Four distinct antigenic regions are present in the primary structure of HIV-1 and HIV-2 proteinases.

OBJECTIVE: The purpose of this study was to assay reactivity of antibody-positive sera to different parts of the HIV-1 and HIV-2 proteinase (PR) proteins. DESIGN: Since the majority of HIV-1-antibody-positive sera react to the proteinase, but the antigenic determinants on the protein have not been identified, we attempted to identify these determinants. INTERVENTIONS: We synthesized 18 peptides representing the PR of HIV-1 and HIV-2 in order to map serum reactivity to the PR protein. RESULTS: Both HIV-1- and HIV-2-antibody-positive sera recognized four distinct antigenic regions in the HIV-1 and HIV-2 PR. CONCLUSIONS: Correlation between our results and the crystallographic structure of the protein revealed that the antigenic regions are positioned at the surface of the HIV-1 PR. Although the structure of HIV-2 PR has not yet been characterized, our results indicate that the folding of the HIV-1 and HIV-2 PR may be very similar.

Identification of four antibody-binding sites in the envelope proteins of simian immunodeficiency virus, SIVsm.

OBJECTIVE: To identify antigenic regions in the envelope glycoproteins of the simian immunodeficiency virus isolate, SIVsm. METHODS: Thirty-eight peptides were synthesized and used in site-directed enzyme-linked immunosorbent assays with sera from experimentally infected macaques. RESULTS: Four antibody-binding regions were identified, corresponding to the second variable region [V2; amino acids (aa) 170-196], the region homologous to V3 in HIV-1 (aa 313-346), the carboxy terminus of gp120 (aa 514-537) and the amino terminus of the transmembrane protein (aa 608-638). Serum reactivity to the V2 region was higher in surviving monkeys than in animals with an early development of simian AIDS. The antigenicity of the peptide appears to be conformationally dependent. CONCLUSIONS: The majority of antigenic sites identified in the envelope proteins of SIV correspond to sites identified in HIV-1 and HIV-2, which further supports the use of the simian model in vaccine development. The pattern of reactivity to the V2 region suggests that absence of antibodies directed to this site might correlate with disease progression.

Non-specific precipitin reactions of IgG at low ionic strength.

Precipitin reactions apparently not involving specific antibodies were obtained in immunodiffusion experiments performed at low salt concentration between IgG and the muscle proteins, actin and tropomyosin. The precipitates, which dissolved at physiologic ionic strength, may result from electrostatic interaction of molecules of different net charge and different distribution and number of charged groups.

A radioimmunoassay for determination of anti-actin antibodies.

The reaction of spontaneously occurring human anti-human antibodies and experimentally produced rabbit anti-actin antibodies was investigated in a solid-phase radioimmunoassay (RIA). Three structurally different in vitro forms of actin monomeric G-actin filamentous F-actin and aggregated denatured actin were used as antigens. Human anti-actin antibodies reacted with F- and G-actin but not with aggregated actin, while rabbit anti-actin antibodies gave a strong reaction with all 3 forms of actin indicating differences in antibody specificities. The results of the anti-actin RIA were compared with those obtained by indirect immunofluorescence (IFL) on cryostat sections of rat stomach. The anti-actin RIA discriminated between patients' sera and control sera in most cases, although the indirect IFL test gave more conclusive results. The seemingly low sensitivity of the anti-actin RIA compared with that of indirect IFL test for detection of human anti-actin antibodies is probably due to favourable antigen distribution in tissue, not available in the solid phase. The anti-actin RIA was able to detect anti-actin antibodies in 8 out of 8 immunized rabbits although only two produced antibodies detectable by indirect IFL. The differences in reactivity between the two methods may depend on the presence of aggregated denatured actin in the antigen preparation used for immunization and exposure of the corresponding antigenic determinants of actin on the solid phase.

Comparison of linear antigenic sites in the envelope proteins of human immunodeficiency virus (HIV) type 2 and type 1.

