps were implemented and validated by manufacturers, further reducing the risk associated with known, as well as unidentified, viruses. Since the late 1990s, a different class of transmissible agent, referred to as prions, has been identified as a new risk for disease transmission. However, prion diseases are very rare, and prion transmission through plasma-derived proteins has not been reported to date. The prion-related risk is minimized by deferring donors with certain key risk factors, and by the manufacturing processes that are capable of removing prions. Advances in science and pathogen safety-related technology, compliance with good manufacturing practices by manufacturers, and increasingly stringent regulatory oversight, has meant that plasma-derived proteins have been developed into today's highly effective therapeutics with very low risk of disease transmission.     

Parameters Affecting the Immunogenicity of Recombinant T Cell Epitopes Inserted into Hybrid Proteins

In the past few years a considerable number of studies have focused on the mechanisms of antigen presentation by classical major histocompatibility complex (MHC) class I and class II encoded molecules. Among different approaches, the engineering of recombinant chimeric genes and proteins has provided new tools to analyze the parameters influencing the intracellular processing of antigenic determinants. This review will summarize and discuss the different models of recombinant genes and molecules that have been used to analyze the influence of the molecular environment of a T cell determinant on its efficient processing and MHC presentation. This approach may also represent an interesting tool for developing new vaccine strategies for inducing T cell responses against pathogens.     

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund''s adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.     

Citrullinated Proteins in Arthritis; their Presence in Joints and Effects on Immunogenicity

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in RA patients and a suggestive involvement in the pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. The aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in DA rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (RSA) and collagen type II (CII). Our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. Unimmunized animals or time points before clinical signs of arthritis were negative. By morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. A specific Cit-RSA T-cell response was observed in animals challenged by citrullinated RSA, no response was recorded when RSA was used as a stimulus. The IgG analysis reveals not only a response towards the modified protein but also cross-reactivity to native RSA. No T-cell or B-cell response was noted in animals injected with unmodified RSA. Cit-CII induced a disease with higher incidence and earlier onset than did the native counterpart. We conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. As inflammation proceeds, citrulline is detected specifically in the joints. All other organs investigated were negative. We also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties.     

Immunogenicity of DNA Vaccines Expressing Tuberculosis Proteins Fused to Tissue Plasminogen Activator Signal Sequences

Novel tuberculosis DNA vaccines encoding native ESAT-6, MPT-64, KatG, or HBHA mycobacterial proteins or the same proteins fused to tissue plasminogen activator (TPA) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. While all eight plasmids induced specific humoral responses, the constructs expressing the TPA fusions generally evoked higher antibody responses in vaccinated hosts. Although most of the DNA vaccines tested induced a substantial gamma interferon response in the spleen, the antigen-specific lung responses were 2- to 10-fold lower than the splenic responses at the time of challenge. DNA vaccines encoding the ESAT-6, MPT-64, and KatG antigens fused to TPA signal sequences evoked significant protective responses in mice aerogenically challenged with low doses of Mycobacterium tuberculosis Erdman 17 to 21 days after the final immunization. However, the protective response induced by live Mycobacterium bovis BCG vaccine was greater than the response induced by any of the DNA vaccines tested. These results suggest that the tuberculosis DNA vaccines were able to elicit substantial immune responses in suitably vaccinated mice, but further refinements to the constructs or the use of alternative immunization strategies will be needed to improve the efficacy of these vaccine candidates.     

Immunogenicity of IpaC-Hybrid Proteins Expressed in the Shigella flexneri 2a Vaccine Candidate SC602

