Human Immunodeficiency Virus Type 1envClones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

Abstract

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is believed to require the induction of both virus-specific CD8 ϩ T cells and neutralizing antibodies (Abs) (56, 63). Neutralizing Abs are of particular interest because their presence at the time of intravenous, vaginal, and oral routes of live virus challenge has been shown to prevent AIDS virus infection in nonhuman primates (31,65,66,69,84,105). Due to the extraordinary degree of genetic diversity of HIV-1 and the structural complexity of its envelope glycoproteins (Env) (54, 118), designing an effective vaccine is difficult. These same properties of the virus also make it difficult to assess vaccine-elicited neutralizing Abs in a way that is meaningful and informative. Many candidate vaccines have included Env for the purpose of generating an effective neutralizing Ab response, but to date, none appear to elicit a response with the desired specificity and cross-reactivity (5,6,13,68). This poor immunogenicity is incompletely understood but might be explained by either the particular design or low valency of prototype immunogens (14).A number of new candidate immunog...

Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime–protein boost HIV-1 vaccine in healthy human volunteers

“…Serum and PBMC samples were collected at Study Weeks 0, 2,4,6,12,14,16,20,22,24,28,30,32,36 and 52 to measure antibody and CMI responses. All volunteers were recruited and enrolled in the Clinical Vaccine Research Unit, UMMS.…”

Immunogenicity of recombinant human immunodeficiency virus type 1-like particles expressing gp41 derivatives in a pre-fusion state

“…Neutralization was measured as a function of reductions in luciferase reporter gene expression after a single round of infection in TZM-bl cells as described [26,53]. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu.…”

“…An assay stock of HIV-1 MN was prepared in H9 cells. Assay stocks of the Env-pseudotyped virus, SF162.LS, and the full-genome clone, ADA, were prepared by transfection in 293T cells [26]. All virus stocks were made cell-free by 0.45-micron filtration and were stored at −70°C until use.…”

“…Recently, it was shown that 2F5 and 4E10 mAbs were more potent in inhibiting viruses from acutely infected individuals than viruses from chronically infected patients [25]. However, experiments revealed that Env -pseudotyped viruses were more sensitive to neutralization than were their classical peripheral blood mononuclear cell (PBMC)-grown viruses, against which these antibodies manifest less potent activity with reduced breadth [23,26]. The molecular bases of these observed differences between pseudovirus and PBMC-derived virus remains to be fully explained [27].…”

Identification and specificity of broadly neutralizing antibodies against HIV

“…The ability to analyze mAb and serum activity against large panels of viruses was demonstrated20 and subsequently used to evaluate large numbers of HIV‐infected donors in the International AIDS Vaccine Initiative (IAVI) Protocol G and C studies to identify those with exceptionally potent and broad sera,8 map the specificities underlying these responses,7, 21 and then isolate bnAbs from these individuals 22, 23, 24, 25, 26, 27, 28. Independently, the standardization of the TZM‐bl neutralization assay and the definition of neutralization sensitivity tiers29, 30, 31 allowed much more rigorous serum analysis.…”