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JOURNAL OF VIROLOGY, Dec. 2004, p. 13232–13252
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 23
Comprehensive Cross-Clade Neutralization Analysis of a Panel of
Anti-Human Immunodeficiency Virus Type 1
Monoclonal Antibodies
James M. Binley,1† Terri Wrin,2Bette Korber,3Michael B. Zwick,1Meng Wang,1
Colombe Chappey,2Gabriela Stiegler,4Renate Kunert,4Susan Zolla-Pazner,5Hermann Katinger,4
Christos J. Petropoulos,2and Dennis R. Burton1*
Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla,1and ViroLogic Inc.,
South San Francisco,2California; Theory Division, Los Alamos National Laboratory, Los Alamos, and The
Santa Fe Institute, Santa Fe, New Mexico3; Institute of Applied Microbiology, University of Agricultural
Sciences, Muthgasse, Vienna, Austria4; and Department of Pathology, New York University
Medical Center, and Research Center for AIDS and
HIV Infection, VA Medical Center,
New York, New York5
Received 3 May 2004/Accepted 9 July 2004
Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunode-
ficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have
a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from
clade B-infected donors and a clade B HIV?plasma against 93 viruses from diverse backgrounds. Anti-gp120
MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad
interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain)
neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of
4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope
overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12
(directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or
E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but
infrequently neutralized other clades (<7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed
against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV?plasma
neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutral-
izing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design,
our data have implications for passive-immunization studies in countries where clade C viruses are common,
given that only MAbs b12 and 4E10 were effective against viruses from this clade.
Neutralizing antibodies (NAbs) against viral envelope pro-
teins (Env) provide the first line of adaptive immune defense
against human immunodeficiency virus type 1 (HIV-1) expo-
sure by blocking the infection of susceptible cells (64, 89, 94,
108). The efficacy of vaccines against several viruses has been
attributed to their ability to elicit NAbs (21, 150). However,
despite enormous efforts, for HIV-1 there has been limited
progress toward an effective immunogen (21, 84, 90).
HIV-1 is among the most genetically diverse viral pathogens
described to date. There are three main branches of the HIV-1
phylogenetic tree, the M (main), N (new), and O (outlier)
groups. Group M viruses are the most widespread, accounting
for ?99% of global infections. This group is presently divided
into nine distinct genetic subtypes, or clades (A through K),
originally based on short sequences mostly in the Env gene (80,
122) but more recently based on full-length sequences. Env is
the most variable HIV-1 gene, with up to 35% sequence di-
versity between clades, 20% sequence diversity within clades,
and up to 10% sequence diversity in a single infected person
(61, 130). Clade B is dominant in Europe, the Americas, and
Australia (60). Clade C is common in southern Africa, China,
and India and presently infects more people worldwide than
any other clade (80). Clades A and D are prominent in central
and eastern Africa. However, many viruses are difficult to
classify into clades due to the common intermixing of cocircu-
lating viruses that leads to interclade recombinants (52, 82).
Some recombinant forms have in fact given rise to important
epidemic lineages, called circulating recombinant forms
(CRFs). The two most common of these are CRF01 (AE),
discovered in Thailand, which was initially classified as clade E,
though later it was found to be clade E only in Env and clade
A in other parts of the genome, and CRF02, an AG recombi-
nant form common in Western Africa (122). Globally, clades A
through D and the CRF01 AE and CRF02 AG recombinants
account for ?90% of global infections.
Although clades provide a useful means to categorize HIV-1
based on genetic relationships, the relevance of clades in dis-
tinguishing virus neutralization sensitivities remains unproven.
* Corresponding author. Mailing address: IMM2, Department of
Immunology, The Scripps Research Institute, 10550 N. Torrey Pines
Rd., La Jolla, CA 92037. Phone: (858) 784-9298. Fax: (858) 784-8360.
E-mail: firstname.lastname@example.com.
† Present address: Torrey Pines Institute for Molecular Studies, San
Diego, CA 92121.
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There is so far no consistent evidence that HIV?sera prefer-
entially neutralize autologous viruses more effectively than
they do viruses from other clades (6, 10, 43, 58, 59, 71, 75, 90,
93, 102, 145), although some studies have suggested stronger
intraclade neutralizing responses for clades C (17) and AE
(CRF01) (75). The overall difficulty of determining clade-
associated neutralizing immunotype groups may be because
sequence differences that determine genetic clades do not in-
fluence neutralization epitopes (71) or that limited sampling
and high background noise activity complicate cross-clade neu-
tralization analyses (92, 101, 152).
