Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.
HR peptide and compare these data with inhibition by a C-HR peptide. Using intact envelope glycoprotein (Env) under fusogenic conditions, we show that the N-HR peptide preferentially binds receptor-activated Env and that CD4 binding is sufficient for triggering conformational changes that allow the peptide to bind Env, results similar to those seen with the C-HR peptide. However, activation by both CD4 and chemokine receptors further enhances Env binding by both peptides. We also show that a nonconservative mutation in the N-HR of gp41 abolishes C-HR peptide but not N-HR peptide binding to gp41. These results indicate that there are two distinct sites in receptor-activated Env that are potential targets for drug or vaccine development.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) mediates virus attachment and fusion to target cells. Binding of the surface subunit (gp120) of Env to the CD4 and chemokine cellular receptors triggers conformational changes in the oligomeric Env complex that activate the membrane fusion activity of the transmembrane subunit (gp41). A detailed understanding of these structural changes in Env would create new opportunities to prevent and treat HIV infection.A leading model of HIV entry proposes substantial refolding of Env, in which Env transitions from a metastable, native (prefusion) conformation through a prehairpin fusion intermediate to a thermostable, six-helix bundle structure (Fig.
We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 g. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADPribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1 [IL-1] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.Worldwide rates of immunization for diphtheria, pertussis, tetanus, polio, measles, and tuberculosis have increased dramatically over the last 20 years. Barriers to mass immunization vary widely among economic markets, but common constraints include the requirement for trained personnel, the association of vaccines with needle-related diseases and injuries, and coldchain and transport issues. Development of less-invasive and more readily administered vaccines has thus become a priority for public health agencies and is associated with an emergence of new technologies in the areas of vaccine delivery vehicles and routes of administration (1,15,19,25).Researchers have recently described transcutaneous immunization (TCI), a needle-free method for delivering vaccines to the host by simple application of adjuvant and antigen to the exterior skin surface (15,16,17,18,19,29). Our previous studies demonstrated that cholera toxin (CT) applied to the skin could be used as an adjuvant to elicit serum immunoglobulin G (IgG) responses against itself and coadministered antigens, including bovine serum albumin, diphtheria toxoid (DTx), hen egg lysozyme, tetanus fragment C, and tetanus toxoid (TTx). The humoral responses elicited by TCI are boosted by sequential applications, and antibodies are detectable in lung and stool exudates (16, 17), suggesting the potential for eliciting mucosally relevant immunity by this method. Moreover, we have reported that application of CT...
The pathogenesis of hepatitis A virus (HAV) infection was studied in owl monkeys following oral administration of the wild-type HM-175 strain of HAV. Stools were collected daily and blood and pharyngeal swabs twice weekly for viral isolation, and animals were necropsied at various intervals after inoculation. Organs were examined for the presence of virus by isolation in cell culture and for viral antigens by immunofluorescence. Monkeys excreted HAV in the stools for 1-4 days after inoculation, presumably due to the residual unabsorbed inoculum. No virus was found in stools for the next 2-3 days. HAV re-appeared on days 4-7 and then persisted through day 39. Viremia occurred on the 10th day and continued until day 35. Virus was isolated occasionally from throat swabs 1 or 2 weeks after it was detected in stools and blood, and there was no evidence that HAV replicated in the pharyngeal tissues. Animals acquired anti-HAV antibody by the 4th week, and alanine aminotransferase (ALT) was elevated 5-5.5 weeks after inoculation. HAV was isolated from liver 5 days after inoculation; however, viral antigens were first detected in Kupffer cells of the liver at 14 days and in hepatocytes at 21 days. HAV antigen was detected in epithelial cells of the intestinal crypts and in the cells of the lamina propria of the small intestine 3 days postinoculation and thereafter until the 5th week, suggesting that these cells might represent an additional site of HAV replication.
We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5 binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We also demonstrate conformational changes in the C heptad of gp41.The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) consists of a surface (gp120) subunit that attaches virus to target cells and a noncovalently associated transmembrane protein (gp41) (Fig. 1A) that mediates fusion between virus and target cell membranes. These subunits are synthesized as a fusion-inactive precursor (gp160) that is cleaved in the synthetic pathway to generate mature Env. gp120 binding to CD4 and chemokine receptors on the host cell triggers fusion-inducing conformational changes in gp41, leading to entry of the viral nucleocapsid into the host cell cytoplasm.The mechanism of gp41-mediated membrane fusion is not fully understood. A widely accepted model of HIV entry postulates that gp41 undergoes major refolding steps as it mediates membrane fusion (Fig. 1B, reviewed in reference 5). In this model, gp41 transitions from its native, metastable conformation as it exists on the surface of virus or infected cells to a final fusion-active conformation consisting of a thermostable six-helix bundle structure (Fig. 1C). This structure forms when two heptad repeat regions in the gp41 ectodomain self assemble into a trimer of hairpins, where the N-and C-terminal heptad repeats align in an antiparallel manner in each hairpin monomer (4,35,37). The N heptads form a triple-stranded coiled coil in the internal layer of the six-helix bundle, and the C-heptad repeat helices form the external layer. It is believed that gp120 binding to receptors loosens the association of gp120 with gp41, which in turn releases the fusion peptide at the N terminus of gp41 to insert into the target membrane. Subsequent folding of this fusion-intermediate conformation into the six-helix bundle structure probably facilitates fusion by bringing membranes together as Env adopts a more thermodynamically stable conformation.We investigated gp41 conformational changes by analyzing how two gp41 monoclonal antibodies (MAbs) with epitopes in the C heptad of gp41 bind Env under various conditions (Fig. 1A). The first antibody, 2F5, well known for being one of the few broadly neutralizing and protective HIV antibodies (20, 28), binds to a core epitope containing the residues ELDKWA at the C terminus of the C heptad (24, 27). The second antibody, D50, also binds a linear peptide from the C heptad but is not neutralizing (7). D50 was generated in mice immunized with a secreted, uncleaved, oligomeric form of Env (8). In enzyme-linked immunosorbent assay experiments (data not shown), we confirmed that both MAbs bind the same C-heptad gp41 peptide (DP-178/T20, residues 638 to 673 of the HXB2 Env) but not an overlapping C-heptad gp41 pe...
Background: N-terminal, heptad repeat (HR1) peptides of HIV envelope glycoprotein form coiled-coil oligomers that inhibit viral entry, but the targets are unclear. Results: An HR1 peptide stabilized as a trimer preferentially selects one resistance pathway, whereas the same unrestrained peptide selects two pathways. Conclusion: Stabilizing the trimer affects development of resistance. Significance: These findings inform inhibitor design and provide insights into virus entry.
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