The occurrence of dominant linear antigenic sites in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2) was evaluated. Twenty-five peptides representing different regions of HIV-2, strain SBL-6669, were synthesized. For comparison the corresponding peptides of HIV-1, strain BRU, were also prepared. The peptides were tested in enzyme-linked immunosorbent assay (ELISA) with human sera from individuals with proven HIV-1 or HIV-2 infection and simian sera from animals infected with HIV-2 or simian immunodeficiency virus of sooty mangabay monkey origin (SIVsm). Four major antigenic regions were identified. Peptides representing parts or the whole V3 (neutralizing loop) region and an additional stretch of amino acids located at the carboxy terminal of this region showed considerable reactivity. This reaction was predominantly type specific, but some heterotypic reactivity was also seen. Peptides representing the carboxy terminal 21 amino acids of the V3 region of the type-related viruses HIV-2 and SIVsm allowed selective identification of strain-specific antibodies. A second major antigenic region was found close to the carboxy terminal end of the large glycoproteins. This region was cross-reactive between the two types. The two additional dominating antigenic regions were located in the amino terminal region of the transmembrane glycoprotein. One region has previously been shown to be a uniquely antigenic type-specific site. The other region was also type-specific, but was identified only in HIV-2, amino acids Glu634-Lys649. Excellent facilities are available for the design of not only type-unique site-specific serological tests but potentially also type-cross-reactive and strain-specific assays.

Analysis of the V3 loop in neutralization-resistant human immunodeficiency virus type 1 variants by direct solid-phase DNA sequencing.

To study the molecular basis for the emergence of human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to neutralization by autologous sera, the DNA sequence of the envelope V3 loop was determined in HIV-1 isolates derived from four patients with primary HIV-1 infection and sequentially thereafter. The gene fragment encoding the V3 loop of gp120 was amplified by a nested polymerase chain reaction (PCR) and subsequently sequenced by a novel solid phase DNA sequencing approach allowing direct sequencing of the viral genome without the need for previous cloning. The results show that all patients have HIV-1 with unique primary sequence of the V3 loop and antibodies to this structure are produced at seroconversion. The structural analysis also demonstrates that the mechanism for virus escape from neutralization in vivo is complex. Thus, in one patient the emergence of neutralization-resistant viruses coincided with the introduction of several amino acid changes in the V3 loop, while in two other patients the V3 loop remained completely unchanged. These findings suggest that an understanding of the interaction between the humoral immune response and HIV-1 may require detailed structural studies of the entire envelope.

Immune responses to pollutant mixtures from indoor sources.

Indoor air pollution occurs as an undesirable consequence of urbanization, energy conservation, indoor bioaerosol contamination, and use of synthetic materials and new technologies, and has become a worldwide concern. It is important to comprehend not only the diversity of pollutant hazards but also to develop novel methods and approaches that establish dose-response relationships, cause-and-effect relationships, and clinical relevance. Coincident with heightened public concern over indoor air pollution and its health consequences, a revolution in immunology has occurred. The immune system is recognized as an essential defensive and homeostatic mechanism. Unfortunately, the immune apparatus is exquisitely sensitive to toxic damage. Equally important, among the disciplines available to assess the health impact of indoor air pollutants, immunology has the capability to provide sensitive and specific tools that may accurately measure relevant clinical effects at tissue, cellular, and molecular levels. Furthermore, exciting new insights into shared communications networks between the immune, endocrine, and central nervous systems may provide future explanations for the myriad human complaints associated with indoor air pollutants.

Amniotic fluid lamellar body count: cost-effective screening for fetal lung maturity.

OBJECTIVE: To create a highly specific cascade testing scheme for fetal lung maturity using the lamellar body count, lecithin/sphingomyelin ratio (L/S), and phosphatidylglycerol. METHODS: A nondedicated hematology analyzer (Sysmex NE 1500, Toa Medical Electronics, Los Angeles, CA) was used to determine the lamellar body counts of 209 unspun amniotic fluid specimens. Maximally specific lamellar body count cutoffs for biochemical maturity and immaturity were determined using receiver operating characteristic curves. Biochemical lung maturity was defined as either a mature L/S ratio or phosphatidylglycerol. Biochemical lung immaturity was defined as both an immature L/S ratio and an immature phosphatidylglycerol. RESULTS: A lamellar body count of less than 8000 (n = 17) was 100% specific for biochemical lung immaturity (positive predictive value = 100%, negative predictive value = 86%). A lamellar body count of greater than 32,000 was 98% specific for biochemical lung maturity (positive predictive value = 99%, negative predictive value = 63%). CONCLUSION: Testing only specimens where the lamellar body count was greater than 8000 and less than or equal to 32,000 for the L/S ratio and phosphatidylglycerol would preclude the need for 76% of all L/S and phosphatidylglycerol assays. Because the lamellar body count is quick, simple, and universally available, it could serve as an extremely cost-effective screening test for fetal lung maturity.