Distinct grouping of primary isolates into neutralization im-
munotypes may be feasible using a few rare broadly neutraliz-
ing human monoclonal antibodies (MAbs) that have been iso-
lated from HIV?clade B-infected human donors (35, 108).
These MAbs neutralize many primary HIV-1 isolates, includ-
ing some from different clades, indicating that certain elements
of Env structure are well conserved (20, 22, 56, 57, 98, 109, 138,
139). Four relatively conserved epitopes have been defined by
a set of five neutralizing human MAbs. Two MAbs recognize
epitopes located on the gp120 surface unit of the Env spike:
MAb b12 is directed against an epitope overlapping the CD4
binding site (5, 19, 22, 121), and MAb 2G12 recognizes a
unique epitope in a carbohydrate-rich region on the outer
domain of gp120 (14, 23, 62, 126, 129, 139). Three MAbs
recognize epitopes located on the membrane-proximal exter-
nal region of the gp41 transmembrane protein: MAb 2F5 has
been mapped to a region overlapping the conserved sequence
ELDKWA (98, 106, 114). MAbs 4E10 and Z13 recognize an
epitope involving the sequence NWF(D/N)IT located adjacent
and carboxy terminal to the 2F5 epitope (14, 153). Passive-
transfer studies in macaques have shown that these MAbs can
provide protection against HIV-1 challenge (4, 73, 74, 79, 107).
The very existence of these MAbs provides hope that a vaccine
able to elicit broadly neutralizing antibodies may be possible in
the future. Unfortunately, all attempts to elicit antibodies with
specificities similar to those of these MAbs have so far failed
(7, 27, 37–39, 69, 78, 96, 119, 132, 140). Since neutralizing
antibodies against these epitopes do not appear to constitute a
significant fraction of the plasma response against Env gener-
ated during HIV-1 infection or by vaccination, their epitopes
appear to be poorly immunogenic (18). Typically, immune sera
from vaccine trials to date at best neutralize only the matched
vaccine strain and a few neutralization-sensitive primary iso-
lates (30, 32, 111, 141).
Some other anti-Env MAbs are of significant interest. These
include MAbs against the gp120 V3 loop (9, 28, 46, 144). The
V3 loop is highly immunogenic and was once considered to be
the principal neutralizing determinant of HIV-1. However, it
has since been shown that many primary HIV-1 isolates resist
neutralization by sequestering the V3 loop, rendering it inac-
cessible to MAbs (13). The V3 loop is highly variable and,
barring some notable exceptions, tends to induce type-specific
antibodies with limited breadth (9, 28, 47, 49, 50). Despite its
sequence variation, the V3 loop retains certain conformational
features that may preserve its ability to interact with chemo-
kine receptors during infection (31, 53, 131, 137, 147). MAb
447-52D is an unusual V3 loop-specific MAb in that it recog-
nizes the well-conserved GPGR motif at the tip of the V3 loop
of clade B isolates. This specificity lends it greater breadth than
most V3 MAbs described to date (47, 133).
Another group of MAbs recognize discontinuous epitopes
that overlap the conserved coreceptor binding site of gp120
and that are preferentially exposed by gp120 binding to CD4.
This group of MAbs is represented by MAbs X5 and 17b (66).
As whole immunoglobulin Gs (IgGs), these MAbs have very
limited neutralizing activity against primary isolates. However,
Fab and single-chain Fv fragments neutralize primary isolates
more effectively (66). Thus, their activity against primary vi-
ruses appears to be limited by steric constraints that are less
significant for smaller IgG fragments.
Many studies have evaluated the neutralization activities of
the above-mentioned MAbs (33–36, 45, 48, 49, 72, 95, 114, 115,
138). However, these all made only a limited assessment of
cross-clade neutralization: at most, only three or four isolates
of each non-B clade were analyzed. Here, we have analyzed the
neutralization activities of b12; b6 (a MAb overlapping a CD4
binding site); 2G12; 2F5; 4E10; X5; 447-52D; 58.2 (a V3 loop
MAb); a five-MAb cocktail of b12, 2G12, 447-52D, 2F5, and
4E10; and a broadly cross-reactive HIV?donor plasma against
a total of 93 viruses, including 61 non-clade B isolates. De-
pending on availability, we included viruses from as many dif-
ferent parts of the world as possible, so that neutralizing ac-
tivity could be collated on a geographic basis as well as on a
genetic basis. To make this large study manageable, we used a
sensitive and highly reproducible single-round pseudovirus in-
fectivity assay. We found that MAbs b12, 2G12, 4E10, and 2F5;
the five-MAb cocktail; and the HIV?plasma exhibited various
degrees of cross-clade neutralization. Neutralization by the V3
loop-specific MAbs was mostly restricted to clade B viruses,
and MAbs b6 and X5 neutralized only sensitive clade B viruses.
