Biomedical subjects

N Sullivan

Publications and source records attributed to N Sullivan.

At least 19 recordsLinked to original sources

PubMed2005

Thiamine deficiency in dogs due to the feeding of sulphite preserved meat.

A 6-year-old dog, a 4-year-old dog and three 7-week-old puppies were diagnosed with thiamine deficiency caused by feeding sulphite treated meat. The 6-year-old dog presented with a history of inappetence, weight loss and vomiting that rapidly progressed to signs of multifocal intracranial disease including mental dullness, paresis, seizures, spontaneous nystagmus and strabismus. Thiamine pyrophosphate effect was elevated at 58% and magnetic resonance imaging revealed bilaterally symmetrical hyperintensity of the caudate nucleus and rostral colliculi. The dog recovered with thiamine supplementation. The 4-year-old dog and three 7-week-old puppies also presented with rapidly progressive multifocal central nervous system signs including ataxia, paresis, increased muscle tone, seizures, nystagmus and exophthalmos. The 4-year-old dog made a rapid recovery with thiamine supplementation. Euthanasia and necropsy of a puppy revealed malacia of multiple brainstem nuclei and oedema of the cerebral cortex. These findings were consistent with thiamine deficiency.

Animal Feed↗

PubMed2001

Anosmia in dementia is associated with Lewy bodies rather than Alzheimer's pathology.

OBJECTIVES: To assess olfactory function of patients with dementia. Odour detection ability is impaired in clinical Parkinson's disease. Evidence of impaired detection in patients with clinically diagnosed Alzheimer's disease is inconsistent. No studies of olfaction have been neuropathologically validated. METHODS: The olfactory function of 92 patients with dementia and 94 controls was assessed using a simple bedside test as part of the Oxford Project To Investigate Memory and Ageing (OPTIMA). Neuropathological assessment was made of cortical Lewy bodies and substantia nigra (SN) cell counts and of Alzheimer's disease in all 92 patients, 22 of whom had SN Lewy bodies and 43 of whom had only Alzheimer's disease. RESULTS: Patients with Lewy bodies were more likely to be anosmic than those with Alzheimer's disease or controls. Patients with Alzheimer's disease were not more likely to be anosmic than controls. Nor was anosmia associated with degree of neurofibrillary tangles, as assessed by Braak stage. Among subjects with Lewy bodies, overall cortical Lewy body scores and Lewy body density in the cingulate were higher in those who were anosmic. Consensus clinical criteria for dementia with Lewy bodies had a sensitivity of 64% and specificity of 89%. In the absence of definite Alzheimer's disease, the criteria had sensitivity of 100%. In patients with definite Alzheimer's disease, anosmia was slightly more sensitive (55%) than the consensus criteria (33%). However, the addition of anosmia to the consensus criteria did not improve their overall performance. CONCLUSION: Dementia with Lewy bodies is associated with impaired odour detection. Misdiagnosis may have accounted for some previous reports of impaired odour detection in Alzheimer's disease. Simple but more sensitive tests of anosmia are required if they are to be clinically useful in identifying patients with dementia with Lewy bodies.

Aged↗

PubMed2001

White matter damage is associated with matrix metalloproteinases in vascular dementia.

BACKGROUND AND PURPOSE: Vascular disease causes multi-infarct dementia (MID) or Binswanger's disease (BD), the latter of which is a progressive form of vascular dementia (VaD) associated pathologically with fibrinoid and hyaline changes in brain arterioles with injury to the white matter. Clinically, BD patients have long-standing hypertension with disturbances of gait and intellect. Because matrix metalloproteinases (MMPs) are important in cerebral infarction, we hypothesized that disturbances in the MMPs may be involved in VAD: METHODS: Brain tissues from 5 patients with VaD of the BD or multi-infarct type (MID) were immunostained with antibodies to glial fibrillary acidic protein (GFAP), a microglial/macrophage cell marker (PG-M1), gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Control tissues were from 8 elderly patients: 4 with strokes without dementia and 4 without neurological diseases. RESULTS: PG-M1+ cells appeared around infarcts in patients with strokes without dementia and in patients with VAD: In 2 of the 3 BD patients, PG-M1 cells were prominent near damaged arterioles and scattered diffusely in white matter. MMP-2 was seen normally in perivascular macrophages and in astrocytic processes near blood vessels and was present in patients with strokes in reactive astrocytes. MMP-9 was rarely seen. MMP-3 was seen in PG-M1+ microglial/macrophage cells around the acute infarctions. In BD, MMP-3 persisted in tissue macrophages and disappeared in long-standing white matter gliosis. CONCLUSIONS: These observations suggest that MMPs may participate in the damage to the white matter associated with VAD: Microglia/macrophage-induced damage, which is amenable to treatment, may be a factor in the progressive forms of VAD:

