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Antibody autoreactivity against bactericidal/permeability-increasing protein (BPI) is strongly associated with Pseudomonas aeruginosa infection in cystic fibrosis (CF), non-CF bronchiectasis (BE), and chronic obstructive pulmonary disease (COPD). We examined the pathogen-specific nature of this autoreactivity by examining antibodies to BPI in bacteremia patients. Antibodies to BPI and bacterial antigens were measured in sera by ELISA from five patient cohorts (n = 214). Antibody avidity was investigated. Bacteremic patient sera (n = 32) exhibited IgG antibody autoreactivity against BPI in 64.7% and 46.7% of patients with positive blood cultures for P. aeruginosa and Escherichia coli, respectively. Autoantibody titers correlated with IgG responses to bacterial extracts and lipopolysaccharide (LPS). A prospective cohort of bacteremic patient sera exhibited anti-BPI IgG responses in 23/154 (14.9%) patients with autoreactivity present at the time of positive blood cultures in patients with Gram-negative and Gram-positive bacteria, including 8/60 (13.3%) patients with Staphylococcus aureus. Chronic tissue infection with S. aureus was associated with BPI antibody autoreactivity in 2/15 patients (13.3%). Previously, we demonstrated that BPI autoreactivity in CF patient sera exhibits high avidity. Here, a similar pattern was seen in BE patient sera. In contrast, sera from patients with bacteremia exhibited low avidity. These data indicate that low-avidity IgG responses to BPI can arise acutely in response to bacteremia and that this association is not limited to P. aeruginosa. This is to be contrasted with chronic respiratory infection with P. aeruginosa, suggesting that either the chronicity or the site of infection selects for the generation of high-avidity responses, with biologic consequences for airway immunity.

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene–oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene–oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.

CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response to Borrelia burgdorferi appears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality of B. burgdorferi infection-induced CD4 T FH cells. We report that CD4 T cells were effectively primed and T FH cells induced after B. burgdorferi infection. These CD4 T cells contributed to the control of B. burgdorferi burden and supported the induction of B. burgdorferi -specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependent B. burgdorferi protein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells. In vitro T-B cocultures demonstrated that T cells isolated from B. burgdorferi -infected but not B. burgdorferi -immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responses in vivo . The data further suggest that B. burgdorferi infection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy.

Clinical immunity to pregnancy associated-malaria (PAM) in multigravida women has been attributed to antibodies that recognize VAR2CSA on the infected erythrocyte (IE) surface. The size and complexity of VAR2CSA have focused efforts on selecting one or more of its six Duffy binding-like (DBL) domains for vaccine development. Presently, however, there is no consensus as to which DBL domain(s) would be most effective in eliciting immunity. This is because antibodies to a number of the DBL domains have been found to block the adhesion of VAR2CSA-expressing erythrocytes to chondroitin sulfate A (CSA)—a major criterion for evaluating vaccine candidacy. Opsonization of IEs by cytophilic antibodies that recognize VAR2CSA represents an important yet understudied effector mechanism in acquired immunity to PAM. To date, no studies have sought to determine the targets of those antibodies. In this study, we found that IgGs from multigravida Malian women showed (i) higher reactivity to recombinant DBL domains by enzyme-linked immunosorbent assay (ELISA), (ii) more binding to VAR2CSA-expressing IEs, and (iii) greater opsonization of these IEs by human monocytic cells than IgGs from malaria-exposed Malian men and malaria-naive American adults. Preincubation of IgGs from multigravida women with recombinant DBL2χ, DBL3χ, or DBL5ε domains significantly diminished opsonization of VAR2CSA-expressing IEs by human monocytes. These data identify the DBL2χ, DBL3χ, and DBL5ε domains as the primary targets of opsonizing IgGs for the first time. Our study introduces a new approach to determining the antigenic targets of opsonizing IgGs in phagocytosis assays.

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Brucellaspp. are intracellular bacteria that cause the most frequent zoonosis in the world. Although recent work has advanced the field ofBrucellavaccine development, there remains no safe human vaccine. In order to produce a safe and effective human vaccine, the immune response toBrucellaspp. requires greater understanding. Induction ofBrucella-specific CD8+T cells is considered an important aspect of the host response; however, the CD8+T-cell response is not clearly defined. Discovering the epitope containing antigens recognized byBrucella-specific CD8+T cells and correlating them with microarray data will aid in determining proteins critical for vaccine development that cover a kinetic continuum during infection. Developing tools to take advantage of the BALB/c mouse model ofBrucella melitensisinfection will help to clarify the correlates of immunity and improve the efficacy of this model. Two H-2dCD8+T-cell epitopes have been characterized, and a group of immunogenic proteins have provoked gamma interferon production by CD8+T cells. RYCINSASL and NGSSSMATV induced cognate CD8+T cells after peptide immunization that showed specific killing in vivo. Importantly, we found by microarray analysis that the genes encoding these epitopes are differentially expressed following macrophage infection, further emphasizing that these discordant genes may play an important role in the pathogenesis ofB. melitensisinfection.

The dispersed gene family 1 ( DGF-1 ) is the fifth largest gene family in the T rypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N - glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.

Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC 50 ) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC 50 of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.

Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages. Fil: Medina, Marcela Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Vintiñi, Elisa Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Raya, Raul Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquímica, Química y Farmacia. Instituto de Bioquímica Clinica Aplicada. Cátedra de Bioquímica Clinica I; Argentina

The generation of protective humoral immune responses against the receptor-binding domain (domain IV) of protective antigen [PA(dIV)] ofBacillus anthracisrepresents a plausible approach against anthrax toxin. In the current study, we have developed a naked DNA vaccine encoding calreticulin (CRT) linked to PA(dIV) ofBacillus anthracis[CRT/PA(dIV)]. We transfected a human embryonic kidney cell line (HEK 293) with CRT/PA(dIV) DNA and performed Western blotting and confocal microscopy analysis. We found that linkage of CRT to PA(dIV) targets PA(dIV) to the endoplasmic reticulum, resulting in secretion of the chimeric CRT/PA(dIV) protein. We then evaluated the ability of CRT/PA(dIV) DNA to generate PA(dIV)-specific antibody responses and protective immunity against lethal anthrax toxin (PA plus lethal factor) challenge. We found that mice immunized with CRT/PA(dIV) DNA were capable of rapidly inducing significantly higher PA(dIV)-specific antibody responses than mice immunized with PA(dIV) DNA alone. Furthermore, we observed that this enhanced antibody response generated by CRT/PA(dIV) DNA was CD4 dependent, since CD4 knockout mice demonstrated a significant reduction in antibody responses. In addition, analysis of the titers and avidity maturation of the induced PA-specific antibodies revealed that vaccination with CRT/PA(dIV) DNA vaccine accelerated the avidity maturation of antibodies to PA(dIV) compared to vaccination with PA(dIV) DNA. Importantly, the enhanced antibody responses correlated to protective immunity against lethal anthrax toxin challenge. Thus, DNA vaccines encoding CRT linked to PA(dIV) may dramatically enhance PA-specific protective antibody responses. Our results have significant clinical applications for biodefense against anthrax toxin.

Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such asBacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.

Antibodies of the immunoglobulin A (IgA) class react with capsular polysaccharides ofStreptococcus pneumoniaeand support complement-dependent opsonophagocytosis (OPC) of the organism by phagocytes. We characterized the biologic impact of the molecular forms of human monoclonal capsule-specific IgA (monomeric IgA [mIgA], polymeric IgA [pIgA], and secretory IgA [SIgA]) on OPC and susceptibility to cleavage by IgA1 protease. The efficiency of SIgA in support of OPC ofS. pneumoniaewas comparable to that of pIgA, and both forms exceeded that of mIgA by a fivefold margin. This structure-function relationship was associated with three factors. First, the avidities, or functional affinities, of both pIgA and SIgA for pneumococcal capsules exceeded those of mIgA. Second, both pIgA and SIgA required less complement to achieve similar levels of bacterial OPC than did mIgA, indicating that secretory component does not hinder the effect of complement. Third, both pIgA and SIgA mediated agglutination of the organism, whereas mIgA did not. All three forms of capsule-specific IgA showed comparable susceptibilities to cleavage and functional inhibition by bacterial IgA1 protease, demonstrating that secretory component does not prevent the proteolytic degradation of IgA1 by IgA1 protease. IgA1 cleavage results in formation of identical Fab fragments for each of the molecular forms, thereby abolishing the contribution of multivalence of pIgA and SIgA. In summary, the polymeric forms of IgA (both pIgA and SIgA) provide a substantial advantage in binding, agglutination, and OPC of the organism.

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders—J1, J3, and J7—were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.

Streptococcus pneumoniaeis a human bacterial pathogen responsible for serious infections including pneumonia. The currently licensed polysaccharide vaccine provides 60 to 80% protection in young adults, but in the elderly the vaccine efficacy is drastically reduced despite normal antibody levels. We hypothesized that the reduced vaccine efficacy in the elderly results from altered variable gene family usage. We have analyzed the light chain gene usage in 20 young (20 to 30 years of age) and 20 elderly (65 to 86 years of age) adults in response to pneumococcal polysaccharide 4 (PPS4) and PPS14. We generated a variable light chain library using B cells specific for PPS4 and PPS14 from each vaccinated individual. We determined complete sequences and somatic mutation frequencies in all isolated variable light chain fragments. Six gene families, κ1, κ2, κ3, κ4, λ1, and λ3, were identified in response to PPS4 and PPS14 in both age groups. Comparison of young and elderly adults demonstrated significant differences in κ4, λ1, and λ3 gene usage in response to PPS4 and PPS14. With aging, there was a significant increase in κ4 gene usage and a significant decrease in λ1 and λ3 gene usage in response to both PPS4 and PPS14. Although both Vκ1 and Vλ3 gene products demonstrated extensive mutations, there was no age-related difference in mutational frequency per gene family. These findings suggest an age-related change in light chain gene usage in response to PPS4 and PPS14.

Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 μg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

The licensure of new pneumococcal conjugate vaccines (PCVs) relies on immunogenicity data. When defining correlates of protection, vaccine efficacy data must be included. In the FinOM Vaccine Efficacy Trial, the PncOMPC vaccine showed an efficacy profile similar to that of the licensed PncCRM vaccine despite different antibody responses after primary and booster vaccinations. We determined antibody kinetics and avidities in a subgroup of infants participating in the FinOM trial. A total of 166 infants in three vaccine groups were immunized at 2, 4, 6, and 12 months of age with 7-valent PCV, PncCRM or PncOMPC, or hepatitis B vaccine. Concentrations of serum immunoglobulin G (IgG) against pneumococcal capsular polysaccharides were determined at 2, 4, 6, 7, 12, 13, and 24 months of age, and the avidity index (AI) to serotypes 6B, 19F, and 23F were determined at 7, 12, 13, and 24 months of age by enzyme immunoassay. Both PCVs were highly immunogenic, but they demonstrated different kinetics of antibody response; the concentration of IgG against serotypes 6B, 19F, and 23F declined faster after the third and fourth doses of vaccine in the PncCRM group than in the PncOMPC group. For both PCVs, the mean AI of anti-6B and -23F, but not of anti-19F, increased during the follow-up, which is in line with serotype-specific protection in the FinOM trial. Our data suggest that the kinetics and avidities of antibodies should be considered, in addition to antibody responses, when defining correlates of protection.

