Iodine-129, Iodine-127 and Cesium-137 in seawater from the North Sea and the Baltic Sea.

In this study, new data are presented for the iodine isotopes (127I, 129I and their isotopic ratios) and Cesium (137Cs) in water samples of the North Sea and the Baltic Sea in 2005 and 2009. This study supplements and extends the study of Michel et al. (2012). Iodine isotopes were separated from their matrix by using an anion exchange method and were determined by applying ICP-MS and AMS. 137Cs in seawater was determined after cesium ion exchange procedure enrichment by gamma-spectrometry. The concentrations of 127I in seawater of the North and Baltic Sea are fairly constant in each Sea with averages of (44 ± 2) and (21 ± 1) ng g-1, respectively, depending on the salinity. However, large variations of 129I concentrations in these areas were detected, which decreased along the French, Belgian, Dutch, German, and Danish shores. 129I/127I isotope ratios in the Baltic Sea are about 10 times lower than in the North Sea in 2009. The highest isotopic ratios (2.7 × 10-6) was detected in the English Channel east of the nuclear reprocessing plant at Cap de la Hague. The results confirm the result of our early study that the sources of 129I in the North Sea are primarily the nuclear reprocessing facilities at Sellafield (UK) and La Hague (F), and that in the Baltic Sea the inflow of water from North Sea through the Danish Straits dominates the occurrence of 129I. In 2009, the activity concentration of 137Cs was at least 6 times higher in the Baltic Sea (37 Bq m-3) than in the North Sea (5.9 Bq m-3), due to release of 137Cs from sediments in the Baltic Sea, which were contaminated by the Chernobyl accident and - to a minor degree - the atmospheric explosions of atomic bombs. The results are discussed by comparing the results of our previous work and the current study demonstrating the continuing disequilibrium of 129I/127I atomic ratio in the environmental compartments.

Retrospective dosimetry of Iodine-131 exposures using Iodine-129 and Caesium-137 inventories in soils--A critical evaluation of the consequences of the Chernobyl accident in parts of Northern Ukraine.

The radiation exposure of thyroid glands due to (131)I as a consequence of the Chernobyl accident was investigated retrospectively based on (129)I and (137)Cs inventories in soils in Northern Ukraine. To this end, soil samples from 60 settlements were investigated for (129)I, (127)I, and (137)Cs by AMS, ICP-MS and gamma-spectrometry, respectively. Sampling was performed between 2004 und 2007. In those parts of Northern Ukraine investigated here the (129)I and (137)Cs inventories are well correlated, the variability of the individual (129)I/(137)Cs ratios being, however, high. Both the (129)I and (137)Cs inventories in the individual 5 samples for each settlement allowed estimating the uncertainties of the inventories due to the variability of the radionuclide deposition and consequently of the retrospective dosimetry. Thyroid equivalent doses were calculated from the (129)I and the (137)Cs inventories using aggregated dose coefficients for 5-year old and 10-year-old children as well as for adults. The highest thyroid equivalent doses (calculated from (129)I inventories) were calculated for Wladimirowka with 30 Gy for 5-years-old children and 7 Gy for adults. In 35 settlements of contamination zone II the geometric mean of the thyroid equivalent doses was 2.0 Gy for 5-years-old children with a geometric standard deviation (GSD) of 3.0. For adults the geometric mean was 0.47 Gy also with a GSD of 3.0. In more than 25 settlements of contamination zone III the geometric means were 0.82 Gy for 5-years old children with a GSD of 1.8 and 0.21 Gy for adults (GSD 1.8). For 45 settlements, the results of the retrospective dosimetry could be compared with thyroid equivalent doses calculated using time-integrated (131)I activities of thyroids which were measured in 1986. Thus, a critical evaluation of the results was possible which demonstrated the general feasibility of the method, but also the associated uncertainties and limitations.

Iodine-129, iodine-127 and caesium-137 in the environment: soils from Germany and Chile.