The influence of specific antibodies and complement on the motility and the viability of Entamoeba histolytica trophozoites.

The distribution of peroxidase-labeled normal or specific rabbit immunoglobulin (Ig) in Entamoeba histolytica trophozoites was studied by transmission electron microscopy. Small amounts of normal Ig became attached to the cell surface but did not redistribute. Internalized specific Ig was firmly bound to the inner membranes of phagocytosis vacuoles, while aggregates of normal Ig were dispersed over the vacuolar lumen. The distribution of fluorescein isothiocyanate-labeled Ig in living amebae was correlated with the cell motility at different times. Immobilization coincided with the cellular metabolic processing of internalized material. Remobilization occurred when the Ig was degraded and antibodies again bound to the reappearing surface antigens. E. histolytica activated complement by the classical pathway. Fresh guinea pig serum alone did not produce lysis which, however, did occur when it was added together with normal rabbit Ig. Normal rabbit Ig may constitute a complement-fixing substrate and activate complement by the classical pathway. Immobilization of the amebae by cytochalasin B (10 micrograms/ml) increased the susceptibility to cytolytic effects of specific antibodies or complement, or both. Pharmacological inhibition of cell motility might augment the immunological defence of the host against amebic infection.

Antigenicity of Entamoeba histolytica strain nih 200: a survey of clinically relevant antigenic components.

The molecular weights of the major polypeptides of Entamoeba histolytica NIH 200 soluble whole extract, and its five fractions eluted from a Sephadex G-200 column, were in the range 150,000-9,000 daltons according to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of SDS-PAGE analysis of 125I surface-labeled amebae revealed more than 12 bands, eight of which corresponded to those recovered in soluble extract. Considering the pronounced phagocytosis and the rapid cell membrane turnover of the ameba, it seems likely that the same antigenic components would be present both on and within the ameba at different stages. The first four gel-filtered fractions were immunogenic and showed non-identical precipitates with sera from 12 Swedish and 12 Ethiopian patients with verified amebic disease. Seven polypeptides in the whole extract reacted with human anti-ameba IgG covalently bound to CNBr activated Sepharose 4B. Immune replica (Western blotting) of SDS-PAGE of whole extract produced by human anti-ameba antisera demonstrated eight bands. The response to the different amebic antigens was not correlated to clinical symptoms, duration of illness or origin of ameba strain. Thus the present results did not support the use of purified fractions rather than the whole soluble extract for diagnostic tests.

Detection of antigen in immune precipitates by silver staining of SDS-polyacrylamide gels.

The silver staining technique of Merril et al. (1981) was used to identify antigens separated by SDS-PAGE after dissociation of immune complexes purified by absorption with staphylococcus A protein. The five and four dominating structural components of measles and RS virus (Mr range 28,000 to 79,000) respectively precipitated with monoclonal antibodies were readily identified, except when the Mr of the virus-specific antigen was in the same range as immunoglobulin heavy and light chains. Specific antigens were identified less effectively after precipitation with polyclonal sera because of a larger background of stainable proteins. This method, which combines the sensitivity and high resolution of silver staining and the specificity of immune complexing with monoclonal antibodies, is rapid and inexpensive.

A new method for measuring D-dimer using immunoturbidometry: a study of 255 patients with suspected pulmonary embolism and deep vein thrombosis.

