Immunization of Mice with Single PspA Fragments Induces Antibodies Capable of Mediating Complement Deposition on Different Pneumococcal Strains and Cross-Protection

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal α-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.Streptococcus pneumoniae is an important human pathogen, being responsible for millions of deaths worldwide every year. The pneumococcal disease burden could be greatly reduced by the use of the current seven-valent conjugate vaccine, but the high cost and restricted serotype coverage limit its widespread use, especially in developing countries. New-generation vaccines containing up to 13 serotypes are expected to increase vaccine coverage, but the serotype replacement in colonization and disease by nonvaccine serotypes observed with the use of the seven-valent conjugate vaccine (8-9, 11) further emphasizes the importance of the development of alternative vaccines. Protein antigens such as PspA (pneumococcal surface protein A) could be used to induce serotype-independent immunity at a low cost (24).PspA is present in all isolated pneumococcal strains and was shown to be an important virulence factor, interfering with complement deposition (19, 21, 25), killing by apolactoferrin (23), and immune adherence to erythrocytes (12). It has been shown to induce protection in mice in carriage, pneumonia, and fatal systemic models (2, 4, 16). Mature PspA is composed of a mosaic structure with four domains: an α-helical N-terminal domain, a proline-rich region, a choline-binding domain, and a short hydrophobic tail (10, 27-28). PspA shows variability in the surface-exposed N-terminal region, and a classification was proposed based on sequence relatedness of the C-terminal portion of the α-helix, the clade-defining region. It has been classified into three families encompassing six clades. Family 1 (Fam1) is composed of clades 1 and 2, Fam2 includes clades 3, 4, and 5, and Fam3, which is rarely isolated, comprises clade 6 (10). Since the degree of similarity seems to be reflected in cross-reactivity, it has been proposed that a broad-coverage vaccine should contain at least one fragment from each of the two major families.Immunization of healthy adults with a single recombinant fragment of PspA in a phase I clinical trial showed the induction of cross-reactive antibodies (14) that were able to induce passive protection in mice challenged intravenously (3). The natural exposure of adults to several pneumococcal strains might be responsible for the cross-reactivity detected, with the immunization with PspA acting as a booster dose.Because of the diversity observed in PspA, it is extremely important to analyze whether each fragment selected to compose a vaccine is indeed able to induce cross-protection. We have previously addressed the degree of cross-reactivity of antibodies to recombinant fragments including the N-terminal and proline-rich regions of PspA proteins from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis of 35 strains isolated in Brazil. As expected, we have observed higher cross-reactivity within the same clade. Within Fam1, anti-PspA1 serum also showed cross-reaction with PspA2-expressing strains, while anti-PspA2 showed reaction restricted to the same clade. Within Fam2, anti-PspA3 serum also showed reactivity restricted to PspA3-expressing strains, while anti-PspA5 and, more strikingly, anti-PspA4 sera showed a broad recognition capacity, being able to react with strains expressing PspA proteins from clades 1 to 5 (7). The ability of sera to recognize a pneumococcal strain by Western blot analysis does not necessarily correlate with their capacity to induce protection in vivo though. In fact, the levels of antibodies to PspA detected by enzyme-linked immunosorbent assay (ELISA) or through surface staining of the bacteria failed to provide a useful correlate of protection (22). Based on the strong evidence supporting the importance of complement in protection against pneumococcal disease, it was proposed that in vitro complement deposition mediated by antibody may be used as a surrogate assay for the prediction of protection induced by surface antigens of pneumococci (15). This work aimed at further characterizing antibodies against the PspA1, PspA2, PspA3, PspA4, and PspA5 N-terminal fragments in terms of their capacity to mediate C3 deposition on the surface of pneumococci expressing PspA proteins from different clades. Moreover, protection of mice against a lethal intranasal challenge with strains expressing PspA from Fam1 or Fam2 was also analyzed. The basis for the broad reactivity observed in the anti-PspA4 serum by Western blot analysis was also further investigated. Of the five PspA fragments analyzed, PspA4 was the only one containing a nonproline (NonPro) block within the proline-rich region. Not all native PspA proteins include this region: of 24 PspA sequences analyzed by Hollingshead and collaborators (10), 14 were shown to have this NonPro block. We have thus examined whether this region would be responsible for increased cross-reactivity.     