Aged↗

PubMed2000

P wave signal-averaged electrocardiogram as a new marker for atrial tachyarrhythmias in postoperative Fontan patients.

OBJECTIVES: This study was undertaken to determine the potential role of P wave signal-averaged electrocardiogram (PSAECG) for risk assessment of atrial tachyarrhythmias (ATs) in patients after Fontan operation. BACKGROUND: Onset of atrial flutter/fibrillation (AFF) in patients who have undergone Fontan operation for univentricular hearts constitutes an unfavorable clinical event associated with a high risk of cardiovascular complications. There is no data available on PSAECG in postoperative Fontan patients to predict potential susceptibility to ATs. METHODS: Twenty-four post-Fontan patients and 15 age-matched healthy subjects were prospectively studied with PSAECG, and the following measurements were made: filtered P wave duration (FPWD), P wave vector integrals (PINTs), root-mean-square voltage for the initial 30 ms (RMSi30), and duration of persistent amplitude signals <4 microV from the beginning of the P wave (Di4). RESULTS: The FPWDs were significantly longer in the study group patients with ATs when compared with the study group patients without ATs (p<0.01) and compared with the controls (p < 0.001). An FPWD cut point of 135 ms resulted in a sensitivity of 71% and a specificity of 81% in differentiating patients with ATs from patients without ATs among the postoperative Fontan patients. The PINT was significantly greater in Fontan patients with AFF and also without AFF when compared with controls (p<0.01, p<0.05, respectively). The RMSi30 and the Di4 were not significantly different between study and control groups. CONCLUSIONS: Signal-averaged P wave duration is significantly prolonged in postoperative Fontan patients. A prolonged signal-averaged P wave duration may be an effective noninvasive marker to predict risk of development of ATs in this patient group.

Adolescent↗

PubMed2000

Expression analysis of cyclins in pituitary adenomas and the normal pituitary gland.

OBJECTIVES: The molecular events involved in pituitary tumour development are still poorly understood. The cyclins play an important role in the control of the cell cycle during cell proliferation and over-expression of the cyclins has been shown in many different tumour types. The aim of this study was to investigate whether, in comparison to the normal gland, ectopic expression of cyclins occurs in pituitary tumours, and whether differences in cyclin expression are seen with different pituitary tumour types or in association with different tumour behaviour. In contrast to work on cyclin D there are no published data on cyclin B, A and E in human pituitary tumours. METHODS: Sixty-seven surgically removed pituitary tumours and 10 specimens of normal human anterior gland were studied using immunohistochemistry to detect the nuclear expression of cyclin A, B, D and E. The microvascular density (as a measure of angiogenesis), Ki-67 labelling index (to assess cell proliferation) and bcl-2 expression had previously been investigated in this cohort. RESULTS: All tumours studied contained cells that immunostained positively for cyclin A, B, D and E. However the proportion of positive cells in each tumour type was different. In contrast, there were no cyclin D positive cells in the normal anterior pituitary gland studied, and labelling indices (LI) for cyclins A, B and E were significantly lower in the normal gland than in pituitary adenomas. The cyclin LIs for A, B, D and E were significantly higher in macroadenomas when compared to microadenomas. Non-functioning pituitary tumours (NFA) generally showed the highest cyclin LI. In particular, both recurrent and nonrecurrent NFA showed significantly higher cyclin D LI than other tumours. The ratio of cells expressing cyclin B compared to those expressing cyclin A was significantly higher in functionless tumours that regrew when compared to NFAs that did not (P<0.05). Cyclin D LI and the overall Ki-67 LI as a measure of cell proliferation were related (R2 = 11.4, P = 0.0033) and bcl-2 positive tumours had significantly higher cyclin D LI compared with bcl-2 negative tumours. There was a weak relationship between angiogenesis and the relative proportion of cells expressing D when compared to those in S phase (D/A ratio) (r2 = 10.5, P = 0.02). CONCLUSIONS: We have demonstrated that ectopic expression of cyclin D and over-expression of cyclins A, B and E, regulating different stages of the cell cycle is common in pituitary adenomas. In addition, cyclin expression was related to size and to pituitary tumour regrowth. The differences between functionless tumours that regrow and those that do not, may be due to reduced bcl-2 expression, increased cell proliferation, more cells at the G2/M stage (B/A ratio) and reduced cell differentiation with more aggressive subsequent tumour behaviour. Cyclin D expression and cell proliferation were related indicating that the cells entering the cycle become 'committed' to cell cycle progression. There was no relative over-expression of individual cyclins, and therefore no evidence of relative increase in cell cycle phase, indicating that the increased cyclin expression is more likely to be due to constant mitogenic stimulation rather than cell cycle regulatory failure. Although nuclear cyclin expression is a good marker of tumour growth and aggressive behaviour, the growth signal that leads to cyclin expression remains to be identified.