MAbs and HIV?human plasma. Seven anti-Env MAbs were derived from
clade B HIV-infected donors. These included the anti-gp120 MAbs b12 (19, 22)
and b6 (5, 19, 121), both directed against an epitope overlapping the CD4
binding site; 2G12, directed against a unique epitope in a carbohydrate-rich
region on the outer domain of gp120 (14, 23, 62, 126, 129, 139); IgG X5 (66, 95),
which recognizes an epitope on gp120 whose exposure is increased when gp120
complexes with CD4; and 447-52D, which was selected with a clade B-derived
peptide and recognizes the GPXR motif at the tip of the V3 loop (28, 45, 47–50,
133). 58.2 is a mouse MAb generated against a clade B Env immunogen and is
directed against the V3 loop HIGPGRAF motif (55). Two anti-gp41 MAbs were
2F5 (14, 29, 62, 97, 98, 106, 114, 115) and 4E10 (14, 134, 153), directed against
epitopes located on the membrane-proximal external region of the gp41 trans-
membrane protein. 2F5 recognizes the conserved sequence ELDKWA (residues
667 to 672, based on the numbering of residues in HXBc2 to facilitate compar-
ison with structural information published on this Env protein) (64, 98, 106), and
4E10 recognizes an epitope involving the sequence NWF(D/N)IT (residues 671
to 676) motif located carboxy terminal to the 2F5 epitope (134, 153). Twenty-
seven HIV?human plasmas from HIV?clade B-infected donors were obtained
from NABI Biopharmaceuticals (Rockville, Md.). These plasmas were available
in large volumes, which is an important criterion for their use as assay controls.
All plasmas were initially tested in neutralization assays against 10 viruses.
Plasma N16 exhibited the most potent and cross-reactive neutralization and was
selected for further use. There is no clinical information available about the
donor of this plasma.
Viruses. Pseudoviruses capable of a single round of infection were produced
by cotransfecting HEK293 cells with a subgenomic plasmid, pHIVluc?U3, that
incorporates a firefly luciferase indicator gene and a second plasmid, pCXAS,
that expresses HIV-1 Env libraries or clones. Recombinant viruses pseudotyped
with Env proteins were harvested 48 h posttransfection. Env gp160 DNAs used
for cloning into pCXAS were derived either from shotgun cloning of viral
quasispecies by reverse transcription-PCR of HIV genomic RNA from HIV?
VOL. 78, 2004CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs13233
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donor plasma or from primary virus that had been cultured in primary peripheral
blood mononuclear cells (PBMCs), as described previously (120). Others were
from well-established molecular clones. An amphotropic control murine leuke-
mia virus (MuLV) Env plasmid was also used to make pseudovirus to control for
nonspecific neutralization.
Uncloned plasma-derived quasispecies were all designated VLGC, with the
following codes that reflect their clades (origins, where known, are given in
parentheses): ABEVLGC1 (Belgium), AxVLGC2 (unknown); AGxVLGC1 and
AGxVLGC2 (both unknown, but CRF_02 recombinants like these are common
in Northern Africa); BBRVLGC1, BBRVLGC2, and BBRVLGC3 (Brazil);
BGPTVLGC3 (Portugal); BFITVLGC1 and BFITVLGC2 (Italy); BFBRV-
LGC3 and FBRVLGC4 (a mosaic Env that appears to be mostly clade F with
traces of clade B sequence) (Brazil); GxxVLGC1 (unknown); GPTVLGC2 (Por-
tugal); JGEVLGC1 (Germany); and JxxVLGC2 (unknown).
For consistency, we used a systematic and standardized eight-character no-
menclature to define viruses by their Env proteins by clade, country of origin,
year isolated, number, and a "c" to indicate a molecular clone. This also assisted
mathematical analysis. Previous names, where known, are given in parentheses,
and references, if available, are cited. Quasispecies derived from primary virus
cultures were as follows. Clade A viruses were ARW93029 (93RW029), ARW92024
(92RW008), ARW92021 (92RW021), ARW92020 (92RW020) (Rwanda) (41),
AUG92031 (92UG031), AUG92037 (92UG037), AUG93077 (93UG077), and
AUG94103 (94UG103) (Uganda) (40, 41). Clade AC recombinants were
ACKE94105 (94KE105) (Kenya) and ACUG93086 (93UG086) (Uganda) (40, 41).