Antibody (Ab) responses to polysaccharides (PS), such asNeisseria meningitidisgroup C PS (MCPS), are characterized as being thymus independent and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled with tetanus toxoid or the diphtheria toxin derivative CRM197, elicit thymus-dependent responses. The present study developed a surface plasmon resonance approach to evaluate Ab responses to MCPS conjugate vaccines, including either O-acetylated (OAc+) or de-O-acetylated (OAc−) forms of the PS. The results were generally consistent with those obtained by enzyme-linked immunosorbent assay and showed that sera from mice immunized with conjugate vaccines contain Abs that bind more effectively to OAc+and OAc−MCPS than sera from mice immunized with fixed bacteria. The data suggest a critical shared or overlapping epitope recognized by all the conjugate vaccine immune sera and strategies for assessing polyclonal Ab avidity.

In order to plan for the wide-scale introduction of meningococcal C conjugate (MCC) vaccine for United Kingdom children up to 18 years old, phase II trials were undertaken to investigate whether there was any interaction between MCC vaccines conjugated to tetanus toxoid (TT) or a derivative of diphtheria toxin (CRM 197 ) and diphtheria-tetanus vaccines given for boosting at school entry or leaving. Children ( n = 1,766) received a diphtheria-tetanus booster either 1 month before, 1 month after, or concurrently with one of three MCC vaccines conjugated to CRM 197 or TT. All of the MCC vaccines induced high antibody responses to the serogroup C polysaccharide that were indicative of protection. The immune response to the MCC-TT vaccine was reduced as a result of prior immunization with a tetanus-containing vaccine, but antibody levels were still well above the lower threshold for protection. Prior or simultaneous administration of a diphtheria-containing vaccine did not affect the response to MCC-CRM 197 vaccines. The immune responses to the carrier proteins were similar to those induced by a comparable dose of diphtheria or tetanus vaccine. The results also demonstrate that, for these conjugate vaccines in these age groups, both standard enzyme-linked immunosorbent assays and those that measure high-avidity antibodies to meningococcal C polysaccharide correlated equally well with assays that measure serum bactericidal antibodies, the established serological correlate of protection for MCC vaccines.

The avidity maturation and immunoglobulin G (IgG) isotype distribution of antibodies after vaccination with a meningococcal B outer membrane vesicle (OMV) vaccine were evaluated as indicators of protective immunity. Pre- and postvaccination sera from 134 healthy toddlers (ages, 2 to 3 years) immunized with a monovalent meningococcal B OMV (serosubtype P1.7-2,4) vaccine adsorbed with AlPO4or Al(OH)3were analyzed by enzyme-linked immunosorbent assay (ELISA) methods. The children were vaccinated three times with intervals of 3 to 6 weeks between vaccinations or twice with an interval of 6 to 10 weeks between vaccinations. A booster was given after 20 to 40 weeks. The avidity index (AI) of antibodies increased significantly during the primary series of vaccinations and after the booster was given. No differences in AIs were found when the results obtained with the two vaccination schedules or with the two adjuvants were compared. After vaccination, IgG1 was the predominant IgG isotype, followed by IgG3. No IgG2 or IgG4 was detected. There was a strong correlation between serum bactericidal activity (SBA) and ELISA titers (r= 0.85 [P< 0.0001] for total IgG,r= 0.83 for IgG1 [P< 0.0001],r= 0.82 for IgG3 [P< 0.0001], andr= 0.84 [P< 0.0001] for the avidity titer). When two subgroups with similar anti-OMV IgG levels were compared before and after the booster vaccination, the higher AI after the booster vaccination was associated with significantly increased SBA. We concluded that avidity maturation occurs after vaccination with a monovalent meningococcal B OMV vaccine, especially after boosting, as indicated by a significant increase in the AI. Vaccination with the monovalent OMV vaccine induced mainly IgG1 and IgG3 isotypes, which are considered to be most important for protection against meningococcal disease. An increase in the AI of antibodies is associated with increased SBA, independent of the level of specific IgG and the IgG isotype distribution. Measuring the AI and IgG isotype distribution of antibodies after vaccination can be a supplementary method for predicting protective immunity for evaluation in future phase III trials with meningococcal serogroup B vaccines.

Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A H C . Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A H C was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to C fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.

Antibody (Ab) responses to polysaccharides (PSs) such asNeisseria meningitidisgroup C PS (MCPS) are characterized as being thymus independent (TI) and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled to tetanus toxoid (MCPS-TT), elicits a thymus-dependent (TD) response. In order to understand the influence of the form of a vaccine (TI versus TD) on the Ab repertoire, we generated monoclonal antibody (MAb) panels from mice immunized and boosted with MCPS or MCPS-TT in different ways. The panels of MAbs were examined for isotype, fine specificity, affinity, and VHgene family usage. The use of MCPS-TT resulted in a shift in the isotype from immunoglobulin M (IgM) and IgG3 elicited in response to the MCPS to primarily IgG1. This isotype shift was accompanied by a change in the fine specificity of the response to the conjugate compared to that of PS. New fine specificities and increased affinity were observed in response to the TD antigen (Ag). Dot blot and Northern analyses of MCPS MAbs revealed that VHgene family usage is dominated by VHJ558, used by 23 of 39 MAbs. VH3609 was seen in three MAbs of restricted fine specificity. VHQ52, VH7183, and VHVGAM3-8 were seen in more than one MAb across these panels, while VH10 and VHX24 were detected only once in response to the TI-2 Ag. All MAbs in the panels utilized kappa light chains, and all functional Jκgenes were expressed.

Immunological intervention, in addition to vector control and malaria chemotherapy, will be needed to stop the resurgence of malaria, a disease with a devastating impact on the health of 300 to 500 million people annually. We have pursued a vaccination strategy, based on DNA immunization in mice with genes encoding two antigens present on the sexual stages of Plasmodium falciparum , Pfs25 and Pfg27, to induce biologically important antibodies that can block development of the parasite in the Anopheles mosquito and thus transmission of the disease. DNA encoding Pfs25 when administered by the intramuscular route, either alone or with DNA encoding Pfg27, had the most potent transmission-blocking effects, resulting in up to a 97% decrease in oocyst numbers in mosquito midguts and a 75% decrease in rate of infection. Immunization with DNA encoding a Pfg27-Pfs25 fusion protein was less effective and DNA encoding Pfg27 elicited antibodies in sera that had only modest effects on the infectivity of the parasite. These results show for the first time that DNA vaccination can result in potent transmission-blocking antibodies in mice and suggest that the Pfs25 gene should be included as part of a multicomponent DNA vaccine.

Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response.

The serotype b-specific carbohydrate antigen (SbAg) of Actinobacillus actinomycetemcomitans Y4 is reported to be the O antigen of lipopolysaccharide, and the highest titers of serum antibody reactive with A. actinomycetemcomitans in early-onset periodontitis (EOP) patients bind SbAg. These high titers of serum antibody reactive with SbAg are associated with a lesser extent and severity of periodontal disease. The aim of this study was to determine if a limited number of genes code for anti-SbAg antibodies as has been shown for immunoglobulin G (IgG) reactive with the type b polysaccharide from Haemophilus influenzae. Serum IgG reactive with the SbAg was prepared from 20 high-titer EOP patients by affinity chromatography. The IgG subclass concentrations were determined, and heterogeneity was analyzed by isoelectric focusing (IEF). IgG2 was the dominant subclass (83% of total IgG) in the anti-SbAg IgG fraction and represented an average of 1.33% of total serum IgG2. The IgG2 reactive with SbAg was isolated from the affinity-purified IgG fraction by affinity chromatography with protein A and subclass-specific monoclonal antibodies. On IEF gels, only 4 to 20 bands were observed in the anti-SbAg IgG fractions, indicating limited heterogeneity. N-terminal amino acid sequence analysis of eight representative anti-SbAg IgG2 preparations indicated that variable heavy and light chains consisted largely of V(H)III and V(kappa)II, respectively. However, a significant fraction of anti-SbAg may use V(H) and V(lambda) genes with blocked N termini. In short, these findings indicate that IgG reactive with SbAg is very much like the antibody reactive with H. influenzae type b polysaccharide. Similarities include IgG2 dominance, limited bands on IEF gels, supporting an oligoclonal response, and use of genes from V(H)III and V(kappa)II regions.

Current porcine pleuropneumonia bacterins afford only partial protection by decreasing mortality but not morbidity. In order to better understand the type(s) of immune response associated with protection, antibody- and cell-mediated immune responses (CMIR) were compared for piglets before and after administration of a commercial bacterin, which confers partial protection, or a low-dose (10(5) CFU/ml) aerosol challenge with Actinobacillus pleuropneumoniae CM5 (LD), which induces complete protection. Control groups received phosphate-buffered saline or adjuvant. Serum antibody response, antibody avidity, delayed-type hypersensitivity (DTH), and lymphocyte blastogenic responses were measured and compared among treatment groups to the lipopolysaccharide (LPS), capsular polysaccharide (CPS), hemolysin (HLY), and outer membrane proteins (OMP) of A. pleuropneumoniae. Peripheral blood lymphocytes and sera were collected prior to and following primary and secondary immunization-infection and high-dose A. pleuropneumoniae CM5 (10(7) CFU/ml) aerosol challenge. Serum antibody and DTH, particularly that to HLY, differed significantly between treatment groups, and increases were associated with protection. LD-infected piglets had higher antibody responses (P < or = 0.01) and antibody avidity (P < or = 0.10) than bacterin-vaccinated and control groups. Anti-HLY antibodies were consistently associated with protection, whereas anti-LPS and anti-CPS antibodies were not. LD-infected animals had higher DTH responses, particularly to HLY, than bacterin-vaccinated pigs (P < or = 0.03). The LD-infected group maintained consistent blastogenic responses to HLY, LPS, CPS, and OMP over the course of infection, unlike the bacterin-vaccinated and control animals. These data suggest that the immune responses induced by a commercial bacterin are very different from those induced by LD aerosol infection and that current bacterins may be modified, for instance, by addition of HLY, so as to stimulate responses which better reflect those induced by LD infection.

The variant surface glycoproteins (VSGs) of African trypanosomes form a dense surface coat on the bloodstream parasites. VSGs are immunodominant antigens that stimulate a rapid antibody response in trypanosome-infected individuals. In the present study, we examined VSG-specific antibodies present not only in sera from infected individuals but also in sera from individuals that had never been exposed to trypanosomes. Native antibodies against different VSGs were detected in sera from uninfected mice, bovines, and humans; the antibodies were revealed to be exclusively directed against variable determinants of the antigens. Further experimentation demonstrated that such native antibodies immunoreact with cellular components of mice and thus are most likely produced by the self-reactive B-cell compartment of the murine immune system.

Variable expression of repeating units of the protective alpha C proteins among clinical isolates of group B streptococci (GBS) may have implications for vaccine development. In this study, alpha C protein genes containing various numbers of repeats (1,2,9, and 16) were cloned in a T7 overexpression vector in Escherichia coli. Expression was induced by isopropyl-beta-D-thiogalactopyranoside, and proteins were purified by anion-exchange, gel filtration, or affinity chromatography or by isoelectric focusing. Rabbits were immunized with purified 1-,2-,9-, or 16-repeat proteins. All proteins appeared to be highly immunogenic. Enzyme-linked immunosorbent assay inhibition with 9-repeat protein as the coating antigen and 9-repeat-antigen-elicited antiserum showed that a 200-fold-higher concentration of 1-repeat antigen than of 9- or 16-repeat antigen was required for 50% inhibition of antibody-antigen binding. The concentration of 2-repeat antigen required for 50% inhibition was intermediate relative to the concentrations of 1- and 9-repeat antigens. These results suggested that antibodies to 9-repeat antigen recognized predominantly a conformational epitope(s) contained in proteins with higher numbers of repeats (9 or 16) but lost considerable binding affinities for an epitope(s) contained in alpha C proteins with fewer repeats (1 or 2). Similar results were obtained with antiserum to 16-repeat antigen. However, antibodies to 1- and 2-repeat antigens recognized 1-,2-,9-,and 16-repeat antigens with equal binding affinities. This finding suggested that 1- and 2-repeat-elicited antibodies recognized an epitope(s) on individual repeats. Loss of repeating units from the alpha C proteins may result in decreased protection because the loss of epitopes (including conformational epitopes) gives the microorganisms the opportunity to escape host antibodies. If 1- and 2-repeat-elicited antibodies bind all alpha C proteins with equal affinity, regardless of their repeat number, they may prevent GBS strains with fewer repeats from escaping host immunity. Protection data obtained with antisera to the proteins with different repeat numbers support this hypothesis: mouse pups challenged with GBS strain A909 were better protected when immunized with 1- or 2-repeat-elicited antiserum (76 and 75%, respectively) than when immunized with 9- or 16-repeat-elicited antiserum (41 and 48%, respectively).

Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

The human immunoglobulin M monoclonal antibody HA-1A was first described as an antibody which bound specifically to the lipid A region of lipopolysaccharide (LPS) (N. N. H. Teng, H. S. Kaplan, J. M. Herbert, C. Moore, H. Douglas, A. Wunderlich, and A. Braude, Proc. Natl. Acad. Sci. USA 82:1790-1794, 1985) and provided significant protection when administered to patients with gram-negative bacteremia and shock (E. J. Ziegler, C. J. Fisher, Jr., C. L. Sprung, R. C. Straube, J. C. Sadoff, G. E. Foulke, C. H. Wortel, M. P. Fink, R. P. Dellinger, N. N. H. Teng, I. E. Allen, H. J. Berger, G. L. Knatterud, A. F. LoBuglio, C. R. Smith, and the HA-1A Sepsis Study Group, New Engl. J. Med. 324:429-436, 1992). Since that original report, questions have arisen in the scientific literature concerning the specificity of this antibody in LPS and/or lipid A binding. Experiments have, therefore, been carried out with a variety of assay formats to determine the capacity of this HA-1A antibody to bind to lipid A and LPS. Direct binding experiments with a sensitive enzyme-linked immunosorbent assay (ELISA) system have established that HA-1A will bind to purified lipid A from both Escherichia coli and Salmonella spp. These results have been confirmed by using a fluid-phase antigen-antibody competitive inhibition assay with purified lipid A and an antibody-antibody competitive inhibition assay with a monoclonal antibody with known specificity for lipid A. The HA-1A monoclonal antibody has also been shown to bind to a panel of R-chemotype LPS by ELISA and, unlike many other previously reported anti-lipid A antibodies, binding of HA-1A to R-chemotype LPS and lipid A is comparable. Although binding of HA-1A to S-LPS (smooth, wild-type LPS) could not be detected by direct ELISA, competitive inhibition experiments with some preparations of S-LPS have been able to show specific HA-1A binding. Collectively, these data confirm the binding specificity of HA-1A for the lipid A component of LPS and provide evidence that this monoclonal antibody manifests a relatively uncommon profile in its capacity to bind lipid A and R-chemotype LPS as well as some preparations of S-LPS.

Porphyromonas gingivalis is a suspected pathogen in rapidly progressive periodontitis (RPP). We have determined the anti-P. gingivalis serum immunoglobulin G (IgG) isotype response and avidity and the subclass titer distributions for 30 RPP patients and 30 age-, sex-, and race-matched healthy subjects by using enzyme-linked immunosorbent assay technology. Patients and control subjects were classified as seropositive if their total IgG response to P. gingivalis was twofold or more than the median response in healthy subjects. The predominant antibody responses for both patients and healthy subjects were IgG2 and IgG3, with a subclass order of IgG2 greater than IgG3 greater than IgG1 greater than IgG4. The avidity of the IgG response was highest for the seropositive healthy subjects and was no different between seronegative and seropositive RPP patients. The subclass antibody responses did not depend on gender, and there were no correlations between titer, avidity, or subclass with disease severity in the RPP patients as measured by pocket depth or bone loss on dental X rays. The seronegative RPP patients exhibited antibody responses that were greater than the responses of seronegative healthy subjects for all four subclasses, while the seropositive RPP patients had higher IgG1 and IgG4 levels than seropositive healthy subjects. These findings are consistent with the hypothesis that both carbohydrate and protein antigens are important in the IgG response to P. gingivalis. The relative predominance of IgG2, a subclass which lacks strong complement fixation and opsonic properties, and the low avidity of patient anti-P. gingivalis IgG antibodies suggest that humoral responsiveness to infection with P. gingivalis may be ineffective in clearing this organism.

Plasmodium falciparummerozoite surface protein (MSP3) is a main target of protective immunity against malaria that is currently undergoing vaccine development. It was shown recently to belong, together with MSP6, to a new multigene family whose C-terminal regions have a similar organization, contain both homologous and divergent regions, and are highly conserved across isolates. In an attempt to rationally design novel vaccine constructs, we extended the analysis of antigenicity and function of region-specific antibodies, previously performed with MSP3 and MSP6, to the remaining four proteins of the MSP3 family using four recombinant proteins and 24 synthetic peptides. Antibodies to each MSP3 family antigen were found to be highly prevalent among malaria-exposed individuals from the village of Dielmo (Senegal). Each of the 24 peptides was antigenic, defining at least one epitope mimicking that of the native proteins, with a distinct IgG isotype pattern for each, although with an overall predominance of the IgG3 subclass. Human antibodies affinity purified upon each of the 24 peptides exerted an antiparasite antibody-dependent cellular inhibition effect, which in most cases was as strong as that of IgG from protected African adults. The two regions with high homology were found to generate a broad network of cross-reactive antibodies with various avidities. A first multigenic construct was designed using these findings and those from related immunogenicity studies in mice and demonstrated valuable immunological properties. These results indicate that numerous regions from the MSP3 family play a role in protection and provide a rationale for the tailoring of new MSP3-derived malaria vaccines.