Soil profiles from Bavaria in southern Germany and from Chile were analysed for (129)I by accelerator mass spectrometry (AMS), for (127)I by inductively coupled plasma mass spectrometry (ICP-MS), and for (137)Cs by gamma-spectrometry. The mean deposition density of (137)Cs in soils from Bavaria was (41×1.5(±1)) kBq m(-2) (geometric mean and geometric standard deviation), originating mostly from the Chernobyl fall-out. The deposition density of (129)I in these soils was (109×1.5(±1)) mBq m(-2). The dominant sources of (129)I in Bavaria are, however, the reprocessing plants La Hague and Sellafield and not the Chernobyl fall-out. The (129)I/(127)I isotopic ratios of the Bavarian soils were between 10(-7) and 10(-10), i.e. 10(2)-10(5) times higher than the ratios observed for the samples from Chile. The (129)I integral deposition densities in Chile, Easter Island and Antarctica were between 0.3 mBq m(-2) and 2 mBq m(-2). In these soils, the observed (129)I/(127)I ratios were about 10(-12). The soils from Chile allow the determination of the (129)I fall-out from the atmospheric nuclear weapons explosions undisturbed from contaminations due to releases from reprocessing plants. An upper limit of the integral (129)I deposition density of the atmospheric nuclear weapons explosions on the Southern Hemisphere (27°S) is about 1 mBq m(-2). Finally, the dependence of the migration behaviour of (137)Cs, (127)I and of (129)I on the soil properties is discussed. It turns out that there is a distinctly different behaviour of (127)I, (129)I, and (137)Cs in the soils exhibiting different sorption mechanisms for old and recent iodine as well as for (137)Cs.

Iodine-129 and iodine-127 in European seawaters and in precipitation from Northern Germany.

In order to obtain a comprehensive survey on the consequences of the marine (129)I discharges from the European reprocessing plants La Hague and Sellafield, the distribution of (129)I and (127)I in surface waters of the North Sea, the English Channel, the Irish Sea, and the Northeast Atlantic was studied using accelerator mass spectrometry for (129)I and ICP-MS for (127)I. Samples of seawater were taken in the German Bight in May, September, and November 2005 and in the entire North Sea and the English Channel in August 2005. Further samples were obtained from the Irish Sea in June and August 2006 and from Arctic waters between Spitsbergen and Southern Norway in September 2005. (129)I is a conservative tracer in seawater. The concentrations of (127)I are relatively constant with exceptions of coastal areas with high biological activity and of areas influenced by influx from rivers and the Baltic Sea. The variability of the (129)I/(127)I isotopic ratios is exclusively determined by admixture of (129)I released from the reprocessing facilities Sellafield and La Hague to the seawater. The (129)I/(127)I ratios were between 4 × 10(-9)and 3 × 10(-6): at least 3 orders of magnitude higher than the natural equilibrium isotopic ratio 1.5 × 10(-12). (129)I/(127)I ratios of a few times 10(-10) were only found in seawater from the Indian Ocean and from the Pacific at Hawaii. Comparison of the results obtained for seawater with those of a measurement of airborne iodine species and with iodine isotopes in precipitation in Northern Germany demonstrates the transfer of (129)I and (127)I from the sea into the atmosphere and the dominating role of the marine discharges for the atmospheric fallout of (129)I in Western Europe. The results are discussed with the goal to estimate the relevance of the marine discharges for the contamination of the continental areas.

Phase transition of displacive type in PbZr(0.94)Ti(0.06)O(3).

In this paper the electrostrictive properties above T(εmax), represented by the field-related M and polarization-related Q coefficients, have been reported for Pb(Zr(1-x)Ti(x))O(3) ceramics with x = 0.03-0.10. Among M(11)(T) and Q(11)(T) dependences, those found for Pb(Zr(0.94)Ti(0.06))O(3) have been clearly distinguished. In this case, the Q(11)(T) dependence is linear in the whole temperature range above T(εmax). Experimental and theoretical analysis of the M(11)(T) dependences has shown that the phase transition to the ferroelectric phase in Pb(Zr(0.94)Ti(0.06))O(3) ceramics seems to be of the displacive type.