Clade B viruses were BUSxx692 (692), BUSx1168 (1168), BUSx1196 (1196),
BUSx5768 (5768; previously P27), and BUSx6101 (6101; previously P15) (United
States) (16); BUSxPAVO (PAVO) and BUSTORNO (TORNO) (Italy) (16);
BTTxx515 (QH0515), BTTxx692 (QH0692), and BTTZ4589 (QZ4589) (Trin-
idad) (26); BHT92593 (92HT593) and BHT92594c (92HT594) (Haiti) (40);
BBR92020 (92BR020), BBR92021 (92BR021), and BBR92028 (92BR028) (Bra-
zil) (41); BUSxxME1 (ME1) and BUSxME46 (ME46) (United States) (24);
BUS92712 (92US712) and BUS93073 (93US073) (United States) (40);
BTH93305 (93TH305) (Thailand; unpublished; previously thought to be clade
E); BUSxxBaL (BaL) (United States) (54); BFRxBXO8 (BX08) (France) (87);
BUSxxx44c (clone 44; derived from a patient designated AC10 [D. Montefiori,
unpublished data]), BUSxJRFLc (JR-FL), and BUSJRCSFc (JR-CSFc; a clone
related to the published JR-CSF sequence) (United States) (103); and
BUSSF162c (United States) (25). Molecular clones of 3 T-cell line-adapted
viruses were BFRxIIIBc (HXB2c; a clone related to the published HXB2 se-
quence) (France) (117), BFRxNL43c (NL4-3) (France) (125), and BUSxxxMNc
(MN) (United States) (105).
Clade C viruses were CIN93101 (93IN101) (85), CIN93905 (93IN905; also
known as 301905) (70), CIN93999 (93IN999; 301999) (70), 21068 (70), CIN11246
(94IN11246-3; also known as 11246-3) (70), and CIN98022 (98IN022) (India)
(123); CMW93959 (93MW959; 301959), and CMW93960 (93MW960, 301960)
(Malawi) (40); CBR98004 (98BR004) (Brazil) (123); CZA97012 (97ZA012)
(South Africa) (123); and CCN98006 (98CN006) and CCN98009 (98CN009)
(China) (123).
Clade D viruses were DUG93082 (93UG082), DUG94118 (94UG118),
DUG92005 (92UG005), DUG92038 (92UG038), and DUG92024 (92UG024)
(Uganda) (40, 41).
Clade AE viruses (CRF01_AE recombinants comprised of almost full-length
clade E Env with small fragments of clade A sequence at the N terminus of gp120
and the C terminus of gp41) were ETH92001 (92TH001), ETH92005 (92TH005),
(92TH022), ETH92024 (92TH024) (3), ETHCMU02 (CMU02) (81), and CMU06
(Thailand) (116).
Clade F viruses were FBR93020 (93BR020) and FBR93029 (93BR029) (Bra-
zil) (40). BFBR93019 (93BR019) is a BF hybrid (Brazil) (40).
Some of the Env sequences are from quasispecies and are therefore of uncer-
tain length in the variable domains. The Env sequence from the ARW92021 Env
quasispecies was so heterogeneous that it could not be determined with certainty.
Accordingly, this sequence was not submitted to the database. A depiction of
novel interclade recombinant Env genes in this panel is shown in Fig. 1, to give
an indication of the sites of recombination between Envs from different clades
that form these hybrids. Many of these recombinants have breakpoints down-
stream of the gp41 transmembrane domain, so that the entire ectodomain is from
a single clade (viruses AGxVLGC1, AGxVLGC2, BGPTVLGC3, BFITVLGC1,
BFITVLGC2, BFBRVLGC3, and BFBR93019). In contrast, the AC hybrids
ACUG93086 and ACKE94105 have mixed gp120/gp41 ecotodomains.
Live primary-virus isolates provided by the National Institutes of Health AIDS
Reference Reagent Program were grown in PBMCs, as described previously (154).
