Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies.

Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity.

On the prereceptor-engaged HIV-1 envelope glycoprotein (Env) spike, epitope access by the membrane-proximal external region (MPER)-directed broadly neutralizing antibodies 2F5 and 4E10 remains unresolved. Data on binding to cell surface Env and entry data using primary isolates suggest inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and slow kinetics of shedding induced by 2F5 and 4E10 indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed in their antibody-bound state (or at least sampled) prior to receptor/coreceptor engagement or if receptor interactions both expose and form the MPER epitopes, presumably in the putative prefusion transitional intermediate. Here, we performed antibody-virus "washout experiments" using both lab-adapted and a panel of clade B primary isolates to analyze MPER accessibility. The neutralization activity of 2F5 and 4E10 against lab-adapted viruses and sensitive and moderately resistant viruses was largely unaffected by relatively rapid antibody-virus washing, suggesting direct interaction with the "static" spike. However, for more neutralization-resistant viruses, the 2F5 and 4E10 antibodies could neutralize only under the "no antibody-virus wash" conditions, implying that the MPER epitopes were not accessible prior to receptor engagement. Accessibility in the washout conditions could be precisely predicted by the relative resistance to neutralization in a standard neutralization format. These data are consistent with a model in which the local MPER antibody epitope conformations may be sampled on the native spike but are occluded to antibody by local steric or distal quaternary constraints adopted by highly resistant HIV-1 isolates.

Transthoracic echocardiographic abnormalities in asymptomatic diabetic patients: association with microalbuminuria and silent coronary artery disease.

AIMS: This study aimed to assess, on routine echocardiography, cardiac left ventricular (LV) disorders, their determinants and their role in the screening process of silent myocardial ischaemia (SMI) in asymptomatic diabetic patients. METHODS: A total of 586 asymptomatic diabetic patients with one or more additional cardiovascular risk factors, but no history of heart failure or myocardial infarction, prospectively underwent rest echocardiography and myocardial scintigraphy. Those with SMI (abnormal scintigraphy) were subsequently screened for angiographic coronary artery disease (CAD). RESULTS: LV hypertrophy, LV dilatation, systolic dysfunction and hypokinesia were found in 33.6, 8.6, 3.2 and 6.1%, respectively, of the study population. SMI was found in 156 (26.6%) patients, 55 of whom had silent CAD. On multivariate analysis, age (OR: 1.03 [1.00-1.05], P=0.02), microalbuminuria (OR: 2.2 [1.4-3.2], P<0.0001) and silent CAD (OR: 2.4 [1.3-4.6], P=0.007) were predictive of LV hypertrophy. Creatinine clearance (OR: 0.97 [0.96-0.99], P=0.002) and silent CAD (OR: 3.7 [1.3-10.0]) were associated with LV dilatation. LV systolic dysfunction was associated with microalbuminuria (OR: 3.8 [1.3-11.4], P=0.02) and silent CAD (OR: 3.8 [1.1-12.6], P=0.03). Hypokinesia was associated with retinopathy (OR: 2.4 [1.1-5.4], P=0.04), microalbuminuria (OR: 2.3 [1.1-5.0], P=0.04) and LV dilatation (OR: 3.0 [1.1-8.1], P=0.03). In patients with SMI, the positive predictive value of LV hypertrophy associated with another echocardiographic abnormality (n=19) for CAD was 63.2%. CONCLUSION: LV hypertrophy was found in one-third of asymptomatic diabetic patients, while LV dilatation, systolic dysfunction or hypokinesia was seen in<10%. The main predictors of LV abnormalities were microalbuminuria and silent CAD. The presence of LV hypertrophy with another abnormality should raise the possibility of the presence of silent CAD.

Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies.

Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.

gp120: Biologic aspects of structural features.