The Plasmodium falciparum major merozoite surface protein (gp195) is a protective antigen against lethal malaria. However, increasing evidence indicates that the efficacy of a malaria vaccine will require a strong adjuvant that is safe for human use. We compared the efficacies of two low-toxicity synthetic immunomodulators, B30-MDP (a lipophilic muramyl dipeptide derivative) and LA-15-PH (a synthetic equivalent of monophosphoryl lipid A), with that of Freund complete adjuvant (FCA) in eliciting an antibody response to gp195. Rabbits were immunized with native gp195 and B30-MDP, LA-15-PH, or the two in combination, with liposomes as the vehicle. Aluminum hydroxide and FCA were used as reference adjuvants. Results showed that adjuvant formulations based on B30-MDP alone or in combination with LA-15-PH induced high antibody titers to gp195, as compared with FCA. LA-15-PH alone was less effective. Aluminum hydroxide induced significantly lower antibody titers. The functional activity of the rabbit anti-gp195 antibodies induced by different adjuvants was evaluated in an in vitro parasite growth inhibition assay previously shown to correlate with anti-gp195 immunity in the Aotus monkey model. All rabbits immunized with B30-MDP-LA-15-PH and two of three rabbits immunized with B30-MDP alone produced sera that strongly inhibited parasite growth. The degree of growth inhibition was similar to that with FCA. The antibody titers of the rabbits receiving B30-MDP-LA-15-PH strongly correlated with the degree of in vitro growth inhibition. Our findings provided strong evidence that adjuvant formulations based on synthetic B30-MDP and LA-15-PH can replace FCA as adjuvants in stimulating protective immunity specific for gp195.

Splenic microarchitecture is substantially altered during acute malaria infections, which may affect the development and regulation of immune responses. Here we investigated whether engagement of host Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adaptor protein MyD88 is required for induction of the changes and whether antibody responses are modified when immunization takes place during the period of splenic disruption. The alterations in splenic microarchitecture were maximal shortly after the peak of parasitemia and were not dependent on engagement of TLR2, TLR4, or TLR9, and they were only minimally affected by the absence of the MyD88 adaptor molecule. Although germinal centers were formed in infected mice, they did not contain the usual light and dark zones. Immunization of mice with chicken gamma globulin 2 weeks prior to acute Plasmodium chabaudi infection did not affect the quantity or avidity of the immunoglobulin G antibody response to this antigen. However, immunization at the same time as the primary P. chabaudi infection resulted in a clear transient reduction in antibody avidity in the month following immunization. These data suggest that the alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibody response during a malaria infection and could impact the development of immunity to malaria or to other infections or immunizations given during a malaria infection.

The search for an efficacious vaccine against malaria is ongoing, and it is now widely believed that to confer protection a vaccine must induce very strong cellular and humoral immunity concurrently. We studied the immune response in mice immunized with the recombinant viral vaccines fowlpox strain FP9 and modified virus Ankara (MVA), a protein vaccine (CV-1866), or a combination of the two; all vaccines express parts of the same preerythrocytic malaria antigen, thePlasmodium bergheicircumsporozoite protein (CSP). Mice were then challenged withP. bergheisporozoites to determine the protective efficacies of different vaccine regimens. Two immunizations with the protein vaccine CV-1866, based on the hepatitis B core antigen particle, induced strong humoral immunity to the repeat region of CSP that was weakly protective against sporozoite challenge. Prime-boost with the viral vector vaccines, FP9 followed by MVA, induced strong T-cell immunity to the CD8+epitope Pb9 and partially protected animals from challenge. Physically mixing CV-1866 with FP9 or MVA and then immunizing with the resultant combinations in a prime-boost regimen induced both cellular and humoral immunity and afforded substantially higher levels of protection (combination, 90%) than either vaccine alone (CV-1866, 12%; FP9/MVA, 37%). For diseases such as malaria in which different potent immune responses are required to protect against different stages, using combinations of partially effective vaccines may offer a more rapid route to achieving deployable levels of efficacy than individual vaccine strategies.

In the Finnish Otitis Media Vaccine Trial, the now-licensed pneumococcal conjugate vaccine containing polysaccharides conjugated to protein CRM197(PncCRM) and the experimental pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine (PncOMPC), showed similar efficacy profiles against acute otitis media despite different antibody concentrations in sera. We now report the opsonophagocytic activities (OPA) in these sera. OPA, antibody concentration, and avidity for serotypes 6B, 19F, and 23F were determined in sera of infants who received either pneumococcal conjugate (PCV) or control vaccine at 2, 4, and 6 months of age and either the homologous or pneumococcal polysaccharide vaccine at 12 months of age. OPA varied by vaccine and serotype. The majority of PCV recipients had positive OPA after the fourth dose, while OPA was undetectable in the control group. Coinciding with the efficacy data, the concentration of antibodies required for 50% killing was low for 6B and high for 19F for both PCVs. Contradictory to the efficacy data, PncOMPC induced lower functional capacity to 23F than PncCRM. OPA correlated with antibody concentration, while avidity and functional capacity of antibodies showed no correlation. The OPA data provide valuable additional information for serotype-specific differences in protection and when evaluating serotype-specific immunogenicity and should thus be considered when defining serological correlates of protection.