The 25 viruses that matched viruses in our pseudovirus panel were ARW92021,
BHT92594c, BUS92712, BUSxxBaL, BUSJRCSF, BUSxJRFL, BUSxxxME1,
BUSxxME46, BTTQZ458, BUSSF162, BFBR92019, CIN93101, CMW93959,
CMW93960, CIN11246, DUG92021, DUG92038, DUG92046, and FBR93020.
Neutralization assays. (i) Single-round pseudovirus neutralization assay. A
recombinant-virus assay involving a single round of virus infection was used to
measure neutralization (110, 120). Recombinant luciferase pseudoviruses were
incubated for 1 h at 37°C with 10 serial fourfold dilutions of MAbs or heat-
FIG. 1. Depiction of the genetic composition of recombinant viruses. Along the top, from left to right, on the gp160 sequence, we annotate the
signal peptide (SP), the gp120 variable loops, the fusion peptide (FP) of gp41, the helical regions HR1 and HR2, the transmembrane domain (TM),
and the leucine zipper domains in the cytoplasmic tail (LZ1 and LZ2). Amino acid positions are depicted along the bottom. Env fragments from
different clades are represented in color. The approximate recombination junctions were gleaned from sequence information from each of these
viruses submitted to the Los Alamos database.
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inactivated plasma, usually starting from 50 ?g/ml (MAbs) or a 1:20 dilution
(plasma). In a variant protocol, virus was incubated with antibody for 18 h before
the mixture was added to target cells. U87 cells expressing CD4 plus the CCR5
and CXCR4 coreceptors were inoculated with virus-antibody (Ab) dilutions in
the absence of added cations. Virus stocks were screened to ensure that they
were functional and yielded a high luciferase reporter light signal in target cell
lysates. Input virus used in each experiment was not standardized because prior
experiments (not shown) had demonstrated that the input virus dose does not
affect the neutralizing 50% inhibitory concentrations (IC50s) of antibodies. Virus
infectivity was determined 72 h postinoculation by measuring the amount of
luciferase activity expressed in infected cells. Neutralizing activity is reported as
the concentration or dilution of each MAb or plasma required to confer 50%
(IC50) or 90% (IC90) inhibition of infection (percent inhibition ? {1 ? [lucif-
erase ? Ab/luciferase ? Ab]} ? 100). To eliminate nonspecific neutralization,
the criterion for genuine neutralization is that the titer must be at least 2.5-fold
higher against HIV-1 than it is against the amphotropic control MuLV. Due to
the large size of this study, each individual virus-Ab combination was in general
tested only once. To ensure that the results were reproducible, the control viruses
JR-CSF (R5-tropic) and NL4-3 (X4-tropic) were run at least six times in all
assays. The reproducibility of the assay within and between runs was assessed by
looking at these controls and was found to be within 2.5-fold.
(ii) PBMC neutralization assay. Traditional neutralization assays were per-
formed using primary virus grown in PBMCs, as described previously (153). Each
experiment was performed in triplicate. Briefly, using 96-well round-bottom
tissue culture plates, 50 ?l of MAb at 100 ?g/ml and 50 ?l of primary-isolate virus
(100 50% tissue culture infective doses) were coincubated for 1 h at 37°C, and
then 100 ?l of phytohemagglutinin-activated target PBMCs (105/ml) was added.
Neutralization was evaluated at a single MAb concentration (50 ?g/ml, according
to the MAb concentration in the virus-MAb mixture before being added to the
plate). After an overnight incubation, the cells were washed twice with tissue
culture medium. On day 4, 100 ?l of the medium was replaced with fresh tissue
culture medium. On day 7, the supernatants were collected and treated with 1%
(vol/vol) Empigen (Calbiochem). Neutralization was measured as a reduction in
p24 production in the supernatants. p24 was assayed using an in-house enzyme-
linked immunosorbent assay, as described previously (88). Production of p24 in
the MAb-containing cultures was compared to p24 production in cultures with-
out MAb run in the same assay. Neutralization was recorded in assays that
resulted in a 10-fold reduction in the mean p24 content. Each virus-MAb com-
bination was assayed at least twice to ensure consistency. The PBMCs used came
from a pool of three anonymous healthy donors and were constant throughout all
neutralization assays.
Two-dimensional hierarchical clustering of viruses and MAbs based on neu-
tralization susceptibility profiles. Viruses were clustered according to their IC50
neutralization susceptibility profiles so that those with shared susceptibilities
were grouped together. The MAbs were also arranged according to their abilities
to neutralize the panel of viruses. Dendrograms to show the clustering patterns
for viruses and for MAbs were then derived. The most common clades in the
study were assigned colors, indicated on the ends of dendrogram branches.