Adenoma↗

PubMed1998

CCR5 has an expanded ligand-binding repertoire and is the primary receptor used by MCP-2 on activated T cells.

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic HIV-1 strains to enter the host cells. In this study, we aim to better understand the ligand-binding profiles of CCR5 and the chemokine-receptor usage on leukocyte cells. We found that MCP-2 could bind to CCR5 transfectants with high affinity and cross-compete effectively with RANTES, MIP-1alpha, and MIP-1beta. MCP-2 is a true agonist for CCR5, eliciting a robust chemotactic response in CCR5 transfectants similar to that of the three known CCR5 ligands and exhibiting cross-desensitization with RANTES in the Ca2+ flux response. MCP-4 also bound to CCR5 with high affinity and was efficiently displaced by other CCR5 ligands. However, MCP-4 only partially displaced the binding of radiolabeled MIP-1alpha and caused a chemotactic response only at high concentrations. Furthermore, MCP-2 inhibited the binding of the M-tropic HIV-1 gp120 envelope glycoprotein to CCR5 and HIV-1 infection of peripheral blood mononuclear cells. More importantly, we found that MCP-2 could bind and elicit chemotaxis in CD3-activated and IL-2-maintained T cells, and most of these functions could be specifically inhibited by the anti-CCR5 mAb 2D7, whereas the responses mediated by MIP-1alpha or MCP-4 were only partially inhibited by 2D7. Thus, although MCP-2 can bind to and signal through CCR1, CCR2b, and CCR5, among which both CCR2 and CCR5 are expressed at high levels on activated T cells, it appears to preferably utilize CCR5 on these cells. In contrast, MIP-1alpha and MCP-4 seem to activate multiple receptors on the same cells.

Animals↗

PubMed1998

Inclusion body disease in two captive Australian pythons (Morelia spilota variegata and Morelia spilota spilota).

Two captive Australian pythons, one carpet and one diamond python, presented with signs of central nervous system dysfunction. The carpet python was agitated. Its head was tilting and it was incoordinated and had convulsions. It was treated with antibiotics and anthelmintics but was eventually euthanased after failing to respond to therapy. The diamond python had flaccid paralysis of the caudal half. It was not treated and became disoriented and died. Hepatocytes from both pythons contained irregular 2 to 10 micron eosinophilic intracytoplasmic inclusion bodies. The brain of the diamond python was not available for examination. Occasional neurones in the carpet python brain contained similar inclusion bodies and other changes suggestive of viral infection. The clinical signs and histopathological findings in both pythons were consistent with boid inclusion body disease.

Animals↗

PubMed1998

CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.

Animals↗

PubMed1998

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies.

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.

Animals↗

PubMed1997

Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection.

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4(+) T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.

Amino Acid Sequence↗

PubMed1997

CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro.