The Oligosaccharide of Gonococcal Lipooligosaccharide Contains Several Epitopes That Are Recognized by Human Antibodies

Recently, we isolated human IgG from normal human sera (NHS) using lipooligosaccharide (LOS) from gonococcal strain JW31R as an affinity ligand. We provided evidence that the oligosaccharide (OS) moiety of LOS was immunogenic in humans and that NHS contains functional antibodies that bind to the branched OS. The present study aimed to identify bactericidal antibodies that bind to partial core OS structures or their adjacent sites expressed in the 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. Using 15253 LOS from serum-resistant gonococcal strain 15253 as an affinity ligand, we isolated IgG2 and found that this preparation contained at least three different species. (i) One IgG2 species recognized a cross-reactive epitope that is expressed on 3,4-branched and 2,3:3,4-dibranched neisserial LOSs. (ii) Another IgG2 species was specific for JW31R LOS from a pyocin-resistant gonococcal strain; this IgG-defined epitope was not shared with the aforementioned branched LOSs. (iii) The third IgG2 species bound to the “Salmonella minnesota” Rb and Re mutant lipopolysaccharides (LPSs); this IgG2 recognizes a KDOα2-4KDO residue at the reducing end of the carbohydrate moiety of each LPS. The IgG2 was also found to be functional and facilitated the killing of strain 15253. The current results show that neisserial LOS contains several epitopes within its OS moiety that are recognized by human antibodies.Lipooligosaccharide (LOS) is a major antigenic and immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae (34). LOS produced by these bacteria consists of an oligosaccharide (OS) moiety and lipid A, and structural variation of the OS leads to LOS heterogeneity (34). As shown in Fig. ​Fig.1A,1A, Neisseria bacteria biosynthesize a core pentasaccharide, N-acetylglucosamine (GlcNAc); two heptose (Hep) and two 2-keto-3-deoxymannooctulosonic acid (KDO) residues are sequentially α-glycosidically linked. GlcNAc is linked to the 2 position of Hep[II], which is linked to Hep[I] via its 3 position. The latter Hep[I] is linked to the 5 position of the terminal KDO of KDO(α2-4)KDO. The structural variation of neisserial OS stems from how glycoses are further biosynthesized from the inner core disaccharide, Hep[II](α1-3)Hep[I], of the pentasaccharide (Fig. ​(Fig.1A).1A). Sequential addition of glycoses to the 4 position of the 3-substituted Hep[I] produces a 3,4-branched OS structure (Fig. ​(Fig.1B),1B), and addition of both to the same Hep[I]-4 position and to the 3 position of the 2-substituted Hep[II] produces a 2,3:3,4-dibranched OS (Fig. ​(Fig.1C)1C) (4, 16, 18, 25, 41, 42). Furthermore, functional groups such as phosphate and acetyl groups are expressed in the GlcNAc(α1-2)Hep[II] portion of the core (Fig. ​(Fig.1A);1A); O-phosphorylation takes place at the 3 and 6 positions of Hep[II] (6, 35), and O-acetylation may occur at the 3 position of GlcNAc (17).Open in a separate windowFIG. 1.Structure of the conserved OS (A) and two major branched OS structures (B and C) biosynthesized by N. meningitidis and N. gonorrhoeae. To distinguish the two Hep residues of the core OS, the Hep linked to KDO and the other Hep were defined as Hep[I] and Hep[II], respectively, as described previously (42).Recently, we reported the isolation of human IgG from normal human sera (NHS) using a 2,3:3,4-dibranched LOS (Fig. ​(Fig.1C)1C) from serum-sensitive gonococcal strain JW31R as an affinity ligand (43). The purified IgG did not bind to any 3,4-branched neisserial LOSs examined and bound only to the ligand JW31R LOS and the truncated form of LOS from serum-resistant strain 15253. Both LOSs contain a disaccharide, Galβ1-4Glc (lactose), on Hep[I], and the structural difference between the two LOSs is the length of the OS linked to Hep[II]; the same disaccharide on Hep[I] is present on Hep[II] of 15253 LOS (41), whereas a tetrasaccharide, an elongated form of lactose, resides on JW31R LOS (5). We have determined that the IgG was specific for the OS moieties of the JW31R and 15253 LOSs but not their lipid A portions.The strain 15253 LOS described above is recognized by murine monoclonal antibody (MAb) 2C7, and an OS epitope recognized by MAb 2C7 is frequently expressed in vitro and in vivo on the surface of the gonococci (10). This MAb requires the strain 15253 OS structure containing lactose on both Hep[I] and Hep[II] for binding, but it does not bind to JW31R LOS (42). Although this binding difference between MAb 2C7 and the above-mentioned anti-JW31R IgG exists, recognition of strain 15253 LOS by the latter IgG showed that a 2C7-like epitope is also recognized by human IgG. This human IgG was also found to be bactericidal against strain JW31R. These previous results provided evidence that the OS moiety of LOS is immunogenic in humans and also showed that NHS contains functional antibodies specific for the branched OS.Other investigators have also reported that NHS contains bactericidal antibodies that recognize neisserial LOS (3, 15) and that the OS moiety is immunogenic in humans. However, very little is known about the OS structures of LOS that are recognized by human antibodies. Humans are the only natural host of the pathogenic neisseriae, and the molecular basis of the recognition specificity of human antibodies to LOS is important for us to understand the immune responses of the host to these bacteria.In the present study, we aimed to isolate and characterize human IgG by using 15253 LOS, which contains the MAb 2C7 epitope (10, 42), as an affinity ligand. We anticipated identifying bactericidal antibodies that bind to partial core OS structures or their adjacent sites expressed in the 3,4-branched and 2,3:3,4-dibranched LOSs. We found that IgG2 isolated by affinity chromatography from NHS bound to the aforementioned branched LOSs and also to Salmonella mutant Rb and Re lipopolysaccharides (LPSs). The IgG2 was also found to be functional and able to facilitate the killing of serum-resistant strain 15253, which expresses the ligand LOS. The current results demonstrated that neisserial OS contains several epitopes that are recognized by human antibodies.     

