HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.     

Autoimmunity and immune system dysregulation in schizophrenia: IgGs from sera of patients hydrolyze myelin basic protein

Several different theories of schizophrenia (SCZ) were discussed; the causes of this disease are not yet clear. Using ELISA, it was shown that titers of autoantibodies against myelin basic protein (MBP) in SCZ patients are ~1.8‐fold higher than in healthy individuals but 5.0‐fold lower than in patients with multiple sclerosis. Several rigid criteria were checked to show that the MBP‐hydrolyzing activity is an intrinsic property of SCZ IgGs. Approximately 82% electrophoretically homogeneous SCZ IgGs purified using several affinity sorbents including Sepharose with immobilized MBP hydrolyze specifically only MBP but not many other tested proteins. The average relative activity of IgGs from patients with negative symptoms was 2.5‐fold higher than that of patients with positive symptoms of SCZ, and it increases with the duration of this pathology. It was shown that abzymes are the earliest statistically significant markers of many autoimmune pathologies. Our findings surmise that the immune systems of individual SCZ patients can generate a variety of anti‐MBP abzymes with different catalytic properties, which can attack MBP of the myelin‐proteolipid shell of axons. Therefore, autoimmune processes together with other mechanisms can play an important role in SCZ pathogenesis. MBP‐hydrolyzing antibodies were previously detected in the blood of 80% to 90% of patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, some similar neuropsychiatric indicators of disease common to SLE, MS, and SCZ were described in the literature. Thus, the destruction of the myelin sheath and the production of MBP‐hydrolyzing antibodies can be a common phenomenon for some different diseases.     

Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase, RNase, and polysaccharide-hydrolyzing activities are associated with IgGs from the sera of patients with multiple sclerosis (MS). Here we present evidence demonstrating that highly purified MS IgGs (but not Igs from the sera of healthy individuals) catalyze specifically hydrolysis of human myelin basic protein (hMBP). In contrast to many known proteases, IgGs do not hydrolyze many other different proteins. Specific inhibitors of acidic and thiol proteases have no remarkable effect on proteolytic activity of IgGs. However, specific inhibitor of serine (PMSF, AEBSF, and benzamidin) and metal-dependent (EDTA) proteases significantly inhibit activity of proteolytic abzymes. Interestingly, the ratio of serine-like and metal-dependent activities of MS IgGs varied very much from patient to patient. The findings speak in favor of the generation by the immune systems of individual MS patients of a variety of polyclonal anti-MBP IgGs with different catalytic properties.     

Substrate specificity of IgGs with peroxidase and oxidoreductase activities from sera of patients with systemic lupus erythematosus and multiple sclerosis

The analysis of IgGs to protect humans from oxidative stress through oxidation of harmful compounds was carried out. We have compared here for the first time peroxidase (in the presence of H₂O₂) and oxidoreductase (in the absence of H₂O₂) activities of IgGs from sera of healthy humans and patients with systemic lupus erythematosus (SLE) and multiple sclerosis (MS). In addition, substrate specificity of SLE and MS IgG preparations in the oxidation of different compounds was analyzed: 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), 3,3′‐diaminobenzidine (DAB), homovanillic acid (HVA), o‐phenylenediamine (OPD), α‐naphthol, 3‐amino‐9‐ethylcarbazole (AEC), p‐hydroquinone (pHQ), and adrenaline. IgGs of healthy humans and SLE and MS patients oxidized DAB, ABTS, and OPD due to their peroxidase and oxidoreductase activities, while other compounds were substrates of IgGs only in the presence of H₂O₂: adrenaline was not oxidized by both activities of IgGs. The average SLE IgGs peroxidase activity increased statistically significant in comparison with abzymes from healthy humans in the order (‐fold): OPD (1.2) <  DAB (1.7) < α‐naphtol (2.2) ≤ AEC (2.4) < ABTS (4.5) < 5‐ASA (10.6), while with oxidoreductase activity: OPD (1.8) ≤ DAB (2.1‐fold) < ABTS (5.0). Only HVA was oxidized by IgGs with peroxidase activity of healthy donors faster than by SLE (1.3‐fold) and MS abzymes (2.4‐fold). In the oxidation of several substrates, only three IgGs of MS patients were used. The data speak of a tendency to increase the peroxidase and oxidoreductase activities of MS IgGs in comparison with healthy donors, but to a lesser extent: OPD (1.1 to 1.2‐fold) ≤ ABTS (1.2 to 1.8‐fold). It was shown that development of SLE and MS leads to increase in peroxidase and oxidoreductase activities of IgGs toward most of classical substrates. Thus, abzymes can serve as an additional factor of reactive oxygen species detoxification protecting of patients with SLE and MS from some harmful compounds somewhat better than healthy peoples.     