Dendrograms were generated using methods described by Hastie et al. (51) and
implemented using the hierarchical clustering tool and the Mcquitty clustering
algorithm in R (version 1.8.1, 2003; The R Foundation for Statistical Computa-
tion) (54a). The actual neutralizing scores were changed to 0, 1, 2, 3, and 4,
encoding undetectable to highly sensitive neutralization, and the vectors of the
scores were used as the basis for clustering.
Nucleotide sequence accession numbers. Sequence data for each of the Envs
have been submitted to GenBank (accession numbers AY669697 to AY669786).
Comparison of neutralization by pseudovirus and tradi-
tional PBMC assays. To verify that the pseudovirus assay
yields meaningful neutralization data, we initially compared
b12, 2G12, X5, 2F5, and 4E10 activities against 25 viruses in
pseudovirus and traditional PBMC neutralization assays (Fig.
2). In the PBMC assay, neutralization was scored as positive if
?90% neutralization was observed at a MAb concentration of
50 ?g/ml. In the pseudovirus assay, the MAb concentration
that gave 90% neutralization was determined, and the data
were coded so that the warmer the color, the more potent the
neutralization. We arbitrarily took 50 ?g/ml as the neutraliza-
tion cutoff.
Overall, we observed fairly concordant patterns in the two
assays: 73% of the 125 assay reactions compared were either
positive in both assays or negative in both assays, and 27% of
the reactions were discordant. Comparing the neutralizations
in PBMC and pseudovirus assays purely on the basis of the
presence or absence of any detectable neutralization, we ob-
served the following concordance: b12, 84%; 2G12, 76%; X5,
72%; 2F5, 76%; and 4E10, 52%. Therefore, for b12, 2F5, and
2G12, the concordance was 75 to 85%, but for X5 it was
somewhat lower and for 4E10 it was considerably lower.
Clearly, then, much of the discordance can be attributed to
4E10, which weakly neutralized 14 of 25 viruses in the
pseudovirus assay, yet only 2 of these viruses were neutralized
in the PBMC assay (IC90? 50 ?g/ml). It appears that the
pseudovirus assay is more sensitive than the PBMC assay for
detecting 4E10 activity. When 4E10 is removed from the over-
all MAb comparison, the data concordance of the remaining
reactions increases from 73 to 78%. In contrast, MAb X5
neutralization is more easily detected in the PBMC assay. This
might be explained by differing levels of CCR5 coreceptor
expression on the target cells, which could affect the window of
opportunity for X5 to neutralize virus after it has engaged
CD4. Experiments to evaluate the discordant X5 results are
ongoing. Since X5 does not neutralize particularly effectively in
either assay, giving many double-negative results, this discor-
dance does not have as large an impact on the overall concor-
dance as does 4E10. Similarly, 2G12 shows 43% discordance in
neutralization against clade B viruses, but this is compensated
for by the 100% double-negative results against the non-B
isolates in the panel. For the remaining two MAbs, b12 and
2F5, the sensitivities of the two neutralization assays appear to
be quite similar.
Effect of virus-MAb incubation time on neutralization. To
determine the most appropriate pseudovirus neutralization as-
say protocol for our analysis, we initially determined the effect
of the virus-MAb incubation time on IC50titers. Comparing 1-
and 18-h incubations with a subset of mostly clade B viruses
(Fig. 3), we observed a mean 6.2-fold increase in neutralization
sensitivity in the 18-h format. However, there was significant
variability in the factor increase that appeared to be highly
virus and MAb dependent. Some virus-MAb mixtures resulted
in dramatically increased neutralization in the 18-h format. For
example, the increase in BFRxNL4-3c neutralization was 73.6-
fold with b12 and 99.4-fold with 2F5 but only 2.5-fold with
2G12. In general, 2G12 showed a disproportionately smaller
increase in neutralization (mean, 2.5-fold increase) compared
to the other MAbs. The dramatically increased neutralization
with 18 h of incubation was not restricted to neutralization-
sensitive viruses like BFRxNL4-3c. For example, BUS92712
was 15-fold better neutralized by 4E10 in the 18-h format.
Since the discrepancies are neither entirely virus nor MAb
specific, it is difficult to be sure what underlies this phenome-
non. It is possible that the discrepancies are related to MAb-
induced virus shedding of gp120 that might progressively in-
crease with longer MAb-virus incubation times (88, 112). We
decided to use the 1-h format for subsequent analysis both to
avoid this caveat and to generate data that can be compared
VOL. 78, 2004 CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs13235
Page 5
more easily to previously published data, where a 1-h virus-
MAb incubation phase is the norm.