Molecular Mapping of the Major Epitopes of BP180 Recognized by Herpes Gestationis Autoantibodies

Herpes gestationis (HG) is an autoantibody-mediated subepidermal bullous dermatosis associated with pregnancy. The primary target of HG autoantibodies is BP180, a 180-kDa hemidesmosomal glycoprotein. We previously showed that autoantibodies and autoimmune T lymphocytes from HG patients recognize the MCW-1 antigenic site (AA 507-520), which is located in the membrane-proximal noncollagenous domain (NC16A) of BP180. Here, we analyzed the sera of 37 HG patients to further define the sites on BP180 that are targeted by autoantibodies. All of the HG sera, but none of the control sera, were immunoreactive with sec180e, a 120-kDa recombinant protein encompassing the entire BP180 extracellular domain. HG sera depleted of reactivity to NC16A no longer reacted with sec180e, indicating that the major HG-associated epitopes on BP180 are restricted to the NC16A domain. The vast majority of the HG sera (34 of 37) reacted with a 7 amino acid peptide corresponding to the N-terminal half of MCW-1 (MCW-1A). Eleven HG sera (including the 3 that failed to react with MCW-1A) recognized one or more of three antigenic sites located within a 15 amino acid stretch immediately downstream of MCW-1A. In summary, we have identified four major HG-associated epitopes clustered within a 22 amino acid region of the BP180 ectodomain. These findings support the hypothesis that an autoimmune response to the BP180 NC16A domain is a crucial step in the pathogenesis of HG.     

Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.Chronic obstructive pulmonary disease (COPD) is currently the fourth leading cause of death in the United States and Europe. The morbidity and mortality associated with exacerbations of COPD together with the associated health care-related costs of $32 billion reported in the United States in 2002 demonstrate that there is a need for a better understanding of the etiology and pathogenesis of these events (6, 36, 43). Bacteria have been isolated in large numbers from the lower respiratory tract during exacerbations, and up to 50% of COPD exacerbations are due to a bacterial agent, primarily nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (31). M. catarrhalis accounts for up to 10% of these infections in adults, and this strictly human pathogen is currently among the three most prominent causes of otitis media in children (13, 15, 28). Some of the primary reasons why M. catarrhalis continues to cause disease can be attributed to the fact that greater than 90% of the clinical isolates express beta-lactamase, there is a high frequency of recurrent disease observed for children that have recovered from infection, and there is a lack of a vaccine (13, 27, 42, 47). Thus, the identification of potential drug targets and vaccine antigens is clearly a priority.One of the major problems hindering the identification of putative vaccine antigens involves the fact that M. catarrhalis is a strictly human pathogen, and the human immune response to this bacterium remains poorly understood. Previous studies investigated the production of new antibodies against different bacterial pathogens in patients suffering from COPD and lower respiratory tract infections. These patients exhibited increased antibody responses to bacterial outer membrane proteins (OMP) and surface-exposed lipooligosaccharides (LOS) after clearing the bacterial strain following an exacerbation (4, 29, 38, 51). Human serum immunoglobulin G (IgG), sputum IgA, or salivary IgA antibodies against M. catarrhalis surface proteins such as UspA1, UspA2, Hag, TbpB, CopB, OMP CD, OMP E, and OMP G1b have been developed (1, 3, 25, 28, 29). In addition, new antibodies to LOS have also been detected in some COPD patients (3, 28, 29).The LOS structure of M. catarrhalis has been well studied. There are three major serotypes, serotypes A, B, and C, previously defined by polyclonal antisera and structural analyses (8-10, 23, 46). The LOS glycosyltransferase (lgt) genes that encode the enzymes required to transfer carbohydrate residues to the M. catarrhalis LOS molecule were previously identified and characterized (11, 33, 41, 49). In addition, the lgt present in a given strain of M. catarrhalis can be used to identify the specific LOS serotype of that isolate using our previously described multiplex PCR method (12). Serotypes A and B are the predominant glycoforms expressed by most clinical isolates analyzed to date (12, 46). In recently reported animal studies, other researchers suggested that detoxified M. catarrhalis LOS has potential as a vaccine antigen in a mouse pulmonary clearance model (16, 19, 52, 53). While these data are both valuable and interesting, it is sometimes difficult to link observations of animals to those of humans (34).Currently, we have constructed a panel of defined LOS mutants that are defective in the expression of each specific glycosyltransferase gene identified in all three major M. catarrhalis LOS serotypes. These truncations are a comprehensive set of mutations with various oligosaccharide (OS) chain lengths representing most, if not all, possible LOS epitopes (11, 33, 41, 49). Purified LOS samples from these mutants were used in enzyme-linked immunosorbent assays (ELISAs) to assess the development of new human antibodies to all LOS epitopes developed following an M. catarrhalis infection. ELISAs were previously employed to determine levels of antibody to Neisseria meningitidis lipopolysaccharide (LPS), including inner core mutations, in patients following disease (35). Thus, this LOS-based ELISA system with the full set of mutations has the potential to determine the regions of the LOS molecule that elicit new antibodies in both children and adults following infection, providing a unique opportunity to analyze the human immune response to these major surface glycolipids in the native host.     

用抗-(抗HBc)抗体在同系小鼠中诱导抗-抗Id抗体

用单克隆抗-(抗HBc)抗体(抗-Id)免疫BALB/c鼠,诱生出具有与抗-HBc相同反应性的抗-抗Id抗体(Ab_3)。这种抗体在ELISA试验中可与HBc-Ag特异性反应,并能抑制单克隆抗-HBc与HBcAg的结合。表明我们建立的抗-H-Bc的抗Id能模拟HBcAg刺激小鼠产生免疫反应。     

三株抗rhIL－6单克隆抗体识别表位的鉴定及IL－6的双单克隆抗体夹心RIA检测方法的建立

采用放射免疫标记抗体竞争结合法，对３株抗ｒｈＩＬ－６单克隆抗体识别的表位进行了鉴定。按识别表位的不同，将３株单克隆抗体分为两组：一组ＳＩ８，ＳＩ９识别相同或相近的表位，另一组ＳＩ７。在表位鉴定的基础上，选择识别不同表位的单克隆抗体，由此获得最佳组合，灵敏度为８０ｐｇ／ｍｌ。通过对肾脏病人血清ＩＬ－６水平检测，所得结果与以ＩＬ－６依赖株Ｂ９生物学检测结果一致，且重复性好，特异性强。     

Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice

Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.Pneumococcal diseases kill more than 1 million children worldwide every year. The situation is worse in developing countries, where 90% of deaths occur. In Latin America, there are at least 1.6 million cases of pneumococcal disease every year, killing 18,000 children (53). While appropriate treatment, including the use of antibiotics and good nutrition, lowers the incidence of pneumococcal diseases, vaccines are the most efficacious way of preventing them. The existing pneumococcal conjugate vaccine dramatically reduces diseases, disabilities, and deaths, but elevated cost and protection restricted to included serotypes have prevented its implementation in large-scale immunization programs in developing countries. For these reasons, there is considerable interest in using conserved pneumococcal protein antigens as vaccines to provide cost-effective broad protection in all age groups. A number of leading candidates have been shown to elicit protection in mice (10, 51); among these antigens, two of the most promising candidates are pneumococcal surface protein A (PspA) and PspC.An additional concern in the development of cost-effective vaccines against pneumococcal disease is the route of immunization. Human vaccines are traditionally administered intramuscularly by needle inoculation, which brings the risk of transmitting blood-borne pathogens such as human immunodeficiency virus and hepatitis viruses (20). Furthermore, the cost of equipment and well-trained personnel for delivering vaccines by parenteral routes is several times higher than the cost of the vaccines themselves. This aspect is extremely important for vaccine implementation in large-scale immunization programs for developing countries. Mucosal delivery of pediatric vaccines has become an explicit goal of the WHO (20). Immunization via mucosal surfaces would greatly increase the ease of vaccination and would be more readily acceptable than parenteral immunization in many populations. Therefore, the move from injection to mucosal application would be very positive from economical, logistical, and safety standpoints. Mucosal immune responses are also important for the prevention of many infectious diseases because they represent the first barrier from the hosts that pathogens must evade.Research into the host immune response to pneumococcal diseases has focused primarily on the role of innate and adaptative humoral immune responses. However, in the last few years, attention has been drawn to cellular immune responses against Streptococcus pneumoniae, with interesting results. The majority of these studies analyzed cellular aspects of innate immunity and proposed that lymphocytes, neutrophils, and macrophages orchestrate effective immune responses without the presence of specific antibodies. In this context, proinflammatory cytokines promote an adequate milieu for pneumococcal clearance (22, 24, 25, 31, 34, 50, 54). A Th1-biased immune response has also been shown to be engaged in the resolution of pneumococcal infection in humans (21). Nevertheless, inflammatory cell influx into the lung and mucosal responses must be regulated to avoid exacerbated tissue injury. This is evidenced in recent studies of the role of γδ T cells and/or anti-inflammatory cytokines, such as interleukin-10 (IL-10), in pneumococcal infection (26, 42, 55).Protective immune responses against invasive pneumococcal disease and colonization were shown using pneumococcal whole-cell vaccines (28, 46) or recombinant proteins as mucosal vaccines (2, 6, 7, 9, 40). In recent approaches, lactic acid bacteria (3, 11, 18, 37), which are able to activate and modulate the innate immune system (35, 42), were used for pneumococcal antigen presentation, with promising results.Very few works compared pulmonary and systemic immune responses induced by pneumococcal antigens using parenteral and mucosal immunizations (13). The present study aims at investigating local and systemic cellular and humoral immune responses required for protection against invasive intranasal (i.n.) challenge with S. pneumoniae strain ATCC 6303 using PspA and PspC antigens administered by both routes, without the use of adjuvants, or presented by Lactobacillus casei through the nasal route.     

Mapping Epitopes of the Plasmodium vivax Duffy Binding Protein with Naturally Acquired Inhibitory Antibodies