Metal ions-dependent peroxidase and oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats

We present evidence showing that a small fraction of electrophoretically homogeneous IgGs from the sera of healthy Wistar rats is bound with several different Me2+ ions and oxidizes 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2O2 and through an oxidoreductase activity in the absence of H2O2. During purification on Protein A-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me2+ ions. Therefore, in the absence of external metal ions, the specific peroxidase activity of IgGs from the sera of different rats varied in the range 1.6-26% and increased up to 13-198% after addition of Fe2+ or Cu2+ ions as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of HRP is 24-fold lower than its peroxidase activity, while oxidoreductase and peroxidase activities of IgGs are comparable. Oxidoreductase activities of different IgGs in the absence of external metal ions varied from 22 to 800%, and in the presence of Fe2+ or Cu2+ ions, from 37 to 1100% in comparison with the HRP oxidoreductase activity (100%). Chromatography of the IgGs on Chelex-100 leads to the adsorption of a small IgG fraction bound with metal ions and to its separation to many different subfractions demonstrating various affinities to the chelating resin and increased levels of the specific oxidoreductase and peroxidase activities. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defense mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activity of antibodies may play an important role in the protection of organisms from oxidative stress and toxic compounds.     

Affinity and catalytic heterogeneity and metal-dependence of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with systemic lupus erythematosus

It was shown using enzyme-linked immunosorbent assay (ELISA) that titers of antibodies against human myelin basic protein (hMBP) in systemic lupus erythematosus (SLE) patients 4.2-fold higher than in healthy individuals, but 2.1-fold lower than in patients with multiple sclerosis (MS). Approximately 86% electrophoretically and immunologically homogeneous SLE immunoglobulin Gs (IgGs) purified using several affinity resins including Sepharose with immobilized hMBP specifically hydrolyze only hMBP but not many other tested proteins. Several rigid criteria were applied to show that the hMBP-hydrolyzing activity is an intrinsic property of SLE IgGs but not from healthy donors. In contrast to MS IgGs, abzymes from SLE patients are more sensitive to ethylenediaminetetraacetic acid and less sensitive to specific inhibitors of serine-like proteases. We present the first evidence demonstrating a significant diversity of different fractions of SLE IgGs in their affinity for hMBP-Sepharose, the ability of IgGs to hydrolyze hMBP at different optimal pHs (5-10) and be activated by different metal ions: Ca(2+) > Mg(2+) ≥ Co(2+) ≥ Fe(2+) ≥ Ni(2+) ≥ Zn(2+) ≥ Cu(2+) ≥ Mn(2+) . Combinations of Ca(2+) + Mg(2+) and Ca(2+) + Co(2) lead to a significant increase in the antibody proteolytic activity as compared with Ca(2+) , Co(2+) , or Mg(2+) ions taken separately. Our findings suggest that the immune systems of individual SLE similar to MS patients can generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons and play an important role in pathogenesis not only MS but also SLE patients.     

Affinity and catalytic heterogeneity of polyclonal myelin basic protein‐hydrolyzing IgGs from sera of patients with multiple sclerosis

Human myelin basic protein (hMBP)‐hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3–10.5). IgGs containing λ‐ and κ‐types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1–IgG4 sub‐classes were analyzed for catalytic activity. IgGs of all four sub‐classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19‐mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti‐hMBP abzymes with different catalytic properties, which can attack hMBP of myelin‐proteolipid shell of axons, playing an important role in MS pathogenesis.     

Characterization of anti-Cryptosporidium IgA antibodies in sera from immunocompetent individuals and HIV-infected patients.