Structure-function correlations of pneumococcal antibodies are important in predicting how changes in the pneumococcus (Pnc)-specific B-cell repertoire will influence humoral immunity against invasive Pnc disease. Using a unique panel of human hybridomas derived from memory B cells after pneumococcal conjugate vaccination, we analyzed the structure-function relationship of nine monoclonal antibodies (MAbs) reactive to Pnc polysaccharides. The avidities of the antibodies correlated with the avidity of donor immune serum (R, 0.7;P< 0.025), and this relationship was particularly strong for immunoglobulin A clones (R, 1;P< 0.0005), suggesting that the MAbs may represent important clones contributing to serological memory. Common heavy-light chain combinations and amino acid replacement mutations were seen for clones with the same serospecificity from different individuals. The two highest-avidity MAbs used Vh3-48, and two MAbs with the same serospecificity, using the same V gene pairings (Vh3-7 and Vk2A17), had similar avidities, suggesting that canonical V gene use makes an important contribution to avidity. Although all clones had mutation levels consistent with their being derived from memory B cells, low levels of replacement mutation were associated with high avidities. This relationship was strongest for Vh genes (R, 0.8;P< 0.01). Opsonophagocytosis was demonstrated for all clones, and there was a trend toward clones using canonical genes with low levels of mutation having high opsonophagocytic activities (R, 0.5). These data suggest that the use of canonical genes in the Pnc antibody response is associated with highly functional antibodies and that most somatic mutations seen in these genes are not antigen selected.

Passive immunization has been successfully employed for protection against bacterial and viral infections for over 100 years. Immunoglobulin Fc regions play a critical role in the clearance of bacterial pathogens by mediating antibody-dependent and complement-dependent cytotoxicity. Here we show that antibody fragments engineered to recognize the protective antigen component of the B. anthracis exotoxin with high affinity and conjugated to polyethylene glycol (PEG) for prolonged circulation half-life confer significant protection against inhalation anthrax despite their lack of Fc regions. The speed and lower manufacturing cost of bacterially expressed PEGylated antibody fragments could provide decisive advantages for anthrax prophylaxis. Importantly, our results suggest that PEGylated antibody fragments may represent a unique approach for mounting a rapid therapeutic response to emerging pathogen infections.

Antibodies directed against Pfs25, a protein present on the surface of zygotes and ookinetes of Plasmodium falciparum , completely block pathogen transmission. We evaluated the immunomodulatory effect of CpG oligodeoxynucleotides (ODN) on the immunogenicity of recombinant Pfs25 (rPfs25) formulated in alum (Al). Immunization of mice with rPfs25 plus CpG ODN improved both the antibody titer (a 30-fold-higher antibody response than that with rPfs25-Al alone) and avidity. Coadministration of CpG ODN dramatically enhanced the titer of immunoglobulin G2A (IgG2a) compared to the titer of the IgG1-dominant response caused by rPfs25-Al alone, and the sera from the CpG ODN-coadministered group completely blocked the transmission of P. falciparum parasites to mosquitoes, as determined by membrane feeding assays. However, transmission-blocking experiments revealed that blocking efficacy was dependent on high-titer antibody levels, independent of isotypes. These results suggest that CpG ODN can be used as an adjuvant to enhance the immunogenicity of rPfs25 as a malaria transmission-blocking vaccine.

It has been previously shown that one of the three meningococcal C conjugate (MCC) vaccines introduced in the United Kingdom proved highly immunogenic after the first dose of a three-dose schedule, with evidence of immune memory after dose 3. Thus, in infants a one- or two-dose schedule of this MCC vaccine, conjugated to tetanus toxoid (TT), may suffice. Healthy infants ( n = 586) were randomized to receive either one (group 1), two (group 2), or three (group 3) doses of MCC-TT vaccine with a 10-μg polysaccharide booster given at 13 to 14 months of age. Serum bactericidal antibody (SBA) levels were measured by utilizing rabbit complement (rSBA), meningococcal C-specific immunoglobulin G (IgG), and avidity indices (AIs). For groups 1, 2, and 3, the percentages of infants with an rSBA level of ≥8 against strain C11 were 98.4, 100, and 99.4%, respectively. Infants in group 1 with prevaccination rSBA titers of ≥8 had post-primary MCC rSBA geometric mean titers (GMTs) significantly lower than those infants with prevaccination rSBA titers of

The relative avidity of serum immunoglobulin G antibody for the fimbria antigen of Actinobacillus actinomycetemcomitans was assessed by diethylamine dissociation enzyme-linked immunosorbent assay in patients with adult periodontitis. High-titer sera from patients not harboring A. actinomycetemcomitans had significantly higher avidities for the fimbria antigen than did high-titer sera from patients with A. actinomycetemcomitans in their periodontal pockets. The elicited antibodies against the fimbria antigen may afford protection against A. actinomycetemcomitans infection.

Streptococcus pneumoniae causes meningitis, bacteremia, pneumonia, and acute otitis media and is responsible for approximately 40,000 deaths per year in the United States (1, 5, 29). The capsular polysaccharide (PS) functions as a virulence determinant, and although 90 or more different pneumococcal capsular PS (PPS) serotypes have been identified worldwide, only a subset of these are responsible for the majority of disease (1, 5, 29). Complement-dependent phagocytosis mediated by anti-PPS antibodies provides protection against pneumococcal disease; accordingly, pneumococcal vaccine development has focused upon the induction of these antibodies (9, 17, 24, 32). The presently licensed pneumococcal vaccine, which consists of a mixture of 23 different PPS serotypes, appears to be efficacious in healthy adults; however, this vaccine is poorly immunogenic in children under 2 to 3 years of age, a population at increased risk for developing invasive pneumococcal disease. New pneumococcal vaccines are being developed with protein-conjugated forms of PPS (18, 20, 32), a strategy that has proven to be successful in generating immunogenic and efficacious pediatric vaccines for Haemophilus influenzae type b (Hib) (11). The evaluation of pneumococcal vaccines involves a serological assessment of anti-PPS antibody responses, with the aim of developing reliable surrogates of vaccine protective efficacy. While the serum anti-PPS antibody concentration is typically considered the primary index of response and protection (32, 33), properties such as avidity (14) and opsonophagocytic activity (9, 30) may be important surrogates as well. Recently, Anttila and colleagues observed avidity differences among PPS-specific antibodies elicited in infants following vaccination with different PPS-protein conjugate vaccines and showed that avidity may be influenced by the type of PPS conjugate used for vaccination (4). The relationship between anti-PPS antibody avidity and protective efficacy has not been investigated to date. Although studies of the human antibody response to Hib PS have directly implicated avidity as a determinant of antibody bactericidal and rat-protective activities (2, 14, 22, 31), it is not known whether these conclusions are generally applicable to antibody responses to the various capsular serotypes of pneumococci. This issue is particularly important since, unlike the situation with immunity to Hib, which is mediated principally by bactericidal antibody, opsonophagocytosis functions as the primary effector mechanism in immunity to pneumococci. In the present report, we determined the avidities of immunoglobulin G2 (IgG2) antibodies specific for PPS 6B and 23F elicited in adults following PPS vaccination and examined the relationship between avidity and protective activities. We studied antibodies to PPS capsular serotypes 6B and 23F because these two serotypes of pneumococci are a frequent cause of disease, are components of experimental conjugate vaccines presently under evaluation in clinical trials (18, 20, 32), and are structurally disparate. PPS 6B is a straight-chain negatively charged polymer consisting of repeating units of galactose-glucose-rhamnose-ribitol phosphate, whereas PPS 23F is a branched-chain negatively charged polymer of glucose-galactose-rhamnose with glycerol phosphate and rhamnose attached to the galactose unit via esterification.

