Affinity purification and refined structural characterization of terminal deoxynucleotidyltransferase.

A total of 56 stable murine hybridoma monoclones that produce homogeneous antibodies against human or calf terminal deoxynucleotidyltransferase have been established. All of the antibodies exhibited specific binding to various Mr forms of terminal transferase and eight possessed neutralizing activity. Results are presented that permitted characterization of ten of these antibodies with respect to their immunoglobulin class, their recognition of calf or human terminal-transferase Mr species by immunoblotting techniques and their recognition of distinct antigenic sites. Terminal transferase was purified in a single step by using an immunoaffinity column constructed with a monoclonal antibody exhibiting a high binding affinity for the enzyme. Single monoclonal antibodies were also used to bind selectively to terminal-transferase antigen in tissue slices and individual cells.     

Antibodies against Pseudomonas aeruginosa toxin in preparations of normal human immunoglobulin

Some lots of commercial normal human immunoglobulin have been found to contain antibodies neutralizing the action of P. aeruginosa exotoxin. The content of antibodies in human immunoglobulin preparations correlates to a certain degree with their protective activity determined in the passive protection test in white mice. Certain lots of normal human immunoglobulin have been found to possess protective activity, but contain no specific antitoxins. The clinical testing of these immunoglobulin preparations used for treating patients with Pseudomonas infections has yielded promising results.     

Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma

The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.     

Neutralization of bacterial viruses by antibodies of young animals

The development of the avidity of 19S and 7S neutralizing antibodies was studied after the immunization of young rabbits with ΦX 174 bacteriophage. Both 19S and 7S antibodies formed during the entire course of the immunization process were able to neutralize bacteriophage ΦX 174 without the participation of thermolabile serum components: neither heat inactivation at 56°C nor addition of piglet complement into the neutralizing system influenced the titer in the neutralization test. In both immunoglobulin classes (IgM and IgG) the first antibodies formed possessed a low avidity that increased in the course of the immunization process so that in the secondary response antibodies of both 19S and 7S type were formed possessing a high binding activity towards the specific antigen. Following the immunization with a high dose of the phage antigen (10¹⁰ PFU of ΦX 174 phage) 19S antibodies were formed at a relatively fast rate, the avidity of which did not further increase during immunization. When a low antigen dose was used for immunization the avidity of antibodies significantly increased in the course of immunization.     

Experience with the diagnosis of dysentery in adults by determining antibodies to Shigella in the saliva]

Concentration of immunoglobulins and titres of antibodies to Sh. sonnei, Sh. flexneri and enteropathogenic E. coli 0111 was determined in mixed saliva and the blood serum of patients suffering from Sonne dysentery, acute intestinal diseases of unknown etiology, and healthy individuals. The sum total immunoglobulin concentration in mixed saliva proved to be 53--81 times less than in the blood serum, but in the first substrate there was 53--75, and in the second--15% of immunoglobulin A. There proved to be distinct changes in the specific IgA-antibodies in the saliva of patients with Sonne dysentery. A preponderant accumulation of IgG-antibodies was noted in the blood serum. Elevation of both types of antibodies was maximal during the second week of the disease. Sonne dysentery was diagnosed in 80% of the patients by recording the intensity of shifts in the specific antibodies in the saliva, and in 63%--in the blood. The expediency of immunological testing of saliva for the diagnosis of dysentery is substantiated.     

Neutralization of tier-2 viruses and epitope profiling of plasma antibodies from human immunodeficiency virus type 1 infected donors from India

Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19–27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.     

Neutralization of sendai virus by the IgG subclass antibodies of the guinea pig

A comparative study was made of the neutralizing activities of IgG subclasses IgG1 and IgG2, fractionated from guinea pig antisera against Sendai virus. The yields of IgG2 from the antisera were about 16 times as much as those of IgG1. The neutralizing activity of IgG2 per unit weight was four times as high as that of IgG1. This neutralizing activity of both IgG subclasses was enhanced about 10 times by addition of antibodies to the L-chain of guinea pig immunoglobulin. It is suggested that, in the complement-dependent neutralization of the virus, IgG1 and IgG2 activate the complement through the alternative and the classical pathway, respectively.     

A new type of anti-ganglioside antibodies present in neurological patients

High titers of anti-GA1 antibodies have been associated with neurological syndromes. In most cases, these antibodies cross-react with the structurally related glycolipids GM1 and GD1b, although specific anti-GA1 antibodies have also been reported. The role of specific anti-GA1 antibodies is uncertain since the presence of GA1 in the human nervous system has not been clarified. A rabbit was immunized with GD1a and its sera were screened for antibody reactivity by standard immunoassay methods (HPTLC-immunostaining and ELISA). Anti-GD1a antibodies were not detected but, unexpectedly, anti-GA1 IgG-antibodies were found. Antibody binding to GA1 was inhibited by soluble GA1 but also by GD1a. These results indicate that the rabbit produced antibodies that recognize epitopes present on the glycolipids, that are absent or not exposed on solid phase adsorbed GD1a. We investigated the presence of these unusual anti-ganglioside antibodies in normal and neurological patient sera. Approximately, 10% of normal human sera contained low titer of specific anti-GA1 IgG-antibodies but none of them recognized soluble GD1a. High titers of IgG-antibodies reacting only with GA1 were detected in 12 patient sera out of 325 analyzed. Of these, 6 sera showed binding that was inhibited by soluble GD1a and four of them also by GM1. This new type of anti-ganglioside antibodies should be considered important elements for understanding of the pathogenesis of these diseases as well as their diagnosis.     

Mechanisms of antibody-mediated protection against lymphocytic choriomeningitis virus infection: mother-to-baby transfer of humoral protection.