Human antibody response to Cryptosporidium parvum has been previously shown as involving immunoglobulin (Ig)M and IgG isotypes. The interest in anti-cryptosporidial IgA antibody response has been recently stimulated by studies on the therapeutic effects of secretory IgA antibodies to Cryptosporidium in animal models and in patients. In the present study, isotypes of serum anti-Cryptosporidium antibodies have been characterized in donors of the following categories: (a) healthy adults, (b) healthy children, (c) immunocompetent children with transient cryptosporidial diarrhea, (d) HIV-infected patients without clinical and parasitological evidence of Cryptosporidium infection and (e) AIDS patients with cryptosporidial diarrhea. Antibodies were detected using C. parvum oocysts purified by density gradient centrifugation from bovine faeces. The IgA antibodies were revealed using alpha-chain specific antibodies. Indirect immunofluorescence analysis with oocysts was used as control. Although high levels of serum antibodies of the IgA class were detected in some donors in the group of healthy adults, elevated values were consistently found in HIV-infected patients. Higher values were found in HIV patients with clinical cryptosporidiosis. The presence of a secretory component in serum IgA antibodies in these patients has been documented. Data indicate that IgA serum antibodies are produced as well as IgM and IgG antibodies upon contact with the parasite, and suggest that elevated IgA serum antibodies to Cryptosporidium are not associated with protection in HIV patients.     

Detection of high levels of immunoreactive human beta-1 interferon in sera from HIV-infected patients

We have examined human interferon beta-1 (HuIFN-beta 1) levels in the sera from patients with various diseases by using a newly developed highly sensitive enzyme-linked immunosorbent assay system (ELISA). High levels of HuIFN-beta 1 were detected in the sera from Human Immunodeficiency Virus-I (HIV-I) infected patients, especially of asymptomatic carriers. The serum level of HuIFN-beta 1 may be a good indicator of HIV-I infection and of other immunological disorders.     

Binding modes of IgG from pemphigus autoimmune sera onto guinea pig keratinocytes and the fate of bound IgGs

Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Anti-interferon-gamma antibodies in sera from HIV infected patients

High serum levels of antibodies to interferon-gamma (IFN-gamma) have been found in patients infected with human immunodeficiency virus (HIV). A radioimmunoassay (RIA) with a recombinant IFN-gamma protein or an affinity purified IFN-gamma preparation as antigens, was developed to detect the specific anti-IFN-gamma antibodies. Reactivity of sera to IFN-gamma was confirmed by Western blot analysis. These antibodies, however, do not seem to recognize the active site of the molecule, since they do not neutralize the antiviral IFN-gamma activity in a biological assay. These results enforce the hypothesis of the role of autoimmunization during the course of the disease.     

Tetrameric and homodimeric camelid IgGs originate from the same IgH locus

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as V(H)H and C(H)H, respectively, and which differ from conventional V(H) and C(H) counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both V(H)H and V(H) genes followed by a unique D(H)-J(H) cluster and C region genes, which include both C(H)H and C(H) genes. Although this general gene organization greatly resembles that of other typical mammalian V(n)-D(n)-J(n)-C(n) translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM(+) stage, similar to the case for conventional IgG.     

Analysis of sera from breast cancer patients by radioimmunoassays

Summary The antibody reactivity of human breast cancer sera was evaluated by means of radioimmunoassays and established breast cancer cell lines. When tested against the MDA-MB 231 cell line, 30 of 324 sera had detectable antibody reactivity. All the positive sera, however, reacted with other cell lines as well, generally including cultures initiated from sites other than breast cancers, and often including animal cell cultures. In competition radioimmunoassays the positive sera fell into various groups, indicating that a diversity of antigens was being detected. Two patients' sera identified antigens that were expressed on breast cancer cells but that were not expressed on an assortment of other cell types. Sera like these two, which identify potentially important tumor markers, could serve as valuable reagents for the analysis of the tumor-assiciated antigens of human breast cancer cells.     

Complement-dependent cytotoxicity of sera obtained from subacute sclerosing panencephalitis patients

Human lymphoid cells (NC-37) persistently infected with either measles virus (Schwarz and TYCSA strains) or subacute sclerosing panencephalitis (SSPE) virus (Halle and Mantooth strains) were destroyed in the presence of complement by anti-measles sera as well as by sera from SSPE patients. The cytotoxic activity was demonstrated in both IgG and IgM fractions of measles convalescent sera, but only in IgG fraction of SSPE sera. Measles convalescent sera completely lost the cytotoxic activity to all the cell lines, when absorbed with any one of the cell lines, indicating that the viral surface antigens of these cell lines infected with measles or SSPE virus are identical. On the other hand, the cytotoxic activity of SSPE sera could not be readily absorbed with these cells. Thus, the affinity of SSPE sera for the viral surface antigens might be lower than that of measles convalescent sera.     