Continuous subcutaneous insulin infusion leads to immediate, stable and long-term changes in metabolic control.

BACKGROUND: Evaluations of continuous subcutaneous insulin infusion (CSII) usually focus on one pre- and one post-CSII measurement to assess metabolic therapy outcome. AIM: Extending this research, the aim of the present study was to provide a more fine-grained analysis of achieved glycaemic control. METHODS: In 52 patients with type 1 diabetes (mean age of 37.85 years at CSII begin; s.d. +/- 12.41), haemoglobin A(1c) (HbA(1c)) levels were assessed every 3 months over a period of 5 years (1 year before and 4 years after the introduction of CSII). Mixed models were utilized to describe changes in glycaemic control. RESULTS: The pre-post course showed that already in the first quarter, a statistically significant lower HbA(1c) level was obtained [7.30%, in contrast to 8.21% at the last quarter with intensified conventional therapy (ICT)]. In the following 15 quarters, the mean HbA(1c) levels remained constantly lower than that with ICT. Overall, the aggregated mean HbA(1c) level of patients with CSII therapy was 7.19%, in contrast to 8.08% with ICT; thus, an overall decrease by 11% was achieved. In addition, individual differences in blood glucose level and age of diabetes onset as a predictor for therapy success were analysed. CONCLUSIONS: The data show an immediate, stable and long-term effect of CSII on HbA(1c). In addition, a significant relationship between metabolic control and age of diabetes onset was found, as well as a reduction of variance in HbA(1c) levels between subjects after change to CSII.

Type-specific epitopes targeted by monoclonal antibodies with exceptionally potent neutralizing activities for selected strains of human immunodeficiency virus type 1 map to a common region of the V2 domain of gp120 and differ only at single positions from the clade B consensus sequence.

Only a few monoclonal antibodies (MAbs) have been isolated that recognize conserved sites in human immunodeficiency virus type 1 (HIV-1) Env proteins and possess broad neutralizing activities. Other MAbs directed against targets in various domains of Env have been described that are strongly neutralizing, but they possess limited breadth. One such MAb, 2909, possesses a uniquely potent neutralizing activity specific for a quaternary epitope on SF162 Env that requires the presence of both the V2 and the V3 domains. We now show that replacement of the SF162 V3 sequence with consensus V3 sequences of multiple subtypes led to attenuated but still potent neutralization by 2909 and that the main determinants for the type specificity of 2909 reside in the V2 domain. A substitution at position 160 completely eliminated 2909 reactivity, and mutations at position 167 either attenuated or potentiated neutralization by this antibody. Different substitutions at the same positions in V2 were previously shown to introduce epitopes recognized by MAbs 10/76b and C108g and to allow potent neutralization by these MAbs. Two substitutions at key positions in the V2 domain of JR-FL Env also allowed potent expression of the 2909 epitope, and single substitutions in YU2 V2 were sufficient for expression of the 2909, C108g, and 10/76b epitopes. These results demonstrate that the minimal epitopes for 2909, C108g, and 10/76b differed from that of the clade B consensus sequence only at single positions and suggest that all three MAbs recognize distinct variants of a relatively conserved sequence in V2 that is a particularly sensitive mediator of HIV-1 neutralization.

Factors determining the breadth and potency of neutralization by V3-specific human monoclonal antibodies derived from subjects infected with clade A or clade B strains of human immunodeficiency virus type 1.