We have investigated the capacity of live attenuated Shigella flexneri strains to act as vectors for the induction of local and systemic antibody responses against heterologous epitopes. The S. flexneri IpaC antigen was selected as a carrier protein into which the C3 neutralizing epitope of the poliovirus VP1 protein was inserted in eight sites distributed along IpaC. The resulting IpaC-C3 hybrid proteins were expressed from recombinant plasmids in the S. flexneri 2a vaccine candidate, SC602. Their production was similar to that of wild-type IpaC. All of the hybrid proteins but one were secreted as efficiently as wild-type IpaC. Immunization of mice with each of the recombinant SC602 derivatives reveals that one construct is able to induce serum and local anti-C3 antibodies, showing that at least one permissive site of insertion within IpaC can be defined. Furthermore, mouse-to-mouse variability in the anti-C3 response indicates that the amount of hybrid proteins produced in the host by SC602 should be improved for optimal use of S. flexneri live attenuated strains as mucosal vectors for foreign epitopes.Live attenuated vectors are one of the most efficient delivery systems for stimulation of the mucosa-associated immune system (for a review, see reference 10). They have therefore been extensively used to express foreign antigens and epitopes selected from pathogens against which the induction of a local immune response is required for protection (for a review, see reference 23). Usually, foreign epitopes are inserted within a carrier protein that is expressed in the live vector.Shigella flexneri live attenuated strains have been developed as candidates for vaccines against shigellosis, an invasive disease of the human colon (22). The capacity of such strains to act as mucosal vectors has been recently reported (16). Local and systemic antibody responses to fimbriae and CS3 fibrillae of enterotoxigenic Escherichia coli were generated in guinea pigs or mice following immunization with these antigens expressed in CVD1203, an S. flexneri 2a live attenuated strain that confers protection against Shigella keratoconjunctivitis in the guinea pig model (15).The purpose of the present study was to investigate whether S. flexneri vaccine strains could be used as immunization vectors to express heterologous epitopes of eukaryotic origin and, in turn, elicit local and systemic antibody responses to foreign sequences. The IpaC antigen, previously reported as a potential carrier protein (1), was selected for the insertion of the neutralizing C3 epitope of the VP1 protein of poliovirus (26). This 11-residue-long sequence has been previously used as a reporter epitope (5, 25). As immunogenicity of a B-cell epitope depends on its flanking sequences within the hybrid protein (25), the C3 epitope was inserted into eight different sites within the IpaC coding sequence. SC602, an S. flexneri 2a vaccine strain attenuated both in its capacity to move intra- and intercellularly and in its survival in tissues (1), was used as vector. This strain is safe and protective in the macaque monkey model (8) as well as in human volunteers (9). The IpaC-C3 hybrid proteins were expressed from recombinant plasmids within SC602 to retain the functionality of the wild-type (wt) IpaC, thus maintaining the invasiveness of the live attenuated vector and ensuring efficient stimulation of local immunity. Immunogenicity of the IpaC-C3 proteins expressed in SC602 was assessed following an immunization protocol that allows the induction of local and systemic anti-IpaC antibody responses in mice (1).     

Pasteurisation and Homogenisation of Milk Enhances the Immunogenicity of Milk Plasma Proteins in a Rat Model

Groups of rats were immunised intraperitoneally with 0.5 ml of raw milk, skim milk, pasteurised milk or pasteurised/homogenised milk. On day 14, animals were boosted with identical preparations, and serum samples were collected on day 28 for the measurement of IgG antibodies directed against milk plasma proteins. Samples were assayed for antibodies to casein, beta-lactoglobulin, bovine gamma globulin and bovine serum albumin. Significantly higher concentrations of antibodies of all specificities were detected in the serum of animals immunised with pasteurised/homogenised milk compared with animals immunised with raw or skim milk. Higher concentrations of antibodies were also seen in the group immunised with pasteurised/homogenised milk than the group immunised with pasteurised milk, although significant differences were only seen in the response to casein (p < 0.01). Additional animals were twice immunised with either raw milk fortified with keyhole limpet hemocyanin (KLH), or pasteurised/homogenised milk which had been fortified with KLH prior to processing. Such treatment significantly enhanced the immunogenicity of the KLH (p < 0.05). These results raise the possibility that homogenisation may similarly effect the way in which the human immune system interacts with cows' milk proteins in the gastrointestinal tract, and suggest that milk processing may be an important variable to consider in studies of human allergy to cows' milk proteins.     

Prediction of Epitopes in Closely Related Proteins Using a New Algorithm

Latrophilin 1 (presynaptic receptor) binds α-latrotoxin from black widow spider venom and regulates neurotransmitter release from nerve endings. The study of the mechanism of action of this receptor is impeded by the existence of closely related latrophilins 2 and 3. A profile of differences detecting the most differing and identical sites in several proteins was developed in order to obtain highly specific antibodies for differentiation between isoforms of related proteins. In addition, we used an algorithm for prediction of immunogenic sites of the protein, based on the basic vector method. The peptides selected using this algorithm were used for immunization of animals. The resultant sera exhibited the estimated specificity and high affinity for the corresponding receptor forms.     

Therapeutic Effects of Mycobacterial Secretory Proteins Against Established Asthma in BALB/c Mice

Purpose

Live/killed mycobacteria and culture supernatants can suppress asthmatic reactions. This study investigated whether mycobacterial secretory proteins have therapeutic effects on asthma.

Methods

Mycobacterium bovis bacille Calmette-Guérin (BCG; 2×10⁵ CFUs) and mycobacterial secretory proteins (Ag85 complex, 38-kDa protein or MPB70; 4 or 20 µg) were administered intraperitoneally to female BALB/c mice with established airway hyperresponsiveness. One week after treatment, the mice underwent a methacholine challenge test, and then inflammatory cell numbers in bronchoalveolar lavage fluid (BAL) and around bronchi (<500 µm), and cytokine levels in splenocyte supernatants, were assessed.