D-Dimer testing has been suggested as a non-invasive method for the exclusion of pulmonary embolism (PE) and deep vein thrombosis (DVT). In this study, we compared a new method, the Miniquant D-dimer (Biopool International, Ventura, California, USA) to other previously validated D-dimer methods used for the purpose. Patients who were undergoing a definitive diagnostic study for thromboembolism had a blood sample drawn at that time. A whole-blood D-dimer (SimpliRed; Agen Biomedical Ltd, Brisbane, Australia) test was performed, and residual plasma was frozen and later analyzed using two enzyme-linked immunosorbent assay (ELISA) methods (D-dimer Gold; Agen, and Asserachrome D-Di; Stago International, Parsippany, New Jersey, USA) and the Miniquant D-dimer. Once all samples were analyzed, the correlation and accuracy of the Miniquant was compared with the ELISA method using Spearman's regression and Dunn's multiple paired comparison. All D-dimer methods were compared with radiographic studies. The data was analyzed collectively and segregated into in-patient (n = 112) and out-patient (n = 143) populations. The Miniquant D-dimer sensitivity, specificity and negative predictive value (NPV) for all patients were 95, 21, and 94% for DVT, and 100, 26, and 100% for PE. This new D-dimer method demonstrates acceptable sensitivity in patients with PE and DVT and, based on the high NPV of this method, it can be used for the exclusion of thromboembolism.

Transfusion-associated microchimerism.

Blood transfusion is a newly recognized cause of microchimerism, the stable persistence of a minor population of allogeneic cells. Relatively recent advances in polymerase chain reaction technology have spawned new information about the frequency and aetiology of transfusion-associated microchimerism (TA-MC). Although conceptually related to fetal-maternal microchimerism, TA-MC is a distinct and separate entity. Evidence of TA-MC has been strongest among patients with severe traumatic injuries who receive relatively fresh blood products shortly after an episode of massive haemorrhage. The presence of a focal deficit in the cellular immunologic repertoire prior to transfusion that happens to match a blood donor's human leucocyte antigen type also appears to be an important predisposing factor. TA-MC seems to be common (affecting approximately 10% of transfused injured patients), enduring (lasting years to decades) and pronounced (involving up to 5% of circulating leucocytes and multiple immunophenotypic lineages suggestive of haematopoietic engraftment). Further study of this topic may reveal important information regarding potential clinical consequences of TA-MC, as well as basic haematologic and immunologic processes.

Purification of hepatitis B surface antigen by affinity chromatography.

Hepatitis B surface antigen (HBsAg), was purified from plasma by affinity chromatography on matrix-bound sulphated carbohydrates such as heparin or dextran sulphate. Further purification by precipitation with polyethylene glycol (PEG) 6000 resulted in a highly purified HBsAg preparation. The overall recovery amounted to about 70% of the total antigen content of the starting plasma. Electron microscopic data revealed mainly 22 nm spherical particles accompanied by few or no filaments. The process is simple, rapid and lends itself readily to large-scale applications.

Further characterization of the Ca2+-dependent F-actin-depolymerizing protein of human serum.

The F-actin-depolymerizing factor (ADF) of human serum was purified and identified as a 93000-daltons protein. The concentration of ADF was calculated to 120-150 micrograms/ml in four normal sera and about half as much in three sera from leukemia patients treated with cytostatic drugs. In the presence of Ca2+ ADF shortened actin filaments into fragments, the size of which was correlated to the actin: ADF molar ratio, as judged by electron microscopy. The severing of F-actin was not necessarily followed by an increase in the quantities of monomeric actin, as determined by a DNase I inhibition assay and a sedimentation assay. The findings indicated that ADF shortens filamentous actin by breaking bonds between adjacent actin molecules thereby forming stable ADF-actin complexes, without a monomeric net release. The effect of ADF on F-actin was rapid and was reversed upon chelation of Ca2+. ADF cross-reacted immunologically and exhibited similarity in reaction mechanism with gelsolin, the Ca2+-dependent F-actin-severing protein from macrophages. This implies that the proteins are both structurally and functionally related. The physiological role of ADF may be to handle actin released at cell destruction, probably by forming ADF-G-actin 1:1 complexes thereby preventing formation of actin filaments.

Two neutralizing domains in the V3 region in the envelope glycoprotein gp125 of HIV type 2.