The neutralizing activities of anti-V3 antibodies for HIV-1 isolates is affected both by sequence variation within V3 and by epitope masking by the V1/V2 domain. To analyze the relative contribution of V3 sequence variation, chimeric Env genes that contained consensus V3 sequences from seven HIV-1 subtypes in the neutralization-sensitive SF162 Env backbone were constructed. Resulting viral pseudotypes were tested for neutralization by 15 anti-V3 MAbs isolated from humans infected with viruses of either subtype B (anti-V3(B) MAbs) or subtype A (anti-V3(A) MAbs). Pseudovirions with the subtype B consensus V3 sequence were potently neutralized (IC(50) < 0.006 microg/ml) by all but one of these MAbs, while pseudovirions with V3 subtypes A, C, F, H, AG, and AE were generally neutralized more effectively by anti-V3(A) MAbs than by anti-V3(B) MAbs. A V1/V2-masked Env version of SF162 Env with the consensus B V3 sequence was also neutralized by these MAbs, although with considerably lower potency, while similarly masked chimeras with V3 sequences of subtype A, C, or AG were weakly neutralized by anti-V3(A) MAbs but not by anti-V3(B) MAbs. Mutations in the V1/V2 domain of YU-2 Env increased the sensitivity of this highly resistant Env to a pool of anti-V3(B) MAbs several thousand-fold. These results demonstrated (i) the exceptional sensitivity of representative V3 domains of multiple subtypes to neutralization in the absence of epitope masking, (ii) the broader neutralizing activity of anti-V3(A) MAbs for viruses containing diverse V3 sequences, and (iii) the generality and dominant effect of V1/V2 masking on restriction of V3-mediated neutralization.

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120.

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120.

Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.

Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6.

Human immunodeficiency virus-type I (HIV-1) infection elicits antibodies (Abs) directed against several regions of the gp120 and gp41 envelope glycoproteins. Many of these Abs are able to neutralize T-cell-line-adapted strains (TCLA) of HIV-1, but only a few effectively neutralize primary HIV-1 isolates. The nature of HIV-1 neutralization has been carefully studied using human monoclonal Abs (MAbs), and the ability of such MAbs to act in synergy to neutralize HIV-1 has also been extensively studied. However, most synergy studies have been conducted using TCLA strains. To determine the nature of Ab interaction in HIV-1 primary isolate neutralization, a panel of 12 anti-HIV-1 human immunoglobulin G (IgG) MAbs, specific for epitopes in gp120 and gp41, were used. Initial tests showed that six of these MAbs, as well as sCD4, used individually, were able to neutralize the dualtropic primary isolate HIV-1(89.6); MAbs giving significant neutralization at 2 to 10 microg/ml included 2F5 (anti-gp41), 50-69 (anti-gp41), IgG1b12 (anti-gp120(CD4bd)), 447-52D (anti-gp120(V3)), 2G12 (anti-gp120), and 670-D (anti-gp120(C5)). For studies of reagent interaction, 16 binary combinations of reagents were tested for their ability to neutralize HIV-1(89.6). Reagent combinations tested included one neutralizing MAb with sCD4, six pairs consisting of two neutralizing MAbs, and nine pairs consisting of one neutralizing MAb with another non-neutralizing MAb. To assess the interaction of the latter type of combination, a new mathematical treatment of reagent interaction was developed since previously used methods could be used only when both reagents neutralize. Synergy was noted between sCD4 and a neutralizing anti-gp120(V3) MAb. Antagonism was noted between two pairs of anti-gp41 MAbs (one neutralizing and one non-neutralizing). All of the other 13 pairs of MAbs tested displayed only additive effects. These studies suggest that Abs rarely act in synergy to neutralize primary isolate HIV-1(89.6); many anti-HIV-1 Abs act additively to mediate this biological function.

Effect of soluble CD4 on exposure of epitopes on primary, intact, native human immunodeficiency virus type 1 virions of different genetic clades.

We have used a virus-binding assay to examine conformational changes that occur when soluble CD4 (sCD4) binds to the surface of intact, native, primary human immunodeficiency virus type 1 virions. The isolates examined belong to seven genetic clades (A to H) and are representative of syncytium-inducing and non-syncytium-inducing phenotypes. Conformational changes in epitopes in the C2, V2, V3, C5, and CD4 binding domain (CD4bd) of gp120 and the cluster I and II regions of gp41 of these viruses were examined using human monoclonal antibodies that are directed at these regions. The studies revealed that sCD4 binding causes a marked increase in exposure of epitopes in the V3 loop, irrespective of the clade or the phenotype of the virus. Sporadic increases in exposure were observed in some epitopes in the V2 region, while no changes were observed in the C2, C5, or CD4bd of gp120 or the cluster I and II regions of gp41.

