Detection of early secretory antigenic target-6 antibody for diagnosis of tuberculosis in non-human primates

Tuberculosis is one of the most economically devastating, zoonotic infections of captive non-human primates. The limitations of the tuberculin skin test, which is currently used to diagnose tuberculosis in living non-human primates, make it necessary to find new, simple, and economical diagnostic methods. We describe use of an enzyme-linked immunoassay to detect IgG antibodies against early secretory antigenic target (ESAT)-6, a small protein secreted by virulent tubercle bacilli, in paired (pre- and post-outbreak) sera from 57 non-human primates involved in an outbreak of Mycobacterium bovis infection in a research colony. Of 25 animals with tuberculosis lesions at necropsy, 22 (88%) had high serum levels of the ESAT-6 antibody. The ESAT-6 antibody was found in 16% (5/32) of post-outbreak sera from animals in which tuberculosis could not be confirmed at necropsy. The strong association between the ESAT-6 antibody and tuberculosis in non-human primates documented in this study, together with the robustness of the serologic assay, make the ESAT-6 ELISA a valuable tool for diagnosis of tuberculosis in captive non-human primates.     

Defining the protective antibody response for HIV-1

The development of an effective HIV-1 vaccine would be greatly facilitated by knowledge regarding the type and quantity of antibodies that are protective. Since definitive immune correlates are established only after a vaccine has been shown to be effective in humans, animal models are often used to guide vaccine development. Experimental lentivirus infection of non-human primates has shown that neutralizing antibodies can protect against infection. Most specifically, studies of passive antibody transfer in the chimeric SIV/HIV-1 immunodeficiency virus (SHIV) model have provided quantitative data on the level of protective antibody required. While direct extrapolation to humans is difficult, these data likely provide important insights into the protection afforded by antibodies against HIV-1. When used alone, high levels of neutralizing antibodies are required to completely block infection. However, even modest levels of antibody can provide partial protection and affect disease course. Understanding the exact level of protective antibody becomes even more complex in the setting of active immunization and coexisting cellular immunity. Despite this uncertainty, recent findings from lentiviral animal models strongly suggest that neutralizing antibodies will contribute to protection against HIV-1. Based on these data, a major goal of HIV-1 vaccine strategies is the induction of neutralizing antibodies against circulating primary HIV-1 strains.     

From rodent reagents to human therapeutics using antibody guided selection

Guided selection is a method of producing a human version of a rodent or any other non-human antibody. The process is a serial transition from rodent to human via rodent-human chimaerics, through to a panel of human antibodies with similar characteristics to those of the starting rodent antibody. The guided selection process can be undertaken using either phage display or ribosome display, and chimaeric antibodies can be made either in series or parallel, with or without the retention of the original rodent CDR3s. Guided selection has successfully been used for the generation of a number of human versions of rodent antibodies, including HUMIRA, an inhibitor of tumour necrosis factor-alpha which is approved for the treatment of moderate to severe rheumatoid arthritis in over 40 countries.     

Immunogenicity of engineered antibodies

Administration of a therapeutic antibody can lead to an anti-antibody response (AAR). Much effort has been applied to engineering antibodies with as little as possible non-human structure to minimize such responses. Here, we review reported AAR to murine, mouse-human chimeric, and humanized antibodies. Replacement of mouse immunoglobulin constant regions with human ones effects the largest immunogenicity reduction. Humanization of variable domains effects a further decrease.     