HIV-1 particles are decorated with a network of densely arranged envelope spikes on their surface. Each spike is formed of a trimer of heterodimers of the gp120 surface and the gp41 transmembrane glycoproteins. These molecules mediate HIV-1 entry into target cells, initiating the HIV-1 replication cycle. They are a target for entry-blocking drugs and for neutralizing Abs that could contribute to vaccine protection. The crystal structure of the core of gp120 has been recently solved. It reveals the structure of the conserved HIV-1 receptor binding sites and some of the mechanisms evolved by HIV-1 to escape Ab responses. The gp120 consists of three faces. One is largely inaccessible on the native trimer, and two faces are exposed but apparently have low immunogenicity, particularly on primary viruses. We have modeled HIV-1 neutralization by a CD4 binding site monoclonal Ab, and we propose that neutralization takes place by inhibition of the interaction between gp120 and the target cell membrane receptors as a result of steric hindrance. Knowledge of gp120 structure and function should assist in the design of new drugs as well as of an effective vaccine. In the latter case, circumventing the low immunogenicity of the HIV-1 envelope spike is a major challenge.

Antibodies in human infectious disease.

Investigation of human antibody responses to viral pathogens at the molecular level is revealing novel aspects of the interplay of viruses with the humoral immune system. In viral infection, at least two types of human antibody responses exist: a response to mature envelope on virions that is neutralizing and a response to immature forms of envelope (viral debris) that is not. Many pathogens have, to varying degrees, evolved envelopes to minimize antibody responses against epitopes exposed on the virion. In this article, we review recent studies on human immunodeficiency virus type 1, Ebola virus, and respiratory syncytial virus. Prion diseases are diseases of protein conformation. We have generated a large panel of antibodies recognizing the cellular prion protein (PrP(c)), some of which also react with the abnormally folded infectious prion protein (PrP(Sc)). These antibodies are being used to gain insight into both the molecular events leading to the formation of infectious PrP and the physiologic role played by PrP in normal and prion-infected cells.

Neutralizing antibodies have limited effects on the control of established HIV-1 infection in vivo.

Neutralizing antibodies can protect against challenge with HIV-1 in vivo if present at appropriate concentrations at the time of viral challenge, but any role in the control of established infection is unclear. Here, we show that high serum concentrations of neutralizing monoclonal antibodies, either singly or as a cocktail, have little sustained effect on viral load in established HIV-1 infection in hu-PBL-SCID mice. In some instances, virus replication of neutralization-sensitive virus continues even in the presence of high levels of neutralizing antibody. In most instances, neutralization escape occurs in a few days, even from a cocktail of three antibodies that recognize distinct epitopes. The results imply that humoral immunity is unlikely to play a significant role in the control of established HIV-1 infection in humans.

Highly potent RANTES analogues either prevent CCR5-using human immunodeficiency virus type 1 infection in vivo or rapidly select for CXCR4-using variants.

The natural ligands for the CCR5 chemokine receptor, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES (regulated on T-cell activation, normal T-cell expressed and secreted), are known to inhibit human immunodeficiency virus (HIV) entry, and N-terminally modified RANTES analogues are more potent than native RANTES in blocking infection. However, potent CCR5 blocking agents may select for HIV-1 variants that use alternative coreceptors at less than fully inhibitory concentrations. In this study, two N-terminal chemical modifications of RANTES produced by total synthesis, aminooxypentane (AOP)-RANTES[2-68] and N-nonanoyl (NNY)-RANTES[2-68], were tested for their ability to prevent HIV-1 infection and to select for coreceptor switch variants in the human peripheral blood lymphocyte-SCID mouse model. Mice were infected with a CCR5-using HIV-1 isolate that requires only one or two amino acid substitutions to use CXCR4 as a coreceptor. Even though it achieved lower circulating concentrations than AOP-RANTES (75 to 96 pM as opposed to 460 pM under our experimental conditions), NNY-RANTES was more effective in preventing HIV-1 infection. However, in a subset of treated mice, these levels of NNY-RANTES rapidly selected viruses with mutations in the V3 loop of envelope that altered coreceptor usage. These results reinforce the case for using agents that block all significant HIV-1 coreceptors for effective therapy.