Immunoreactivity of intact virions of human immunodeficiency virus type 1 (HIV-1) reveals the existence of fewer HIV-1 immunotypes than genotypes.

In order to protect against organisms that exhibit significant genetic variation, polyvalent vaccines are needed. Given the extreme variability of human immunodeficiency virus type 1 (HIV-1), it is probable that a polyvalent vaccine will also be needed for protection from this virus. However, to understand how to construct a polyvalent vaccine, serotypes or immunotypes of HIV must be identified. In the present study, we have examined the immunologic relatedness of intact, native HIV-1 primary isolates of group M, clades A to H, with human monoclonal antibodies (MAbs) directed at epitopes in the V3, C5, and gp41 cluster I regions of the envelope glycoproteins, since these regions are well exposed on the virion surface. Multivariate analysis of the binding data revealed three immunotypes of HIV-1 and five MAb groups useful for immunotyping of the viruses. The analysis revealed that there are fewer immunotypes than genotypes of HIV and that clustering of the isolates did not correlate with either genotypes, coreceptor usage (CCR5 and CXCR4), or geographic origin of the isolates. Further analysis revealed distinct MAb groups that bound preferentially to HIV-1 isolates belonging to particular immunotypes or that bound to all three immunotypes; this demonstrates that viral immunotypes identified by mathematical analysis are indeed defined by their immunologic characteristics. In summary, these results indicate (i) that HIV-1 immunotypes can be defined, (ii) that constellations of epitopes that are conserved among isolates belonging to each individual HIV-1 immunotype exist and that these distinguish each of the immunotypes, and (iii) that there are also epitopes that are routinely shared by all immunotypes.

Polymerase chain reaction-based assay for antibody-mediated neutralization of HIV-1 reveals a population of nonneutralized virus undetected by conventional p24 assay.

To be successful with strategies involving passive immunization or the generation of neutralizing antibodies against HIV, it is crucial that we improve our understanding of the process of antibody-mediated HIV neutralization. We have used a neutralization assay based on polymerase chain reaction (PCR) that is more rapid and sensitive than the conventional p24 neutralization assay based on enzyme-linked immunosorbent assay (ELISA). PCR assays permit measurement of the number of infectious events and can detect small amounts of HIV-1 only a few days postinfection. In these studies, the human anti-V3 monoclonal antibody 694/98-D was used to neutralize the infectivity of the laboratory isolate HIVIIIB for CEM-SS cells. 8E5/LAV cells, which contain a single integrated copy of proviral DNA per cell, served as a standard to determine the amount of HIV-1 copies in infected CEM-SS cells. Evaluation of antibody-mediated neutralization was possible at 2 to 3 days postinfection, at a time when p24 readouts were not conclusive. We achieved >95% neutralization of HIVIIIB, and of its molecular clone HXB2, using the monoclonal antibody 694/98-D. This degree of neutralization is probably highly significant in vivo. Nevertheless, a small amount of both HIVIIIB and HXB2 ( approximately 5%) escapes neutralization and can consistently be detected after a few days by this sensitive assay. Experiments with different anti-HIV monoclonal antibodies and viruses showed that the assay could be applied to anti-V3 as well as anti-CD4 binding domain antibodies as well as HIV laboratory strains or primary isolates.

Gamma heavy chain disease in man: independent structural abnormalities and reduced transcription of a functionally rearranged lambda L-chain gene result in the absence of L-chains.