Non-human primate immune libraries combined with germline humanization: An (almost) new,and powerful approach for the isolation of therapeutic antibodies

Panning of libraries constructed from immunised non-human primates (NHP) has not been widely used, even though this has proven to be a successful approach for the isolation of human-like antibody fragments with affinities in the nanomolar to the picomolar range. As recently demonstrated, after initial isolation of antibodies with such high affinities, germline humanization may be applied to these Fabs or scFvs to increase the similarity of their framework regions with those encoded by human germline genes. ‘Germlinized’ antibody fragments may be converted to full size IgGs; indications are given that these IgGs could be better tolerated in clinical use than human antibodies. The use of the combination of NHP immune libraries and germline humanization thus may compete with use of libraries of human origin, whether naïve or immune, and with synthetic libraries. In this report, the various approaches will be compared, and advantages of the two-step NHP-based method, as well as corresponding intellectual property aspects, will be discussed.Key words: non-human primate, antibody, naïve library, synthetic library, immune library, affinity, epitope, tolerance, patent     

Generation of human antibody fragments against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>using a phage display chain shuffling approach

Background  

Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.     

Transferring the Characteristics of Naturally Occurring and Biased Antibody Repertoires to Human Antibody Libraries by Trapping CDRH3 Sequences

Antibody repertoires are characterized by diversity as they vary not only amongst individuals and post antigen exposure but also differ significantly between vertebrate species. Such plasticity can be exploited to generate human antibody libraries featuring hallmarks of these diverse repertoires. In this study, the focus was to capture CDRH3 sequences, as this region generally accounts for most of the interaction energy with antigen. Sequences from human as well as non-human sources were successfully integrated into human antibody libraries. Next generation sequencing of these libraries proved that the CDRH3 lengths and amino acid composition corresponded to the species of origin. Specific CDRH3 sequences, biased towards the recognition of a model antigen either by immunizing mice or by selecting with phage display, were then integrated into another set of libraries. From these antigen biased libraries, highly potent antibodies were more frequently isolated, indicating that the characteristics of an immune repertoire is transferrable via CDRH3 sequences into a human antibody library. Taken together, these data demonstrate that the properties of naturally or experimentally biased repertoires can be effectively harnessed for the generation of targeted human antibody libraries, substantially increasing the probability of isolating antibodies suitable for therapeutic and diagnostic applications.     

Non-human primate immune libraries combined with germline humanization

Panning of libraries constructed from immunised non-human primates (NHP) has not been widely used, even though this has proven to be a successful approach for the isolation of human-like antibody fragments with affinities in the nanomolar to the picomolar range. As recently demonstrated, after initial isolation of antibodies with such high affinities, germline humanization may be applied to these Fabs or scFvs to increase the similarity of their framework regions with those encoded by human germline genes. ‘Germlinized’ antibody fragments may be converted to full size IgGs; indications are given that these IgGs could be better tolerated in clinical use than human antibodies. The use of the combination of NHP immune libraries and germline humanization thus may compete with use of libraries of human origin, whether naïve or immune, and with synthetic libraries. In this report, the various approaches will be compared, and advantages of the two-step NHP-based method, as well as corresponding intellectual property aspects, will be discussed.     

Memory immune response generated in Cercopithecus aethiops against meningococcal polysaccharide serogroup C conjugate vaccine

The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model.     

Antigen recognition by serum antibodies in non-human primates experimentally infected with Mycobacterium tuberculosis

Tuberculosis is a significant threat to non-human primates and their caretakers. The diagnosis of tuberculosis in living non-human primates is currently based on the tuberculin skin test, which is cumbersome and sometimes inaccurate. Development of an accurate serodiagnostic test requires identification of the key antigens of Mycobacterium tuberculosis involved in antibody production. When sequential serum samples obtained from 17 cynomolgus, rhesus, and African green monkeys up to seven months since experimental infection with M. tuberculosis Erdman were screened for antibody against purified proteins of M. tuberculosis, three highly seroreactive antigens were identified. One protein, ESAT-6, reacted with sera from all infected animals. Two additional proteins, alpha-crystallin and MTSA-10, were recognized by sera from approximately 90% of infected animals. Time course analysis of antibody production indicated that the earliest response was usually to ESAT-6 alone or to ESAT-6 and other antigen(s). These results provide experimental evidence of the potential value of ESAT-6 as an antigen for use in serodiagnosis of tuberculosis in non-human primates.     