Pleural effusions and sera from patients with benign or malignant diseases

In pleural effusions and sera from 66 patients copper and zinc were quantified by inductively coupled argon plasma-mass spectrometry after mineralizations in a closed-pressurized microwave unit with a mixture of concentrated nitric acid and 30% hydrogen peroxide. Total protein, pH, leukocyte count, lactate dehydrogenase, glucose, C-reactive protein, ceruloplasmin, and alpha1-antitrypsin were determined in many of the effusions. All but four effusions had concentrations of copper (range 58-1720 microg/kg) and zinc (range 27-1001 microg/kg) that were lower than the concentrations in the corresponding sera. Very high concentrations of zinc (1930-6470 microg/kg) were characteristic for thoracic empyemata. In the scatterplots of serum copper versus effusion copper, serum zinc versus effusion zinc, and serum copper/effusion copper versus serum zinc/effusion zinc no clearly delineated regions were noticeably useful for identifying malignant effusions. Similar plots of the concentrations of copper or zinc versus the eight clinical laboratory parameters or plots of clinical parameter versus clinical parameter failed to be of diagnostic value. Statistically highly significant correlations (p < or = 0.05, n > 45, r2 > 0.25) were observed for 9 of 28 pairs of the clinical parameters, for total protein and copper in the effusions and zinc in the effusions and for ceruloplasmin and copper in the effusions. Among the patients suffering from benign or malignant effusions, 52% had zinc concentrations in the sera below the low limit of the normal range (600 microg/kg). Supplementation of such patients with zinc should be considered.     

甲真菌病患者分离真菌多样性分析

为了探寻甲真菌病患者病甲部位的真菌多样性,以期对临床治疗提供参考,结合菌种培养的形态学研究和rDNA ITS序列分析的方法,对临床拟诊为甲真菌病患者的病甲分离培养菌株进行物种鉴定,并以丰富性指数(Ma)、多样性指数(H′)、均匀度指数(J)等指标分析指甲和趾甲分离真菌的多样性。2010年2月至9月在北京大学第三医院皮肤科的1 568例拟诊甲真菌病患者中,641例培养阳性,包括趾甲561例和指甲80例。检测到的皮肤癣菌占59.13%(其中红色毛癣菌为96.04%),酵母菌17.00%,其他真菌23.87%。分离自趾甲的Candida galli、Lewia infectoria和Paraconiothyrium hawaiiense,以及分离自指甲的Cyberlindnera jadinii确定为中国新记录种。趾甲分离菌的物种多样性指数H′、丰富度指数Ma和均匀度指数J均高于指甲分离菌。研究结果表明,该医院就诊甲真菌病患者可分离培养的真菌以皮肤癣菌的红色毛癣菌为主要物种,且趾甲分离菌的菌种多样性较指甲的更为明显。     

Isolation of a TRAIL antagonist from the serum of HIV-infected patients

Virus-host interactions are characterized by the selection of adaptive mechanisms by which to evade pathogenic and defense mechanisms, respectively. In primary T cells infected with HIV, HIV infection up-regulates TNF-related apoptosis inducing ligand (TRAIL) and death-inducing TRAIL receptors, but blockade of TRAIL:TRAIL receptor interaction does not alter HIV-induced cell death. Instead, HIV infection results in a novel splice variant that we call TRAIL-short (TRAIL-s), which antagonizes TRAIL-R2. In HIV patients, plasma TRAIL-s concentration increases with increasing viral load and renders cells resistant to TRAIL-induced death. Knockdown of TRAIL-s abrogates this resistance. We propose that TRAIL-s is a novel adaptive mechanism of apoptosis resistance acquired by HIV-infected cells to avoid their elimination by TRAIL-dependent effector mechanism.     

Non-apical membrane antigen 1 (AMA1) IgGs from Malian children interfere with functional activity of AMA1 IgGs as judged by growth inhibition assay

Background

Apical membrane antigen 1 (AMA1) is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA). However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition.

Methods

Total IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG). From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children.

Results

AMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA.

Conclusion

Children living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1-based vaccine in the target population.