Human Ig heavy chain diseases of the alpha and gamma classes are characterized by the absence of light chain production as well as the disease-defining abnormalities in the heavy chain protein. Prior studies have suggested concomitant structural defects in productively rearranged L-chain genes as the reason for the absent L-chain proteins. We have found that the single rearranged lambda L-chain gene in the OMM heavy chain disease (HCD) cell line has a mutation in the splice donor site at the 3' end of the J exon, resulting in direct splicing of the 3' end of the leader to the acceptor site of the constant region. The cells contain an mRNA consisting of the leader-coding region joined directly to the constant region. The V-region exon is skipped and the shortened mRNA is translated into a truncated protein containing no V-region amino acids. We have also noted that, in contrast to most normal and neoplastic Ig-producing cells, the OMM cells produce an excess of heavy to light chain mRNA as well as protein. The excess is independent of the structural gene abnormality and is due to a low level of L-chain transcription, which can be increased by fusing the HCD cell to the murine myeloma cell line NS-1 or transfecting the defective OMM L-chain gene into a murine plasma cell. The latter data suggest that the OMM cells either lack a transcription factor present in mature plasma cells or have a functional repressor of L-chain transcription.

Conserved and exposed epitopes on intact, native, primary human immunodeficiency virus type 1 virions of group M.

We have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates.

Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells appears to be triggered when two heptad repeat regions in the ectodomain of gp41 associate, converting the prefusogenic form of gp41 to a fusogenic form. Peptides from these two heptad repeat regions, designated N51 and C43, form a coiled coil consisting of an alpha-helical trimer of heterodimers which approximates the core of the fusogenic form of gp41. To understand the antigenic structures of gp41 in these two configurations, and to examine the specificity of anti-gp41 antibodies produced by HIV-1-infected individuals, human anti-gp41 monoclonal antibodies (MAbs) were tested for their reactivity against N51, C43, and the complex formed by these peptides. Of 11 MAbs, 7 reacted with the complex but with neither of the parent peptides. These MAbs reacted optimally with the N51-C43 complex prepared at a 1:1 ratio and appeared to recognize the fusogenic form of gp41 in which the two heptad repeat regions are associated to form the coiled coil. The existence of antibodies from HIV-infected humans that exclusively recognize the N51-C43 complex constitutes the first proof that the coiled-coil conformation of gp41 exists in vivo and is immunogenic. Two of the 11 MAbs were specific for the hydrophilic loop region of gp41 and failed to react with either peptide alone or with the peptide complex, while the remaining 2 MAbs reacted with peptide C43. One of these two latter MAbs, 98-6, also reacted well with the equimolar N51-C43 complex, while reactivity with C43 by the other MAb, 2F5, was inhibited by even small amounts of N51, suggesting that the interaction of these peptides occludes or disrupts the epitope recognized by MAb 2F5. MAbs 98-6 and 2F5 are also unusual among the MAbs tested in their ability to neutralize multiple primary HIV isolates, although 2F5 displays more broad and potent activity. The data suggest that anti-gp41 neutralizing activity is associated with specificity for a region in C43 which participates in complex formation with N51.

Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120.

HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120.

Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins.

To understand the differential expression of epitopes on monomeric and oligomeric forms of the envelope glycoproteins, nine human monoclonal antibodies (mAbs) were derived from the cells of human immunodeficiency virus-infected subjects by selection with soluble oligomeric gp140 (o.140). These nine mAbs and 12 human mAbs selected with V3 peptides, viral lysates, and rgp120, specific for the V2, V3, C5, CD4-binding domain (CD4bd), and gp41, were tested in a binding assay to compare the exposure of these regions on monomeric gp120 or gp41 and on o.140. None of the 21 mAbs were oligomer specific. However, mAbs to V3 and CD4bd were "oligomer sensitive," whereas mAbs to V2 and the distal epitope of C5 tended to be "monomer sensitive" (i.e., to react better with the oligomer or monomer, respectively). The majority of anti-gp41 mAbs reacted similarly with monomer and oligomer. Although the uncleaved o.140 used in this study differs from the cleaved gp120/41 oligomer found on the native virus particle, these results suggest that new epitopes are not introduced by oligomerization of viral envelope proteins, that such oligomer-specific epitopes, if they exist, are not highly immunogenic, and/or that they are not efficiently selected using soluble o.140.

A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex.

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.