The purposes of this study were to map the targets for neutralizing Abs in the HIV-2 glycoproteins with particular emphasis on the role of the V3 region. Sera obtained from guinea pig immunized with peptides representing five immunogenic regions of the envelope proteins were used in cross-neutralization experiments with nine HIV-2 isolates. Broad cross-neutralizing activity was elicited by immunization with two peptides representing the central and COOH-terminal portions of the HIV-2 V3 loop. Murine mAbs were established from animals immunized with two corresponding overlapping peptides. Six mAbs showed neutralizing activity against the homologous virus isolate SBL-6669. Peptide absorption experiments were performed to define the target regions for human neutralizing Abs in the HIV-2 envelope glycoproteins. A significant blocking of neutralizing activity of five HIV-2 Ab-positive sera was seen in the presence of two peptides corresponding to the V3 region. Two overlapping deletion sets of peptides, representing amino acids Ser311 and Arg337, were used to identify the role of individual HIV-2 V3 amino acids in the binding of polyclonal and mAbs. Two distinct antigenic sites were identified in this region, the first corresponding to a region including the conserved motif Phe-His-Ser (amino acid 315-317) and the second in proximity of the COOH-terminal cysteine Trp-Cys-Arg (amino acid 329-331). Potentially these two sites can interact to represent a single discontinuous antigenic site. Taken together, these results indicate that V3 is an important neutralizing domain of HIV-2.

Protection against canine distemper virus in dogs after immunization with isolated fusion protein.

Canine distemper virus attachment (hemagglutinin [H] equivalent) and fusion (F) antigens were purified by affinity chromatography with monoclonal antibodies. The purified antigens were used to immunize groups of three dogs. Radioimmune precipitation assays with sera from these animals showed that the F antigen preparation was pure and induced only an F polypeptide-specific antibody response but that the H antigen preparation had a slight contamination by the F antigen. Immunized animals were challenged with virulent canine distemper virus. Two animals in each group developed pronounced humoral and cellular immune responses after challenge. Among these infected animals, only the dogs immunized with H antigen developed symptoms, albeit mild. In contrast, three nonimmunized control animals developed severe disease, with a fatal outcome in two cases. The complete resistance against challenge in two dogs was interpreted to reflect in one case anti-F immunity and in the other case most likely a high level of anti-H immunity. It is suggested that the F antigen may be of particular interest for the development of morbillivirus and possibly other paramyxovirus subunit or synthetic vaccines, because it can induce immunity capable of blocking virus infection and in situations of virus replication prevent the emergence of symptoms.

F-Actin-depolymerizing activity of human serum.

Non-heated human and animal sera contain a factor which exhibited an inhibiting activity on the staining of actin-containing structures by anti-actin antibodies in indirect immunofluorescence experiments. The presence of this factor lowered the viscosity of F-actin preparations and caused, as studied by electron-microscopy, a depolymerization of F-actin filaments as well as inhibition of filament formation of G-actin. The factor was, after its reaction with F-actin, liberated seemingly unaffected, indicating an enzymatic activity. The factor tentatively termed 'F-actin depolymerizing factor' was heat-sensitive and trypsin sensitive but resisted reduction. It was Ca2+ dependent and the staining inhibiting reaction was faster at 30 degrees C and 37 degrees C than at lower temperatures. Gel filtration experiments on Sephadex G-200 suggested a molecular size of the actin depolymerizing factor slightly higher than that of albumin. The electrophoretic mobility was that of gamma 2 globulin. The physiological role of the factor might be to prevent the presence of F-actin filaments within the circulation.

Purification, morphology and antigenic characterization of measles virus envelope components.

Measles virus haemagglutinin (H), fusion (F) and matrix (M) components were purified by affinity chromatography using monoclonal antibodies coupled to CNBr-activated Sepharose. H and M proteins were purified to homogeneity as determined by polyacrylamide gel electrophoresis by a single cycle of adsorption-desorption. The corresponding purification of the F protein required two cycles of adsorption-desorption. After the second adsorption an extra wash with 1 M-guanidine-HCl was employed to remove the contaminating cellular actin. Electron microscopic examination of the purified envelope proteins showed that, at pH 6.0, the H peplomers had a truncated conical shape (width 6.5 to 4 nm, length 16 nm), and the F peplomers had a club-like shape (dimensions of the oval head 6 X 9 nm, length 15 nm). Lengths of both peplomers were measured excluding their undiscernible hydrophobic part. The M component at pH 3.0 appeared as a rounded particle (diam. 8 nm, central accumulation of contrast 1.5 nm) suggested to include four to six M polypeptides. Rabbit hyperimmune sera were prepared against all three purified envelope components. These sera reacted only with the homologous antigen in radioimmunoprecipitation assays. Both antisera against the H and F components neutralized the virus and blocked virus-specific haemolysis, but only anti-H serum inhibited haemagglutination.