Antibody neutralization of HIV-1 and the potential for vaccine design.

Neutralisation by antibody is, for a number of viruses, an in vitro correlate for protection in vivo. For HIV-1 this is controversial. However, the induction of a potent anti-HIV neutralising antibody response remains one of the principal goals in vaccine development. A greater knowledge of the fundamental mechanisms underlying the neutralisation process would help direct research towards suitable vaccine immunogens. The primary determinant of HIV neutralisation appears to be antibody affinity for the trimeric envelope glycoprotein spike on the virion, suggesting that epitope-specific effects are secondary and implying a single, dominant mechanism of neutralisation. Antibody interference with virion attachment to the target cell appears to be a major mechanism of neutralisation by gp120-specific antibodies. This is probably achieved both by antibody-induced dissociation of gp120 from gp41 and by direct inhibition of virus binding to receptor-coreceptor complexes. A gp41-specific antibody neutralises by interfering with post-attachment steps leading to virus membrane fusion. Recent advances in structural analyses of the HIV envelope glycoproteins coupled with data obtained from antibody mapping and neutralisation studies allow a greater understanding of Env function and its inhibition. This in turn should lead to a more rational basis for vaccine design aimed at stimulating highly effective neutralising antibodies.

Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines.

A number of antibodies generated during human respiratory syncytial virus (RSV) infection have been cloned by the phage library approach. Antibodies reactive with an immunodominant epitope on the F glycoprotein of this virus have a high affinity for affinity-purified F antigen. These antibodies, however, have a much lower affinity for mature F glycoprotein on the surface of infected cells and are nonneutralizing. In contrast, a potent neutralizing antibody has a high affinity for mature F protein but a much lower affinity for purified F protein or F protein in viral lysates. The data indicate that at least two F protein immunogens are produced during natural RSV infection: immature F, found in viral lysates, and mature F, found on infected cells or virions. Binding studies with polyclonal human immunoglobulin G suggest that the antibody responses to the two immunogens are of similar magnitudes. Competitive binding studies suggest that overlap between the responses is relatively limited. A mature envelope with an antigenic configuration different from that of the immature envelope has an evolutionary advantage in that the infecting virus is less subject to neutralization by the humoral response to the immature envelope that inevitably arises following lysis of infected cells. Subunit vaccines may be at a disadvantage because they most often resemble immature envelope molecules and ignore this aspect of viral evasion.

Long-term nonprogressive human immunodeficiency virus-1 infection in a kidney allograft recipient.

We report a unique case of a renal transplant patient with a long-term nonprogressive human immunodeficiency virus type-1 (HIV-1) infection and who is asymptomatic despite sustained immunosuppression. Renal function is normal, and HIV infection was probably acquired through blood transfusion before the transplant. Nonprogression may be due either to an effective immune control of HIV replication or to particular genetic aspects of the virus. Several virological investigations were carried out to verify if she is infected with an attenuated virus strain. Results show an unusual combination of high and stable CD4 count, ongoing viral replication and elevated viral loads. Attempts to isolate the virus from plasma were unsuccessful, but isolation was possible from peripheral blood mononuclear cells, and the virus was shown to be non-syncytium-inducing. Sequence analysis of the nef gene revealed no mutation. This exceptional lack of progression of HIV infection under immunosuppressive therapy requires further investigation.

Erratum to "Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design".

