Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162⌬V2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162⌬V2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162⌬V2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162⌬V2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.Analysis of the crystal structure of the gp120 human immunodeficiency virus (HIV) envelope subunit indicated that neutralization epitopes are primarily clustered in one face of this protein, which is naturally occluded within the oligomeric envelope form, i.e., that present on the surface of virions and infected cells (16,37). These structural observations are supported by numerous immunochemical and virological studies (1,24,25,27,28,31,35,38,40).Several reports have indicated that specific modifications (such as deglycosylations and loop deletions) introduced in the envelope glycoproteins of HIV and simian immunodeficiency virus (SIV) may increase the exposure of neutralization epitopes. Wyatt et al. demonstrated that on the background of the HXB2 virus, a laboratory-adapted CXCR4-using (X4-using) virus, deletions of the first, second, and third hypervariable regions (V1, V2, and V3 loops, respectively) of the gp120 envelope subunit increase the exposure of epitopes participating in HIV envelope-CD4 and -coreceptor binding (38,40). Subsequently, it was demonstrated that the simultaneous deletion of the V1 and V2 loops from the envelope of this virus increases it susceptibility to neutralization by anti-V3 loop and certain CD4-induced monoclonal antibodies...

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1 US4 . We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (R h ) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R h of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

Abstract-The Kenaga nomogram was developed by the U.S. Environmental Protection Agency (EPA) in the mid 1970s and has since been used prior to the registration of a pesticide to estimate the maximum potential pesticide residue level on plant material in the food chain of wildlife. The objective of this study was to evaluate the nomogram using field data. Six pesticides representing a variety of pesticide classes were applied to 15 plant species. Five of the six nomogram categories were tested with plant parts representing differences in surface morphology (i.e., glabrous vs. pubescent leaves). The sixth category was a mixed-grass community seeded with three grass species. Pesticide residue levels were determined the day of application and up to 32 d afterward. While the linear nomogram model does not represent the data as well as other models, relatively few data points (10%) collected on the day of pesticide application exceeded the nomogram predictions. The one systemic pesticide tested had degradation rates similar to nonsystemic pesticides in most categories. Present nomogram categories were significantly different from each other in most cases. However, the forage category should be combined with the leaves and leafy-crop category and have higher estimated residue levels than the Kenaga nomogram. A considerable amount of variation occurs in the level of pesticide residue on plant materials even under controlled experimental conditions and therefore nomogram values should be used as an estimate only until actual field residue data are available. The nomogram, with modifications, appears to be a reasonable regulatory device if careful thought is given in selecting the plant category for making residue estimates.

The Kenaga nomogram was developed by the U.S. Environmental Protection Agency (EPA) in the mid 1970s and has since been used prior to the registration of a pesticide to estimate the maximum potential pesticide residue level on plant material in the food chain of wildlife. The objective of this study was to evaluate the nomogram using field data. Six pesticides representing a variety of pesticide classes were applied to 15 plant species. Five of the six nomogram categories were tested with plant parts representing differences in surface morphology (i.e., glabrous vs. pubescent leaves). The sixth category was a mixed-grass community seeded with three grass species. Pesticide residue levels were determined the day of application and up to 32 d afterward. While the linear nomogram model does not represent the data as well as other models, relatively few data points (10%) collected on the day of pesticide application exceeded the nomogram predictions. The one systemic pesticide tested had degradation rates similar to nonsystemic pesticides in most categories. Present nomogram categories were significantly different from each other in most cases. However, the forage category should be combined with the leaves and leafy-crop category and have higher estimated residue levels than the Kenaga nomogram. A considerable amount of variation occurs in the level of pesticide residue on plant materials even under controlled experimental conditions and therefore nomogram values should be used as an estimate only until actual field residue data are available. The nomogram, with modifications, appears to be a reasonable regulatory device if careful thought is given in selecting the plant category for making residue estimates.