Anti- Haemophilus influenzae b polysaccharide (Hib PS) antibodies elicited in elderly subjects following conjugate vaccination expressed a light-chain variable-region (V L )-associated idiotype and had functional activities similar to those previously observed in children and younger adults. These findings indicate that advanced age is not accompanied by shifts in the major V L component of the Hib PS-specific repertoire or by diminution of the protective function of antibodies.

To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H(C). Twenty-eight unique BoNT/A H(C) binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, respectively, compared to toxin control. scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.

An O-antigen-specific murine monoclonal antibody (MAb) directed against an immunodominant epitope expressed on Klebsiella O1, O6, and O8 lipopolysaccharides (LPS) was examined with respect to its binding to nonencapsulated and encapsulated bacterial cells and its ability to protect against lethal murine Klebsiella sepsis. While the MAb (clone Ru-O1, mouse immunoglobulin G2b) bound well to nonencapsulated organisms of the O1 serogroup, binding was significantly, but not completely, abolished by the presence of the K2 capsule. In a model of experimental Klebsiella peritonitis and sepsis induced by a virulent O1:K2 serogroup strain, higher doses of anti-LPS MAb Ru-O1 than of a previously described anticapsular MAb specific for the K2 capsular polysaccharide were needed to provide protection. However, high-dose (40 microg/g of body weight) pretreatment with anti-LPS MAb Ru-O1 significantly reduced bacterial dissemination to various organs as well as macroscopic and histologic pulmonary alterations. Thus, since the number of Klebsiella capsular antigens occurring in clinical material is too large to be completely "covered" by a K-antigen-specific hyperimmunoglobulin preparation, O-antigen-specific antibodies may supplement K-antigen-specific immunoprophylaxis and -therapy of clinical Klebsiella infection.

Cryptococcus neoformans is a human pathogenic fungus which is unusual in two respects: it has a polysaccharide capsule similar to that found in encapsulated bacteria and it can produce melanin. Capsular and melanization phenotypes are associated with virulence. In this study we analyzed the contributions of the capsular polysaccharide, melanization, and antibody binding to the capsule to the cellular charge of C. neoformans. Cell charge was inferred from measurements of zeta potential. The results indicate that (i) C. neoformans cells are significantly more negatively charged than Saccharomyces cerevisiae cells, (ii) the polysaccharide capsule of C. neoformans is responsible for the high negative charge of the cells, (iii) C. neoformans melanin is negatively charged, (iv) melanization in C. neoformans is associated with an increased negative charge per cell, and (v) antibody binding to the capsule of C. neoformans significantly alters the cell charge. These results suggest that alterations in cell charge attributable to polysaccharide capsule formation, melanization, and antibody binding may affect C. neoformans virulence given that macrophage phagocytosis is effected by the zeta potential of microorganisms.

A fibronectin-binding protein (FnBP) adhesin of Staphylococcus aureus possesses three 37- or 38-amino-acid motifs (D1, D2, and D3) that can each bind fibronectin (Fn) with low affinity and that in tandem comprise D1-3, a high-affinity Fn-binding domain. To identify epitopes for the generation of adhesion-blocking antibodies, rabbits were immunized with recombinant D1-3 or with a glutathione S-transferase fusion protein, GSTD1-3. Affinity-purified antibodies from the D1-3 immunization were poor inhibitors of Fn binding to S. aureus and recognized several different epitopes, with a preference for clusters of acidic amino acids that do not contribute to Fn binding. Antibodies generated with GSTD1-3 as an immunogen were more effective inhibitors, but concentrations in excess of 20 microg x ml-1 did not promote more than 50% inhibition. These antibodies were highly specific for amino acids 21 to 34 of D1 (D1(21-34)), which contain a sequence that is essential for Fn binding and are identical to D2 at 12 of 14 residues. Neither antibody preparation recognized D3(20-33) of the D3 motif, where the only homology to D1(21-34) and D2(21-34) comprises a sequence motif, GG(X3,4)(I/V)DF, that is critical to Fn binding. However, antibodies specific for both D1(21-34) and D3(20-33) could be obtained by using synthetic peptides corresponding to these sequences as immunogens. F(ab')2 fragments derived from these antibodies each caused 40 to 50% inhibition of Fn binding to S. aureus, and their ability to bind to purified FnBP was eliminated by competing Fn. However, mixtures of the two F(ab')2 preparations did not provide additive or synergistic inhibition of Fn binding. Therefore, inhibition of Fn binding to S. aureus requires antibodies specific for D1(21-34) and D3(20-33), but a mixture of antibodies specific for both sequences did not provide complete inhibition.