Cross-clade neutralization analysis. (i) Overview. We next
sought to comprehensively assay a collection of antibodies
against a large panel of viruses. In light of the difficulties of
growing large numbers of primary viruses, we took advantage
of the relatively convenient pseudovirus assay. We chose a
genetically and geographically diverse panel of viruses, includ-
ing those for which live virus stocks are available, to enable a
comparison of results to those from traditional PMBC-based
neutralization assays (as discussed above and shown in Fig. 2).
Our panel included some viruses from early in the pandemic in
an attempt to provide data that could be compared with data
already published for these viruses. We also included some
contemporary and recently transmitted viruses that are typi-
cally available as uncloned quasispecies. Overall, we emphasize
that, while the 93 viruses were selected to represent the broad-
est possible cross section of Env diversity from those available
at ViroLogic, our panel should not be interpreted to accurately
represent the percentages of each type of virus circulating
globally. For example, compared to global estimates, our panel
has a relatively high emphasis on clade B viruses and relatively
little emphasis on clade C isolates. In addition, since we are
concerned about selecting naturally occurring Env proteins, we
did not limit our analysis to viruses of "pure" clade pedigree
but instead selected several recombinant viruses in an attempt
to factor in the relatively common populations of interclade
recombinant viruses.
The collection of antibodies we tested included five known
neutralizing MAbs (b12, 2G12, 2F5, 4E10, and 447-52D), three
control MAbs known to have modest neutralizing activities
(58.2, X5, and b6), an equimolar five-MAb cocktail (b12, 2G12,
2F5, 4E10, and 447-52D), and a plasma sample (N16) from a
clade B HIV-1-infected donor. The neutralizing IC50s (and
dilutions, in the case of the N16 plasma) using a 1-h virus-Ab
incubation phase are presented in Fig. 4. The Env-expressing
plasmids encoded either molecular clones (C) or Env quasi-
species cloned by reverse transcription-PCR from virus in
HIV?donor plasma (uncultured quasispecies [UCQ]) or from
virus that had been cultured in primary lymphocytes (quasi-
species [Q]).
Figure 5 summarizes the IC50data, showing the total num-
ber of viruses neutralized by each MAb with an IC50below 50
?g/ml, regardless of potency. 4E10 neutralized all the viruses;
2F5, b12, and 2G12 neutralized 41 to 67%, and the V3 loop
MAbs neutralized ?20% of the total. At IC90(Fig. 6 and 7),
4E10 still neutralized 56% of the viruses, a larger proportion of
isolates than any other MAb. 2F5 also retained neutralizing
FIG. 2. Comparison of neutralization activities of MAbs b12, 2G12, X5, 2F5, and 4E10 in pseudovirus and PBMC neutralization assays against
25 viruses. For the PBMC assay, an IC90of ?50 ?g/ml is depicted by a gray square, and an IC90of ?50 ?g/ml is shown as a white square. In the
pseudovirus assay, IC90data are shown. To assist comprehension, neutralization in the pseudovirus assay has been color coded so that the warmer
the color, the more potent the neutralization: a white box indicates an IC90of ?50 ?g/ml, a green box indicates 50 ?g/ml ? IC90? 10 ?g/ml, a
yellow box indicates 10 ?g/ml ? IC90? 1 ?g/ml, an orange box indicates 1 ?g/ml ? IC90? 0.1 ?g/ml, and a red box indicates an IC90of ?0.1
?g/ml. N.D., not done.
Page 6
activity against a large proportion of the isolates. The neutral-
izing activities of b12, 2G12, and 2F5 outside clade B were
diminished, so that most of the b12 and 2G12 activity was now
concentrated in clade B. V3 loop MAbs were only weakly
effective at IC90. 2F5 and 4E10 still neutralized all AE recom-
binant viruses but were no longer the most broadly neutralizing
MAbs against clade B viruses; the most broadly neutralizing
MAb was now b12. At IC95, we observed that 4E10 still neu-
tralized several more isolates than any other MAb, but its
potency was very low (data not shown).