小鼠抗食蟹猴IgG单克隆抗体的制备

目的制备抗食蟹猴、恒河猴等非人灵长类实验动物免疫球蛋白二级抗体,开展对其传染病血清学快速诊断方法的建立。方法采用饱和硫酸铵盐析、Agarose-Protein G亲和层析技术,从食蟹猴血清中提纯IgG。经SDS-PAGE电泳鉴定,采用常规法免疫C57BL/6小鼠,三次免疫后取脾细胞与Sp2/0-Ag14骨髓瘤细胞通过PEG4000融合制备杂交瘤细胞,利用间接ELISA、Western blot等方法进行筛选、鉴定。结果得到5株阳性杂交瘤,分别命名为2B6、2B7、2D9、3B2、5E4,并且5株杂交瘤分泌的抗体均与恒河猴的IgG或血清发生交叉反应,而与其他物种如东北虎、犬等动物的IgG或血清无交叉反应。结论 5株杂交瘤产生的单克隆抗体（McAb）具有较好免疫活性,且能长期、稳定地分泌抗体。此项研究工作为后续研究食蟹猴、恒河猴传染病血清学诊断方法奠定基础。     

Detection of elevated antibody against calreticulin by ELISA in aged cynomolgus monkey plasma

Calreticulin (Crt) is a molecular chaperone ubiquitously present in the endoplasmic reticulum. In non-human primates, age-related occurrence of anti-Crt antibody has not been reported. We developed an ELISA assay for an anti-Crt antibody and determined the age-related increase in the levels of anti-Crt antibody in three groups of cynomolgus monkeys: juvenile (1.5 yr), young adults (5-10 yr) and aged adults (20-34 yr). Mean ± SD auto-antibody levels at 450 nm in juvenile, young adults and aged groups were 0.23 ± 0.18, 0.30 ± 0.28, and 0.55 ± 0.33, respectively. Statistically significant differences were noted in the autoantibody levels to Crt among the aged group and juvenile or young adults. This is the first report to demonstrate the expression of anti-Crt autoantibody in aged monkeys and indicates that cynomologous monkeys may serve as an appropriate nonhuman primate model for studies of age-related alteration of immune function in elderly humans. Though preliminary, this finding merits further investigation to determine the relationship between immunosenescence and expression of antibodies to Crt.     

Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as "intrabodies"). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued "rediscovery" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.     

Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.     

The DWEYS peptide in systemic lupus erythematosus

Current therapies for treating systemic lupus erythematosus (SLE) mainly rely upon nonspecific and toxic immunosuppression by corticosteroids and cytotoxics. Although biologics hold promise, many agents have yet to prove clinical efficiency in controlled trials, with further limitations related to safety and cost. The primary self-specificity in SLE is double-stranded (ds) DNA. Studying anti-dsDNA antibodies in animal models of lupus and SLE patients identified a neurotoxic and nephrotoxic subset, including the nephritogenic mouse monoclonal anti-dsDNA antibody R4 that crossreacts with a sequence present in subunits of the N-methyl-d-aspartate receptor. In this review, anti-dsDNA antibodies as a pathogenic factor in SLE and recent efforts for the creation of highly specific, nontoxic therapeutics against an extremely pathogenic subset of such antibodies is discussed.     

Selection and characterization of a human monoclonal neutralizing antibody for Clostridium Botulinum neurotoxin serotype B

Botulinum neurotoxins (BoNTs) are causative agents for botulism and are identified as a category A bioterror agents by the Centers for Disease Control and Prevention (CDC). Current antitoxins against BoNTs intoxication have some limitations including side effects or limited supply. As an alternative, neutralizing monoclonal antibodies will play an increasing role as BoNTs therapeutics. To date, no human anti-BoNT/B neutralizing monoclonal antibodies have yet to be reported. Herein, we describe an improved selection approach and characterization of a human monoclonal antibody, F2, which is capable of binding BoNT/B with high specificity and displays neutralizing activity in an in vitro cell-based assay. Through surface plasmon resonance studies, we have determined its association and dissociation rate constants. In sum, our data demonstrate that monoclonal antibody F2 is a promising BoNT/B therapeutic lead for further development.     