Plasmodium vivax Duffy binding protein (DBP) is a merozoite microneme ligand vital for blood-stage infection, which makes it an important candidate vaccine for antibody-mediated immunity against vivax malaria. A differential screen with a linear peptide array compared the reactivities of noninhibitory and inhibitory high-titer human immune sera to identify target epitopes associated with protective immunity. Naturally acquired anti-DBP-specific serologic responses observed in the residents of a region of Papua New Guinea where P. vivax is highly endemic exhibited significant changes in DBP-specific titers over time. The anti-DBP functional inhibition for each serum ranged from complete inhibition to no inhibition even for high-titer responders to the DBP, indicating that epitope specificity is important. Inhibitory immune human antibodies identified specific B-cell linear epitopes on the DBP (SalI) ligand domain that showed significant correlations with inhibitory responses. Affinity-purified naturally acquired antibodies on these epitopes inhibited the DBP erythrocyte binding function greatly, confirming the protective value of specific epitopes. These results represent an important advance in our understanding of part of blood-stage immunity to P. vivax and some of the specific targets for vaccine-elicited antibody protection.Plasmodium vivax is the major cause of malaria in most regions where this disease is endemic outside Africa, and it causes substantial morbidity worldwide (17). Plasmodium microneme proteins, such as Duffy binding protein (DBP), have important roles in the merozoite invasion of reticulocytes during asexual blood-stage infection (1, 5). DBP is a member of the Duffy binding-like erythrocyte binding protein (DBL-EBP) family expressed in the micronemes and on the surface of P. vivax merozoites and is associated with the decisive junction formation step during the invasion process (1). It is this critical interaction of DBP with its cognate receptor that makes DBP an important antimalaria vaccine candidate. The critical erythrocyte binding motif of DBP is in a 330-amino-acid cysteine-rich domain referred to as DBP region II (DBPII) or the DBL domain, which is the minimal domain responsible for binding to Duffy-positive human erythrocytes (2, 6). The central portion of the DBP domain is hypervariable compared to other DBP regions, and polymorphisms occur frequently at certain residues in a pattern consistent with selection pressure on DBP, suggesting that allelic variation functions as a mechanism for immune evasion (9, 15, 24).Naturally acquired antibodies to DBP are prevalent in residents of areas where malaria is highly endemic, but individuals show significant quantitative and qualitative differences in their anti-DBP serological responses (10, 12, 27, 28). Generally, serological responses to DBP and the inhibition of DBP-erythrocyte binding activity increase with a person''s age, suggesting that there is a boosting effect due to repeated exposure through recurrent infection (13, 16, 18). The initial antibody response to a single P. vivax infection is a response to conformational epitopes and is not broadly protective, while an immunity that transcends strain specificity develops only after repeated exposure (10, 28). Repeated exposure of residents of the areas of Papua New Guinea (PNG) where P. vivax is endemic was observed to correlate with development of antibodies that are reactive to linear epitopes in the critical binding region of DBP. In this study, we compared the reactivity of inhibitory human immune sera to the reactivity of noninhibitory immune sera to identify linear epitopes in DBPII that may serve as a target for vaccine-induced protective humoral immunity.     

Anti-Sperm Antibodies in Female Mice: Responses Following Intrauterine Immunization

PROBLEM: The goal of this study was to measure anti-sperm antibodies in serum following intraluminal uterine immunization and to compare this with the formation of antibodies following intraperitoneal (i.p.) or anal immunization. METHOD: Mice were immunized with human sperm, and antibody was measured using an indirect immunobead assay. RESULTS: High levels of anti-sperm antibodies could be measured following intrauterine immunization and i.p. immunization. Anal immunization resulted in lower levels of antibody formation. Antibody formation after intrauterine immunization was less consistent following transcervical insertion than following needle insertion of sperm. CONCLUSION: The data suggest that intrauterine immunization can result in anti-sperm antibody formation under some conditions.     

Passive Immunization with Antibodies against Three Distinct Epitopes on Plasmodium yoelii Merozoite Surface Protein 1 Suppresses Parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an ~42-kDa fragment (MSP-1₄₂) that is derived from the C terminus of MSP-1. MSP-1₄₂ is further cleaved to an N-terminal ~33-kDa polypeptide (MSP-1₃₃) and a C-terminal ~19-kDa polypeptide (MSP-1₁₉) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-1₄₂ but not with either of the constituents MSP-1₃₃ and MSP-1₁₉, B4 recognized an epitope within the N terminus of MSP-1₃₃, and B6, B10, F5, and G3 bound to MSP-1₁₉. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.     

不同佐剂的复合黏膜疫苗鼻内免疫小鼠抗弓形虫感染的保护作用

目的比较IFN-γ和蜂胶作为佐剂鼻内免疫小鼠抗弓形虫攻击的能力,探索两种佐剂联合应用的效果。方法将5-6周龄雌性BALB／c小鼠60只随机分为4个组,每组15只,分别用20雌可溶性速殖子抗原（STAg）、20μg STAg＋40μg蜂胶、20μgSTAg＋1000U IFN-γ或20μgSTAg＋40μg蜂胶＋1000UIFN-γ鼻内免疫小鼠2次．间隔14d。末次免疫后第10天,用RH株弓形虫速殖子4×10^4个／只灌胃攻击,逐日观察小鼠存活情况。攻击后第43天处死全部存活小鼠,计算胸腺、脾指数,计数脑、肝组织速殖子。结果STAg＋IFN-γ组、STAg＋蜂胶＋IFN-γ组胸腺、脾指数显著高于STAg组（P〈0．05）;组织内速殖子虫荷显著降低（P〈0．01）,STAg＋IFN-γ组小鼠脑、肝组织减虫率分别为57．00％和79．06％;STAg＋蜂胶＋IFN-γ组小鼠脑、肝减虫率分别为68．30％和79．06％。STAg＋蜂胶组小鼠胸腺、脾指数有升高趋势,组织内速殖子虫荷降低,但与STAg组比较差异无统计学意义。结论在抗弓形虫感染中,IFN-γ、蜂胶＋IFN-γ的佐剂效果优于蜂胶,IFN-γ、蜂胶＋IFN-γ辅助STAg显著提高小鼠胸腺、脾比重,增强机体免疫能力,有效抵抗弓形虫的感染攻击,脑、肝组织速殖子虫荷显著减少。     