Understanding the antibody response in HIV-1 infection is important to vaccine design. We have studied the antibody response to HIV-1 envelope at the molecular level and determined the characteristics of neutralizing and non-neutralizing antibodies. These antibodies were isolated from phage display libraries prepared from long-term seropositive asymptomatic individuals. The HIV-1 envelope is presented to the immune system in several antigenically distinct configurations: unprocessed gp160, gp120 and gp41 subunits and native envelope, each of which may be important in eliciting an antibody response in HIV-1 infection. The antibodies tested characteristically had poor affinities for native envelope as expressed on the surface of virions or infected cells, but had high affinities against non-native forms of HIV-1 envelope (viral debris). An exceptionally potent neutralizing antibody in contrast, bound native envelope with equivalent or somewhat higher affinity than this. This indicates that the antibody response in HIV-1 infection is principally elicited by viral debris rather than virions, and that these antibodies bind and neutralize viruses sub-optimally. Potential vaccines should be designed to elicit responses against native envelope.

Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design.

Understanding the antibody response in HIV-1 infection is important to vaccine design. We have studied the antibody response to HIV-1 envelope at the molecular level and determined the characteristics of neutralizing and non-neutralizing antibodies. These antibodies were isolated from phage display libraries prepared from long-term seropositive asymptomatic individuals. The HIV-1 envelope is presented to the immune system in several antigenically distinct configurations: unprocessed gp160, gp120 and gp41 subunits and native envelope, each of which may be important in eliciting an antibody response in HIV-1 infection. The antibodies tested characteristically had poor affinities for native envelope as expressed on the surface of virions or infected cells, but had high affinities against non-native forms of HIV-1 envelope (viral debris). An exceptionally potent neutralizing antibody in contrast, bound native envelope with equivalent or somewhat higher affinity than this. This indicates that the antibody response in HIV-1 infection is principally elicited by viral debris rather than virions, and that these antibodies bind and neutralize viruses sub-optimally. Potential vaccines should be designed to elicit responses against native envelope.

Antibody neutralization of HIV-1.

Neutralizing antibodies are a major component of host defense against viruses, and appear to be particularly important in limiting the spread of cell-free virus. Results from vaccine trials in animal models suggest that these antibodies may contribute to protection against human immunodeficiency virus (HIV) infection. Here, Pascal Poignard and colleagues discuss recent developments in this area, with particular emphasis on the measurement, specificity and mechanism of the antibody response, and its significance for vaccine production.

Neutralizing antibodies to human immunodeficiency virus type-1 gp120 induce envelope glycoprotein subunit dissociation.

The spectrum of the anti-human immunodeficiency virus (HIV) neutralizing immune response has been analyzed by the production and characterization of monoclonal antibodies (mAbs) to the viral envelope glycoproteins, gp41 and gp120. Little is known, however, about the neutralization mechanism of these antibodies. Here we show that the binding of a group of neutralizing mAbs that react with regions of the gp120 molecule associated with and including the V2 and V3 loops, the C4 domain and supporting structures, induce the dissociation of gp120 from gp41 on cells infected with the T cell line-adapted HIV-1 molecular clone Hx10. Similar to soluble receptor-induced dissociation of gp120 from gp41, the antibody-induced dissociation is dose- and time-dependent. By contrast, mAbs binding to discontinuous epitopes overlapping the CD4 binding site do not induce gp120 dissociation, implying that mAb induced conformational changes in gp120 are epitope specific, and that HIV neutralization probably involves several mechanisms.

Epitope exposure on functional, oligomeric HIV-1 gp41 molecules.

We have used cells infected with the HIV-1 molecular clone HX10 to study the binding of monoclonal antibodies (mAbs) to different epitopes within the extracellular domain of the HIV-1 transmembrane glycoprotein gp41. Gp41 mAb binding to the infected cells at 4 degrees was variable but weaker than the binding of an anti-gp120/V3 loop mAb and increased substantially for three of the gp41 antibodies at 37 degrees. Treatment of the cells with soluble CD4 (sCD4) at 37 degrees increased gp41 mAb binding to epitopes spanning residues 521-663, implying that these regions had probably been masked by gp120, which following interaction with sCD4 had subsequently dissociated from gp41. By contrast, the binding of a mAb to residues 662-667 which form a neutralization epitope was reduced by sCD4 binding. Another region which has been described as containing a neutralization epitope spans residues 725-750. MAbs to this region bound equally well to noninfected and HIV-infected cells, and binding was not increased in the presence of sCD4. These data strongly imply that this epitope is not exposed on the external surface of the membrane, a finding in accord with the proposed cytoplasmic localization of this region.