Results

BCG and all of the tested secretory proteins significantly improved airway sensitivity compared to baseline values (P<0.05). The secretory protein Ag85 complex significantly suppressed airway reactivity also (P<0.05), while 38-kDa protein significantly suppressed reactivity and maximal narrowing (P<0.05). The number of eosinophils in BAL and around bronchi, and the goblet cell proportion, were also significantly reduced in mice in both the BCG and secretory protein groups compared to the asthma control group. IFN-γ/IL-5 ratios were significantly higher in mice treated with BCG, 4 µg MPB70 or 4 µg 38-kDa protein than in asthma control mice (P<0.05), and were negatively associated with airway hyperresponsiveness, peribronchial eosinophil numbers and goblet cell proportion (all P<0.05). IL-17A was positively correlated with IL-5 (r=0.379, P<0.001), maximal airway narrowing, peribronchial eosinophil numbers and goblet cell proportion (all P<0.05).

Conclusions

Secretory proteins from BCG and M. tuberculosis and live BCG were effective against established asthma, their effects being accompanied by increased IFN-γ/IL-5 ratios. Thus, allergic asthma could be effectively treated with mycobacterial secretory proteins.     

Usefulness and Limitation of Carcinoembryonic Proteins as Therapeutic and Prognostic Markers in Malignant Tumors

Patients with germ cell tumors, hepatomas and other malignant tumors were studied throughout their courses of surgical, radiological and medical treatments with serial determinations of AFP, CEA, HCG and basic fetoprotein (BFP), which was newly found by M. Ishii.
In general, these carcinoembryonic or trophoblastic markers were observed to be well correlated with the fate of the tumor in the body and to be useful in evaluating the effect of treatment or in predicting the prognosis of the patient. Except for a postoperative transient increase in serum BFP and CEA, the levels fell after surgery and rose again with recurrence of the tumor. Although we had some patients with teratocarcinoma, hepatoma or gastric carcinoma, whose serum AFP showed a transient parallelism with their clinical courses, an obvious dissociation in these markers occurred later in their course. Similar dissociations were also observed with these carcinofetal or trophoblastic markers in patients with chemotherapy or radiotherapy for their teratocarcinomas.
We conclude that each carcinoembryonic protein is a useful but limited marker for malignancy but should be used clinically in combination with other tumor markers.     

Immunogenicity of Outer Membrane Proteins in a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis: Influence of Adjuvants on the Immune Response

The immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO(4), Rhodobacter sphaeroides LPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found for Escherichia coli LPS, meningococcal icsB and rfaC LPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcal htrB1 LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.     

Immunogenicity of amyloid

Human amyloid appears to contain two immunogenic components, the P (plasma) component and the F (fibril) component. Antisera to P component react with both autologous and homologous antigens by immunodiffusion and fluorescent antibody techniques; antisera to F component react with autologous antigens by immunodiffusion and fluorescent antibody techniques, but react with homologous antigens only by the fluorescent antibody method. Antisera to both P component and F component fail to react with non-amyloid human tissues as well as amyloid deposits in guinea-pig tissues. These results point to heterogeneity in the antigenic determinants of human amyloid and possibly greater inter-species heterogeneity of amyloid.     

Immunogenicity of immunoliposomes

Multilamellar immunoliposomes were prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), sphingomyelin (SPH) and biotinylated dipalmitoylphosphatidylethanolamine (PEB) in the molar ratio of 1:1:1:0.1 with surface linked avidin-biotinylated sheep (anti-mouse IgG) IgG (AV-sIgG_B) or GK1.5 monoclonal rat (anti-mouse L3T4 antigen) IgG (AV-GK1.5_B). The ability of these immunoliposomes to induce antibody responses against AV, sIgG or GK1.5 was determined. GK1.5_B and sIgG_B elicited a low-level antibody response (5–10 μg/ml serum) after i.v. immunization and boosting. Liposomes (1 μmol) containing GK1.5_B or sIgG_B were more effective than free GK1.5_B or sIgG_B in eliciting antibodies (20–30 and 100–120 μg/ml serum, respectively). Liposomal AV mixed with either sIgG or GK1.5 gave antibody levels comparable to immunization with free GK1.5_B or sIgG_B. Liposomes with surface AV-sIgG_B or AV-GK1.5_B elicited antibodies against AV and high levels against GK1.5 or sIgG. Immunoliposomes possessing surface AV-sIgG_B or AV-GK1.5_B were eliminated from the circulation of normal mice relatively slowly (T_1/2 15.5 and 30 min): in contrast, liposomal AV-sIgG_B or AV-GK1.5_B was rapidly eliminated from the circulation of immunized mice (T_1/2 4.5 and 4.0 min). These results demonstrate that liposomes with surface IgG (immunoliposomes) are immunogenic, and that repeated administration elicits anti-IgG antibodies that result in a significant reduction in blood circulation residence times.