The role of antiviral antibodies in resistance to lymphocytic choriomeningitis virus (LCMV) infection was explored. Immune serum and monoclonal antibodies prevented fatal T-cell-mediated immunopathology following acute LCMV infections. In addition, 10- and 14-day-old mice that received maternally derived anti-LCMV antibodies through nursing were protected from an otherwise lethal LCMV challenge. Detailed investigation of the mechanism(s) by which these antiviral antibodies provided was carried out by using anti-LCMV monoclonal antibodies. Protection correlated directly with the ability of the antibodies to reduce viral titers in the tissues of conventional (K. E. Wright and M. J. Buchmeier, J. Virol. 65:3001-3006, 1991) and nude mice. However, this reduction was not simply a reflection of virus neutralizing activity, since not all antibodies which neutralized in vitro were protective. A correlation was also found between immunoglobulin isotype and protection: all of the protective antibodies were immunoglobulin G2a (IgG2a), while IgG1 antibodies mapping to the same epitopes were not. Protection appeared to be associated with events controlled by the Fc region. Functional F(ab')2 fragments which retained in vitro neutralizing activity were not protective in vivo. Furthermore, this Fc-associated function was not related to complement-mediated cell lysis, since C5-deficient mouse strains were also protected. These results suggest a role for antibody in protection from arenavirus infections and indicate that a distinct immunoglobulin subclass, IgG2a, may be essential for this protection.     

Genetic control of the immune response

(1) Inbred strains of mice when immunized withp-aminobenzoic acid and sulphanilic acid bound by diazo-linkage to the same protein carrier molecule (bovine gamma globulin) differ in their ability to respond by antibody formation. The strains A and CBA/J form only low levels of antibodies to the haptens after immunization; in strains ScSN and B10.LP the same high titers of antibodies to both haptens were found under these conditions. The strain B10.D₂ forms antibodies well to sulphanilic acid, antip-aminobenzoic acid antibodies are formed only in very low quantity. (2) Individual mice of an inbred strain form a homogeneous population in respect of their capability or inability to form a particular antihapten antibody. The individual titers in a given inbred strain vary only slightly. On the contrary the noninbred strain H shows great variability both in quantity and quality of the immune response to the haptens. (3) The crossing of good and poor anti-hapten antibody producing strains shows in F₁; F₂ and B₁ generation, that the ability to produce antibodies againstp-aminobenzoic and sulphanilic acid depends on the genotype of a given individual. The ability to respond is transmitted to the offspring as a dominant trait. (4) There is no difference in the response to the haptens between males and females of the same strain. (5) The antibodies to the haptens in different strains of mice differ in the ratio of 2-mercaptoethanol sensitive and 2-mercaptoethanol resistant antibody. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Neutralizing Antibodies from the Sera of Human Immunodeficiency Virus Type 1-Infected Individuals Bind to Monomeric gp120 and Oligomeric gp140

Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation.     

Thermodynamics of antibody-antigen reactions. 1. The binding of simple haptens to two classes of antibodies fractionated according to affinity

The thermodynamic parameters which characterize the binding of dinitrophenylglycine and dinitrophenylmethoxypoly(ethylene glycol) to selected affinity classes of equine IgG and IgG(T) antibodies were determined by fluorescence quenching and flow calorimetry. The binding enthalpies and entropies were in all cases large and negative, falling in the ranges -14 to -17 kcal/mol and -18 to -25 eu, respectively. The differences in the enthalpies and entropies of binding for different affinity classes and for different haptens are discussed with reference to differences in the structures of the haptens studied and as indications of differences in binding site structure. In addition, the apparent existence of fluorescent side chains which can transfer energy to either hapten binding site in IgG(T) antibodies but not in IgG antibodies is interpreted as indicative of a smaller average interbinding site distance in IgG(T) than in IgG antibodies.     

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.     

Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design

The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.     

Isolation of human monoclonal antibodies that neutralize human rotavirus

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 micro g/ml.     

Low molecular weight immunoglobulins in Rana catesbeiana tadpoles.

Rana catesbeiana tadpoles formed high and low m.w. antibodies in response to immunization with a bacteriophage. Although the neutralizing activity associated with the low m.w. immunoglobulins was relatively weak, the existence of antibodies in this class was clearly demonstrated by radioimmunoelectrophoresis. Moreover, two antigenically distinct variants of the low m.w. antibodies were detected. These were serologically indistinguishable from the two types of low m.w. immunoglobulin previously isolated from the serum of adult frogs of this species.     

Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus.

Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26- to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and all but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin G1 subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the N- or M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE.     

Ebola Virus Can Be Effectively Neutralized by Antibody Produced in Natural Human Infection

The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 microgram/ml as the recombinant Fab fragment and to 50% at 0.3 microgram/ml (90% at 2.6 microgram/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.     

抗狂犬病毒中和抗体研究进展

狂犬病是一种人兽共患传染病,人和动物一旦发病后死亡率几乎百分之百,而有效的暴露后预防措施可以将死亡风险降至零。根据WHO推荐的狂犬病暴露后预防方案,一般狂犬病暴露者需要进行疫苗注射,严重者则需在进行疫苗注射的同时注射抗狂犬病毒中和抗体。常用的中和抗体有马抗狂犬病毒免疫球蛋白和人抗狂犬病毒免疫球蛋白,然而两者都存在引起过敏反应或血液疾病的风险。人源抗狂犬病毒中和抗体则因为具有安全性高、成本低、可量产等优点有望代替免疫球蛋白用于暴露后预防。基因工程抗体技术的发展加速了抗体人源化的进程。就抗狂犬病毒中和抗体的发展历程,不同类型中和抗体的优缺点以及中和抗体的未来研究方向作了综述及展望,以期为新一代狂犬疫苗的研发提供参考。