Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with Kd values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.

HIV-1 subtype C is the most common HIV-1 group M subtype in Africa and many parts of Asia. However, to date HIV-1 vaccine candidate immunogens have not induced potent and broadly neutralizing antibodies against subtype C primary isolates. We have used a centralized gene strategy to address HIV-1 diversity and generated a group M consensus envelope gene with shortened consensus variable loops (CON-S) for comparative studies with wild-type (WT) Env immunogens. Our results indicate that the consensus HIV-1 group M CON-S Env elicited cross-subtype neutralizing antibodies of similar or greater breadth and titer than the WT Envs tested, indicating the utility of a centralized gene strategy. Our study also shows the feasibility of iterative improvements in Env immunogenicity by rational design of centralized genes.

Altered geometry of the neuromuscular junction and a decreased number of acetylcholine receptors appear responsible for the defect of neuromuscular transmission in myasthenia gravis. We have used cultured rat myotubes as a model to study in vitro the potential role of myasthenic globulins in the pathological process. Acetylcholine receptor content was assayed by the extent of 125I -labeled a-bungarotoxin binding, and acetylcholine receptor function was assayed by the sensitivity to acetylcholine iontophoresis. The half-life of the acetylcholine receptor was 18.5 hr in the presence or absence of control sera. Myasthenic sera and globulins produced a gradual reduction in acetylcholine receptors, as assessed by biochemical and electrophysiological techniques. The half-life in the presence of myasthenic sera was 6 hr. The accelerated turnover was unaffected by puromycin but was slowed by lowered temperature (18-20°), interference with energy metabolism (2,4-initrophenol), and interference with cytoskeletal structures (colchicine and cytochalasin B). We found no electrophysiological evidence to suggest globulin blockade of acetylcholine access to the acetylcholine receptor. Our observations suggest that circulating globulins in myasthenia gravis may contribute to the functional defects -of neuromuscular transmission by accelerating the rate of internalization and degradation of surface membrane acetylcholine receptors.Myasthenia gravis (MG) is a remitting and relapsing neuromuscular disease of human beings characterized by muscle fatigability that increases with exertion and improves with rest. Recent studies have demonstrated altered morphology (1), decreased acetylcholine sensitivity (2, 3), and decreased number of acetylcholine receptors at the motor endplate of myasthenic muscle (4), suggesting that abnormalities of the postsynaptic membrane are responsible for the defect in neuromuscular transmission. The immune response to purified acetylcholine receptors in animals produces an experimental disease, experimental autoimmune myasthenia gravis (5), which is similar to human MG by morphologic, electrophysiologic, and clinical criteria (6)(7)(8). This animal model suggests that autoimmune factors involving the acetylcholine receptor may play a major role in the pathogenesis of human MG. Further evidence for an immune origin of human MG is provided by the presence of acetylcholine receptor antibodies in the sera of most myasthenic patients (9, 10).The specific role of antireceptor antibodies in the pathogenesis of MG is not known. The simplest hypothesis would be that the myasthenic globulin directly blocks the acetylcholine site on the acetylcholine receptor. However, no evidence exists to support this hypothesis. Evidence does suggest that serum globulins from a limited number of patients with MG may block a-bungarotoxin binding to the acetylcholine receptor (11,12), but no data demonstrate that these globulins block acetylcholine access to the receptor or acetylcholine sensitivity of the receptor. In fact,...

Twelve synthetic peptides containing hydrophilic amino acid sequences of human T-cell lymphotropic virus type I (HTLV-I) envelope glycoprotein were coupled to tetanus toxoid and used to raise epitope-specific antisera in goats and rabbits. Low neutralizing antibody titers (1:10 to 1:20) raised in rabbits to peptides SP-2 (envelope amino acids [aa] 86 to 107), SP-3 (aa 176 to 189), and SP-4A (aa 190 to 209) as well as to combined peptide SP-3/4A (aa 176 to 209) were detected in the vesicular stomatitis virus-HTLV-I pseudotype assay. Highertitered neutralizing antibody responses to HTLV-I (1:10 to 1:640) were detected with pseudotype and syncytium inhibition assays in four goats immunized with a combined inoculum containing peptides SP-2, SP-3, and SP-4A linked to tetanus toxoid. These neutralizing anti-HTLV-I antibodies were type specific in that they did not inhibit HTLV-H syncytium formation. Neutralizing antibodies in sera from three goats could be adsorbed with peptide SP-2 (aa 86 to 107) as well as truncated peptides containing envelope aa 90 to 98, but not with equimolar amounts of peptides lacking envelope aa 90 to 98. To map critical amino acids that contributed to HTLV-I neutralization within aa 88 to 98, peptides in which each amino acid was sequentially replaced by alanine were synthesized. The resulting 11 synthetic peptides with single alanine substitutions were then used to adsorb three neutralizing goat antipeptide antisera. Both asparagines at positions 93 and 95 were required for adsorption of neutralizing anti-HTLV-I antibodies from all three sera. Peptide DP-90, containing the homologous region of HTLV-II envelope glycoprotein (aa 82 to 97), elicited antipeptide neutralizing antibodies to EITLV-H in goats that were type specific. In further adsorption experiments, it was determined that amino acid differences between homologous HTLV-I and EITLV-II envelope sequences at HTLV-I aa 95 (N to Q) and 97 (G to L) determined the type specificity of these neutralizing sites. Thus, the amino-terminal regions of HTLV-I and-H gp46 contain homologous, linear, neutralizing determinants that are type specific.

Serum acetylcholine receptor antibodies were measured serially in myasthenia gravis patients before and after early extended thymectomy; they received no medication postoperatively. Clinical improvement occurred with little or no change in antibody level. After plasmapheresis without immunosuppressive drug therapy, we also found clinical improvement without decrease in serum antibody level.