Biology Reference
In-Depth Information
latency? Several cell lines such as ACH-2, J1.1, U-1, and OM-10.1 have been
proposed to represent cells latently infected with HIV-1 (Butera et al., 1991,
1994; Clouse, et al., 1989; Perez et al., 1991). These cells produce large amounts
of virus after exposure to inducing agents such as cytokines or phorbol esters.
Do they produce a di¨erent set of viral proteins before activation than after
activation? This question is crucial because it may provide insights on the
mechanisms that the virus uses to survive during antiviral therapy and most
importantly because it may provide targets for eliminating the persistent in-
fected cells. In a population of cells, are there cells that are latently infected and
other cells that produce low levels of virus? A single-cell analysis of the popu-
lation is required to answer this question.
Flow cytometric analysis of HIV-1 proteins with standard ampli®cation
technologies has not been informative. For instance, most investigators who
have assessed antibodies to gp120 have not used ¯ow cytometric analysis but
have instead relied on neutralization assays and enzyme-linked immunosorbent
assay (ELISA). One of the most important di½culties with ¯ow cytometric
analysis for HIV-1 gp120 has been availability of antibodies with su½cient re-
activity to bind to the various isolates of the virus. In one sense, this problem is
also a re¯ection of the poor sensitivity of ¯ow cytometry inasmuch as a more
sensitive technology would be able to detect antibodies that bind with lower
a½nities.
One of the ®rst antibodies used for the assessment of the expression of viral
proteins by ¯ow cytometry was the human monoclonal antibody F105 that
is speci®c for the CD4 binding site of gp120 (Posner et al., 1991). F105 is a
human IgG1 k monoclonal antibody that was obtained by using Epstein-Barr
virus to transform B cells derived from an HIV-1 infected person. This antibody
has been used to assess gp120 expression by ¯ow cytometry. gp120 from all
strains tested, including MN, RF, IIIB, and SF2, were bound by this antibody.
Nevertheless, the histograms obtained were not impressive; it is not even clear
that the antibody could distinguish infected from noninfected cells, an impor-
tant criterion for usefulness. The use of F105 to analyze gp120 expression has
not been proven to be useful.
Other human monoclonal antibodies speci®c for HIV-1 gp120 have been
used in ¯ow cytometric analysis and have given excellent histograms with cell
lines stably infected with HIV-1 (Alsmadi et al., 1997). Uninfected cells or in-
fected cells without primary antibody were used as controls. Unfortunately, an
isotype/subtype control was not included in the analysis. These antibodies were
shown by ¯ow cytometry to recognize gp120 from native primary isolates from
clades B and E as well as various strains such as SF2 and RF. Nevertheless,
gp120 from strain MN was not recognized. These antibodies seem promising;
however, no additional publications with them have been listed in PubMed
since 1997.
Another study with human monoclonal antibodies speci®c for HIV-1 gp120
demonstrated the possibility that these antibodies could be used in ¯ow cyto-
metric analyses to serotype primary isolates (Zolla-Pazner et al., 1995). Two of