工程化人源性抗-HBs Fab片段抗原结合位点的研究

取自己制备的纯化人源性抗 HBs Fab 片段。用表达的PreS, PreS1, PreS2 蛋白及合成的PreS1 (21 47aa)27 肽检测其抗原结合位点。结果显示该Fab 片段在非还原状态下分子量约为54kD, 在还原状态下分解为大小两个片段, 大片段为Fab的Fd 链, 分子量约为28kD; 小片段为Fab 的κ链, 分子量约为26kD。该Fab 片段与PreS及PreS1 蛋白呈现结合反应, 说明其抗原结合位点位于HBsAg 的PreS1 区     

Antibodies against a Class II HLA–Peptide Complex Raised by Active Immunization of Mice with Antigen Mimicking Peptides

Multiple sclerosis (MS) is an autoimmune disease linked to the human leucocyte antigen (HLA) class II genes DRB1*1501, DRB5*0101 and DQB1*0602. T cells reactive towards the DRB1*1501 in complex with various peptides derived from myelin basic protein (MBP), which is the major component of myelin, have been found in the peripheral blood of MS patients. These autoreactive T cells are believed to play a role in the pathogenesis of MS. In this article, antibodies against the HLA complex DR2b (DRA1*0101/DRB1*1501) in complex with the MBP-derived peptide MBP_85-99 have been generated by immunization of NMRI mice with three different antigen mimicking peptides displayed on M13 bacteriophages. The peptides mimick the epitope of a monoclonal antibody specific for the DR2b–MBP_85-99 complex. The mice developed IgG antibodies not only against the peptides injected, but they also developed antibodies against the DR2b complex and specific antibodies against the DR2b–MBP_85-99 complex. These data open up the possibility of designing antigen mimicking peptides for vaccination against MS.     

活性染色质诱导抗核抗体生成及肾损伤

系统性红斑狼疮 (SLE )的病因和诱导抗核抗体 (ANA )生成的激发原迄今不明。本实验试图用ConA活化淋巴细胞的染色质免疫同系BALB/c小鼠 ,寻找诱导ANA生成的真正免疫原 ,阐明SLE发生的激发原是自身活化细胞的核成分 ,而且证明它所诱导的ANA具有致病性。从ConA活化的脾细胞中提取染色质 ,然后免疫同系BALB/c小鼠 ,用ELISA方法测定IgG类抗双链DNA (dsDNA )、抗组蛋白抗体 ,用免疫荧光法检测抗核抗体核型和免疫复合物沉积 ,用免疫印迹法测定抗核抗体谱 ,在光镜下检测肾损伤及电镜下检测肾小球沉积物 ,用考马斯亮蓝法检测尿蛋白含量。结果显示 ,活性染色质能诱导IgG类抗dsDNA、抗组蛋白等多种抗核抗体生成 ,且肾小球有显著免疫复合物沉积和蛋白尿形成。该实验表明 ,活化淋巴细胞的染色质是诱导SLE发生的真正自身免疫原。     

Pneumolysin, PspA, and PspC contribute to pneumococcal evasion of early innate immune responses during bacteremia in mice

The pneumococcal virulence factors include capsule, PspA, PspC, and Ply. Cytometric analysis demonstrated that the greatest levels of C3 deposition were on a Deltaply PspA(-) PspC(-) mutant. Also, Ply, PspA, and PspC expression resulted in C3 degradation in vitro and in vivo. Finally, blood clearance assays demonstrated that there was enhanced clearance of Deltaply PspA(-) PspC(-) pneumococci compared to the clearance of nonencapsulated pneumococci.     