Chemokine receptors serve as coreceptors for HIV entry into CD4+ cells. Their expression is thought to determine the tropism of viral strains for different cell types, and also to influence susceptibility to infection and rates of disease progression. Of the chemokine receptors, CCR5 is the most important for viral transmission, since CCR5 is the principal receptor for primary, macrophage-tropic viruses, and individuals homozygous for a defective CCR5 allele (delta32/delta32) are highly resistant to infection with HIV-1. In this study, CCR5-specific mAbs were generated using transfectants expressing high levels of CCR5. The specificity of these mAbs was confirmed using a broad panel of chemokine receptor transfectants, and by their non-reactivity with T cells from delta32/delta32 individuals. CCR5 showed a distinct pattern of expression, being abundant on long-term activated, IL-2-stimulated T cells, on a subset of effector/memory T cells in blood, and on tissue macrophages. A comparison of normal and CCR5 delta32 heterozygotes revealed markedly reduced expression of CCR5 on T cells from the heterozygotes. There was considerable individual to individual variability in the expression of CCR5 on blood T cells, that related to factors other than CCR5 genotype. Low expression of CCR5 correlated with the reduced infectability of T cells with macrophage-tropic HIV-1, in vitro. Anti-CCR5 mAbs inhibited the infection of PBMC by macrophage-tropic HIV-1 in vitro, but did not inhibit infection by T cell-tropic virus. Anti-CCR5 mAbs were poor inhibitors of chemokine binding, indicating that HIV-1 and ligands bind to separate, but overlapping regions of CCR5. These results illustrate many of the important biological features of CCR5, and demonstrate the feasibility of blocking macrophage-tropic HIV-1 entry into cells with an anti-CCR5 reagent.

Alleles↗

PubMed1997

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.

CD4 Antigens↗

PubMed1997

Improvement of hemispatial neglect with cold-water calorics: an electrophysiological test of the arousal hypothesis of neglect.

Introducing ice-water into the left ear of right-brain-damaged patients attenuates unilateral neglect symptoms. By examining EEG changes over each hemisphere during this procedure, we were able to test a hypothesis concerning the mechanism of cold-water calorics and the attention-arousal hypothesis of hemispatial neglect. We present a case study of an 83-year-old woman with a massive right-hemisphere CVA exhibiting severe hemispatial neglect. Caloric stimulation produced a leftward eye deviation to central position, and a temporary partial remission of neglect symptoms. Significant changes in EEG activation indicated a central mechanism associated with the regularization of eye gaze. Caloric stimulation also produced a significant interaction between EEG frequency band and hemisphere, indicating that while both hemispheres increased in cortical activation, the right hemisphere increase was significantly greater. This supports the activation-arousal hypothesis of neglect over the mutual inhibition model.

Aged↗

PubMed1996

Human antibody responses to HIV type 1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding.

A large panel of human Fab fragments against the gp41 subunit of the HIV-1 envelope glycoprotein was isolated by panning six phage-displayed antibody libraries against recombinant gp41. The libraries were prepared from HIV-1-seropositive donors. Twenty-three Fabs recognizing conformation-dependent determinants on gp41 were isolated. Further selection of libraries against (1) gp41 ligated with Fabs from the initial selection and against (2) a recombinant gp41-containing gp140 protein yielded five additional Fabs. Competition of members of the Fab panel with one another and with previously described antibodies revealed a series of overlapping specificities that were conveniently grouped into three major epitope clusters. The majority of Fabs recognized epitopes involving residues 649-668 (previously known as the cluster II region), numbered using the Los Alamos LAI sequence. A second set of Fabs reacted with an epitope involving residues 584-609 (known as the cluster I region). Another set of Fabs appeared to recognize a third conformational epitope that has been termed the cluster III region. This third Fab epitope group demonstrated some overlap with both clusters I and II in binding assays. None of the Fabs neutralized HIV-1 laboratory strains at biologically significant concentrations. This tends to support the opinion that a vaccine based on the gp41 molecule has the drawback that neutralizing epitopes of gp41 are rare and/or unfavorably presented to the immune system. Analysis of heavy chain sequences revealed common CDR3 motif sequences in several antibodies, which appears to be an interesting consequence of a persistent immune response to conserved antigen structures.

Amino Acid Sequence↗

PubMed1996

The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.