Conformational changes induced in the envelope glycoproteins of the human and simian immunodeficiency viruses by soluble receptor binding.

We have investigated the molecular basis of biological differences observed among cell line-adapted isolates of the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and the simian immunodeficiency virus (SIV) in response to receptor binding by using a soluble form of CD4 (sCD4) as a receptor mimic. We find that sCD4 binds to the envelope glycoproteins of all of the HIV-1 isolates tested with affinities within a threefold range, whereas those of the HIV-2 and SIV isolates have relative affinities for sCD4 two- to eightfold lower than those of HIV-1. Treatment of infected cells with sCD4 induced the dissociation of gp120 from gp41 and increased the exposure of a cryptic gp41 epitope on all of the HIV-1 isolates. By contrast, neither dissociation of the outer envelope glycoprotein nor increased exposure of the transmembrane glycoprotein was observed when sCD4 bound to HIV-2- or SIV-infected cells. Moreover, immunoprecipitation with sCD4 resulted in the coprecipitation of the surface and transmembrane glycoproteins from virions of the HIV-2 and SIV isolates, whereas the surface envelope glycoprotein alone was precipitated from HIV-1. However, treatment of HIV-1-, HIV-2-, and SIV-infected cells with sCD4 did result in an increase in exposure of their V2 and V3 loops, as detected by enhanced antibody reactivity. This demonstrates that receptor binding to the outer envelope glycoprotein induces certain conformational changes which are common to all of these viruses and others which are restricted to cell line-passaged isolates of HIV-1.

Human and simian immunodeficiency viruses: virus-receptor interactions.

The major cellular receptor for the primate immunodeficiency viruses is the CD4 molecule. As well as mediating virion attachment to the cell surface, CD4 is thought to activate the viral fusion pathway. CD4 is not, however, sufficient for viral entry; other molecules are probably involved, and in certain circumstances these may substitute for CD4. Viral tropism and cytopathogenicity are also influenced by receptor interactions.

Cloned human CD4+ cytotoxic T lymphocytes specific for Toxoplasma gondii lyse tachyzoite-infected target cells.

Infection with Toxoplasma gondii is an important cause of morbidity and mortality throughout the world. In immunocompetent hosts, the infection is usually not significant. However, infection occurring in neonates or other individuals with defective cellular immunity (such as recipients of organ allografts or persons with AIDS) may be life threatening. An effective vaccine to prevent toxoplasmosis, or immunotherapy for persons already infected with Tg would be important additions to the therapeutic armamentarium. We cloned toxoplasma-specific CTL from the PBMC of an asymptomatic individual with serologic evidence for prior Tg infection by stimulation with Ag produced from the RH strain of Tg. These CTL were exclusively of the CD3+, CD4+, CD8- surface phenotype, and lysed autologous target cells that had been either pulsed with Toxoplasma Ag, or infected with live tachyzoites. Lysis of target cells was inhibited by incubation of CTL with anti-T cell antibody, or by incubation of target cells with anti-DR antibody or chloroquine. These CTL also lysed target cells either pulsed with Ag derived from C strain Tg or infected with live C strain tachyzoites, indicating cross-reactivity of recognition. Unlike recently reported murine or human CD8+ Tg-specific CTL, which lysed tachyzoites in an extracellular, and hence HLA-unrestricted environment, these CD4+ CTL had no effect on the infectivity of extracellular tachyzoites. CD8+ Tg-specific CTL were not derived from this donor despite several different approaches to their generation. These data confirm previous reports of human Tg-specific CTL, and extend these observations to include CD4+ CTL. These findings suggest that specific immunotherapy directed against Tg, as well as the development of a preventive vaccine, may be possible.