Humoral response of cynomolgus macaques to human soluble CD4: antibody reactivity restricted to xeno-human determinants.

The CD4 cell surface glycoprotein which is expressed primarily by a subset of T lymphocytes plays a key role in normal immune responses. In the immunopathogenesis of AIDS, it serves as the high-affinity receptor for HIV, facilitating viral attachment and entry into CD4+ cells. As such, the truncated soluble form of this molecule (sT4) has been proposed as a therapeutic drug for the treatment of AIDS whereby it would act as decoy for viral entry into cells or facilitate elimination of soluble viral envelope glycoprotein. In a study designed to look at the effect of sT4 on immune function, sT4 was administrated to experimentally naive primates. In this report, we show that administration of sT4 to cynomolgus macaque monkeys over a period of up to 3 weeks results in antibody responses with specificities for human CD4 molecules. Antisera thus generated bound sT4 and cell surface CD4 expressed on human T lymphocytes but failed to bind to cynomolgus lymphocytes. These antibodies caused no apparent adverse effects on normal immune functions of the cynomolgus macaques. We conclude from these data that the antibody response to soluble CD4 in cynomolgus monkeys is directed at determinants present on human CD4 but absent on monkey CD4. The restricted xenogeneic specificity of the antibody response indicates that soluble CD4 may not be highly immunogenic in syngeneic hosts. The present study also shows that these antibodies can block HIV-induced syncytium formation indicating that the antibodies bind to regions on the CD4 molecule close to the HIV-env gp120 binding site. The gp120 binding site, which resides within the N-terminal V1 domain of CD4, encompasses a region which corresponds to the complementarity determining regions (CDRs) of immunoglobulins. The CDR-like regions of CD4-V1 manifest the greatest species divergence, are tolerant to experimental in vitro mutagenesis, and generate the predominant antibody response in mice immunized with human CD4 indicating that differences in the V1 sequence between human and other non-human primates may localize to this regions.     

Prevalence of rotavirus and norovirus antibodies in non-human primates

Rotavirus and norovirus are associated with a substantial burden of diarrheal disease in humans and some animals, but their role in acute viral gastroenteritis in non-human primates has not been established. We examined sera from five species of Old and New World monkeys and chimpanzees for antibodies to rotavirus and norovirus by enzyme immunoassays using RRV and three recombinant human norovirus capsid proteins, respectively. Most (88%) of the 3 Old World monkey species (mangabey, pigtail, and rhesus) and apes were seropositive for rotavirus. Norovirus antibody was prevalent in the three monkey species, with 53% (44/83) and 58% (48/83) seropositive for GI and GII strains, respectively. Eleven (92%) of the 12 chimpanzees tested were seropositive for GI norovirus. Given the high rate of infection with both viruses, the role of these agents in causing acute gastroenteritis in non-human primates and the value of these animals as models of infection and disease need to be assessed.     

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1’s extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination.     

Two-dimensional immune profiles improve antigen microarray-based characterization of humoral immunity

Antigen arrays are becoming widely used tools for the characterization of the complexity of humoral immune responses. Current antibody profiling techniques provide modest and indirect information about the effector functions of the antibodies that bind to particular antigens. Here we introduce an antigen array-based approach for obtaining immune profiles reflecting antibody functionality. This technology relies on the parallel measurement of antibody binding and complement activation by features of the array. By comparing sera from animals immunized against the same antigen under different conditions, we show that identifying the position of an antigen in a 2-D space, derived from antibody binding and complement deposition, permits distinction between immune profiles characterized by diverse antibody isotype distributions. Additionally, the technology provides a biologically interpretable graphical representation of the relationship between antigen and host. Our data suggest that 2-D immune profiling could enrich the data obtained from proteomic scale serum profiling studies.