We examined the ability of chemokine receptors and related G protein-coupled receptors to facilitate infection by primary, clinical HIV-1 isolates. CCR5, when expressed along with CD4, the HIV-1 receptor, allowed cell lines resistant to most primary HIV-1 isolates to be infected. CCR3 facilitated infection by a more restricted subset of primary viruses, and binding of the CCR3 ligand, eotaxin, inhibited infection by these isolates. Utilization of CCR3 and CCR5 on the target cell depended upon the sequence of the third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein. The ability of various members of the chemokine receptor family to support the early stages of HIV-1 infection helps to explain viral tropism and beta-chemokine inhibition of primary HIV-1 isolates.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1.

We have isolated and characterized human monoclonal antibody 2G12 to the gp120 surface glycoprotein of human immunodeficiency virus type 1 (HIV-1). This antibody potently and broadly neutralizes primary and T-cell line-adapted clade B strains of HIV-1 in a peripheral blood mononuclear cell-based assay and inhibits syncytium formation in the AA-2 cell line. Furthermore, 2G12 possesses neutralizing activity against strains from clade A but not from clade E. Complement- and antibody-dependent cellular cytotoxicity-activating functions of 2G12 were also defined. The gp120 epitope recognized by 2G12 was found to be distinctive; binding of 2G12 to LAI recombinant gp120 was abolished by amino acid substitutions removing N-linked carbohydrates in the C2, C3, V4, and C4 regions of gp120. This gp120 mutant recognition pattern has not previously been observed, indicating that the 2G12 epitope is unusual. consistent with this, antibodies able to block 2G12 binding to recombinant gp120 were not detected in significant quantities in 16 HIV-positive human serum samples.

Amino Acid Sequence↗

PubMed1995

Thymoma in a geriatric rabbit with hypercalcemia and periodic exophthalmos.

An 8-year-old rabbit was referred to an ophthalmologist because of intermittent bilateral exophthalmos and prolapse of the nictitating membranes. Both eyes could be retropulsed normally, and the exophthalmos was induced with ventroflexion. The rabbit had moderate hypercalcemia and a large mediastinal mass that could be seen on thoracic radiographs. The rabbit's condition was unchanged for 5 months. It was reexamined because of weight loss and paroxysmal coughing and, at that time, was thin and tachypneic, and had reduced thoracic compliance. Thoracotomy was performed, and a 5-cm-diameter encapsulated mass, subsequently determined histologically to be thymoma, was removed. The rabbit was euthanatized after surgery because of complications. The periodic exophthalmos and hypercalcemia in this rabbit were believed to be paraneoplastic syndromes.

Animals↗

PubMed1995

Neutralizing recombinant human antibodies to a conformational V2- and CD4-binding site-sensitive epitope of HIV-1 gp120 isolated by using an epitope-masking procedure.

As part of the goal of assembling a mixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1 disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epitope can be accessed. From a phage display library derived from an asymptomatic HIV-1 seropositive donor, a panel of recombinant Fabs against the CD4 binding site (CD4bs) of gp120 was retrieved by affinity selection using recombinant gp120 (strain LAI). Two Fabs corresponding to the dominant clones were used to mask the CD4bs epitope(s) before repeating the selection procedure. Four Fabs were then retrieved that had novel heavy chain sequences. Three recognized a novel epitope distinct from that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region (V2 region) dependence indicated by sensitivity to amino acid changes in the V2 loop and by competition with murine anti-V2 mAbs; 2) CD4bs dependence indicated by sensitivity to amino acid changes usually associated with CD4 binding and by inhibition of Fab binding to gp120 by soluble CD4; this dependence seemed to arise via conformational changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a reversal of the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to glycosylated and deglycosylated gp120 and significant, although much reduced, binding to denatured gp120 in contrast with CD4bs Abs, which do not bind to deglycosylated or denatured gp120. One of the novel Fabs efficiently neutralized the MN and LAI strains of HIV-1. These results indicate the presence of a novel neutralizing conformational epitope on gp120 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gp120. The strategy of masking highly immunogenic epitopes with Abs to rescue a broader range of specific Abs from combinatorial libraries should be widely applicable.

Amino Acid Sequence↗