The great difficulty in eliciting broadly cross-reactive neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) isolates has been attributed to several intrinsic properties of their viral envelope glycoprotein, including its complex quaternary structure, extensive glycosylation, and marked genetic variability.

Prophylactic vaccines that prevent diseases caused by viral pathogens typically elicit neutralizing antibodies (Abs), which rapidly clear virus, and/or cellular immune responses, which eliminate virus-producing cells. In a well-studied prototypical mouse retroviral system, both arms of the immune system are needed to control Friend murine leukemia virus-induced viremia and/or splenomegaly and to prevent the transition to a persistent infection status that invariably leads to erythroleukemia and death (35). Because vaccines directed against the human immunodeficiency virus type 1 (HIV-1) have failed to elicit Abs that neutralize primary HIV-1 strains of diverse geographic and/or genetic origins (5,9,31,47,51,77), recent efforts have primarily focused on regimens that stimulate cellmediated immunity. This retargeting of the vaccine effort towards cellular immune responses is based on numerous reports showing that cytotoxic T lymphocytes (CTL) play a major role in controlling both acute and chronic HIV-1 infections (13,43,52,54,64). A direct demonstration of this effect in an animal model comes from experiments showing that depletion of rhesus macaque (Macaca mulatta) CD8 ϩ T cells with monoclonal Abs (MAbs) at the time of primary simian immunodeficiency virus (SIV) and chimeric simian-human immunodeficiency virus (SHIV) infections leads to markedly elevated levels of viremia at the peak of the acute infection and more rapid onset of disease (38,48,67).

Neutralizing antibody (NAb) is a critical component of an immune system that can potentially provide sterilizing protection against human immunodeficiency virus type 1 (HIV-1). Therefore, an in vitro assay that can rapidly, safely, and accurately evaluate the NAb response vaccine candidates elicit, especially against a large number of HIV-1 variants, would be highly valuable. It has been demonstrated that HIV-1 envelope glycoprotein lacking the cytoplasmic domain can pseudotype murine leukemia virus encoding the beta-galactosidase gene and that this pseudovirus can specifically infect CD4(+) cells (Schnierle BS, Stitz J, Bosch V, et al.: Proc Natl Acad Sci USA 1997;94:8640-8645). Because the pseudovirus is not biohazardous and because the infection can be quantitatively determined within 2 days, we examined the feasibility of using the pseudovirus for high-throughput neutralization assays for HIV-1. We have generated viruses pseudotyped with gp140 of six different HIV-1 isolates (LAI, RF, Bal, AD8, 89.6, and DH12). All six pseudoviruses were infectious and exhibited expected coreceptor usage phenotype in HOS-CD4 cells expressing either CCR5 or CXCR4. More importantly, the neutralization sensitivity profile of these pseudoviruses was virtually identical to that observed from more conventional neutralization assays using either HIV-1 or SHIV. All pseudoviruses could be neutralized by broadly reactive human monoclonal antibody IgG1 b12. Our results indicate that the pseudoviruses are ideal for high-throughput evaluation of immune sera for their capacity to broadly neutralize a large number of HIV-1 isolates.