Molecular Analyses of Anti-DNA Antibodies Induced by Polyomavirus BK in BALB/c Mice

In the present experiments, two groups of BALB/c mice (five individuals in each group) were hyperimmunized through four consecutive immunizations with either BK virus (Group 1) or BK dsDNA complexed with methylated BSA (Group 2). All immune sera taken after the fourth immunization from both groups reacted strongly with polyomavirus BK dsDNA as well as with calf thymus dsDNA, and all sera contained antibodies that bound in the Crithidia luciliae assay. This indicates that polyomavirus BK was able to induce antibodies with binding characteristics similar to SLE anti-DNA antibodies. To further characterize these induced anti-DNA responses, 10 monoclonal anti-DNA antibodies (four from Group 1, and six from Group 2) were generated and selected for reactivity with Sl-nuclease digested CT dsDNA. Their specificity for BK and CT dsDNA molecules, as well as their light and heavy chain variable region cDNA nucleotide sequences were analysed to compare them with known SLE derived anti-DNA antibodies. All of the 10 antibodies bound strongly to BK dsDNA, while seven also bound to CT dsDNA in competitive ELISA experiments. V-region analysis revealed that the induced antibodies resembled anti-DNA antibodies characteristic for murine SLE, and all but one contained arginine in the V_H CDR3 region. The arginines present in the monoclonal antibodies originated either from an RF shift from RF1RF3 of the D-genes or from N-sequence additions. Taken together, the data demonstrate that anti-DNA antibodies in response to hyperimmun-ization with polyomavirus BK have the same characteristics as of those occuring spontaneously in SLE. As virus infection/replication in vivo implies expression of immunogenic (non-self) DNA-binding proteins that may render DNA immunogenic, the present results may therefore suggest one physiological mechanism for production of SLE-related anti-DNA antibodies.     

Immunization with Recombinant Pneumolysin Induces the Production of Antibodies and Protects Mice in a Model of Systemic Infection Caused by Streptococcus pneumoniae

Bulletin of Experimental Biology and Medicine - Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized...     

Innate Natural Antibodies. Primary Roles Indicated by Specific Epitopes

Two members of a unique class of natural antibodies have been identified in all of a large cohort of sera from clinically normal humans of broad age distribution. By means of a series of 10–12 mer peptides the epitope for each of those antibodies was characterized with regard to amino acid identity and conformation. Similar epitope specificity was revealed for the IgM isotopes of cord blood and early post natal sera and for IgM and IgG of adult sera, suggesting that the class of natural antibodies represented by the two identified in this study includes those genomically coded for at their effector level of maturation in the B cells of the neonate. Assay of series of specimens from each of four clinically normal adults revealed that those two natural antibodies are present at relatively constant titer, unique to each individual, over four to five and a half year periods. Those observations imply that the primary function of that class of natural antibodies may be related to maintenance of homeostasis and the molecular identity of each of the two epitopes suggests a role, for each, as monitor or control in intracellular traffic. The previous identification of those epitopes in a conserved protein of HIV also provides support for the proposition that a secondary function of natural antibodies, arising from fortuitous coincidence of the identity of the epitopes, may be that of early defense against infectious invaders.     

PspA and PspC minimize immune adherence and transfer of pneumococci from erythrocytes to macrophages through their effects on complement activation

Pneumococcal surface protein A (PspA) and PspC are important virulence factors. Their absence has been shown to allow improved clearance of pneumococci from the blood of mice and to decrease pneumococcal virulence. In the presence of antibody and complement, pneumococci attach to erythrocytes in a process called immune adherence (IA), which facilitates their delivery to, and eventual phagocytosis by, macrophages. It is not known, however, if PspA and PspC affect IA. Using PspA and/or PspC isogenic mutants and complement-deficient mouse sera, we demonstrated that absence of PspA allows greater deposition of C1q and thus increased classical-pathway-mediated C3 deposition. In the absence of both PspA and PspC, there is also a major increase in C1q-independent C3 deposition through the alternative pathway. The latter was observed even though absence of PspC alone did not have a major effect on alternative-pathway-dependent complement deposition. The enhanced complement C3 deposition realized in the absence of PspA alone and in the absence of PspA and PspC resulted in both greatly increased IA to human erythrocytes and improved transfer of pneumococci from erythrocytes to phagocytes. These data provide new insight into how PspA and PspC act in synergy to protect pneumococci from complement-dependent clearance during invasive infection.