Considering that all the MAbs were isolated from clade
B-infected persons, it is interesting to compare the neutraliza-
tion of clade B to that of non-clade B viruses. Each MAb
neutralized (IC50, ?50 ?g/ml) clade B and non-B isolates,
respectively, as follows: b12, 72 vs. 39%; b6, 17 vs. 0%; 2G12,
72 vs. 26%; 447-52D, 45 vs. 7%; 58.2, 31 vs. 2%; X5, 10 vs. 0%;
2F5, 79 vs. 61%; and 4E10, 100 vs. 100%. From this, we can
infer that gp120 MAbs neutralize a lower percentage of non-
clade B viruses than they do clade B viruses but that b12 and
2G12 are relatively cross-clade neutralizing. Gp41 MAbs are,
however, significantly more cross-reactive outside clade B than
the gp120 MAbs. The mean neutralization titers of each MAb
against clade B and non-B viruses were also calculated from
Fig. 4 as follows (values are in micrograms per milliliter): b12,
2.65 and 10.22; b6, 16.48 and not applicable; 2G12, 3.46 and
17.06; 447-52D, 11.90 and 22.42; 58.2, 11.11 and 44.57; X5, 1.39
and not applicable; 2F5, 6.26 and 7.33; and 4E10, 7.37 and 5.40.
Again, it is apparent that each gp120 MAb neutralizes clade B
viruses more effectively (with a range of differences from 1.88-
to 4.93-fold). As mentioned above, this may be because some
clade B viruses in our panel were derived from viruses that
have been more extensively passaged in culture and are there-
fore inherently more neutralization sensitive. However, if this
is the case, it does not appear to have significantly affected the
activities of the gp41 MAbs. X5 has an apparently very high
mean titer against clade B viruses, but this is artificially en-
hanced by the fact that the MAb neutralizes only a few very
sensitive isolates and has no detectable activity against more
resistant isolates.
(ii) Conformational masking of Env epitopes. In Fig. 8, we
present sequence data relevant to the neutralization of MAbs
directed to linear epitopes (4E10, 2F5, 447-52D, and 58.2).
This helps define the minimal epitope for recognition by these
MAbs. It is clear that, while substitutions in the conserved core
epitopes can predict neutralization resistance, the presence of
the correct core sequence cannot predict neutralization. In-
stead, distal regions of Env may influence neutralization po-
tency. For example, the presence of the correct V3 sequence
motifs resulted in 447-52D neutralization of 20 of 32 (62%)
GPGR-containing viruses. Neither 58.2 nor 447-52D neutral-
izes BUSTORNO, BBRVLGC2,
BFBRVLGC3, despite the presence of HIGPGRAF at the tip
of the V3 loop. This is probably because the V3 loop is fre-
quently sequestered in neutralization-resistant isolates (13,
146). Conformational masking is not limited to MAbs directed
to linear epitopes; those that recognize discontinuous epitopes,
like the CD4 binding site and CD4-induced epitopes, have very
well-conserved epitopes that are frequently subject to steric
occlusion. A more detailed analysis of the relationship of Env
sequence polymorphisms to MAb neutralization, particularly
for those that recognize conformational epitopes, will be pre-
sented elsewhere.
(iii) Analysis of the neutralizing activity of each MAb. (a)
4E10. The most remarkable finding from our analysis is that
MAb 4E10 neutralized all 93 viruses with IC50s ?50 ?g/ml in
the pseudovirus assay (Fig. 4). This is consistent with, and
BFBRVLGC1, or
FIG. 3. Effect of MAb-virus incubation time in pseudovirus neutralization assays. Viruses were incubated with MAb for either 1 or 18 h as
indicated, and the IC50s (?g/ml) were determined. The neutralization titers are color coded as in Fig. 2. The differences in IC50, expressed in ratio
form, are also color coded, so that those with bigger discrepancies are shown in warmer colors and vice versa. N/A, not applicable; N.D., not done.
VOL. 78, 2004CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs 13237
Page 7
Page 8
significantly expands, a recent study in which 4E10 neutralized
an entire panel of clade B viruses in a pseudovirus neutraliza-
tion assay (72). The neutralization observed is specific, as con-
firmed by the lack of nonspecific activity against the ampho-
tropic MuLV control. Although panreactive, 4E10 is only
modestly potent; rather than neutralizing a few viruses very
potently, 4E10 consistently neutralizes viruses with an even
potency. For example, it does not neutralize sensitive clade B
isolates as potently as some of the other MAbs (the average
IC50of 4E10 against clade B viruses is 7.4 ?g/ml; for b12, it is
2.6 ?g/ml; for 2G12, it is 3.5 ?g/ml; for 2F5, it is 6.3 ?g/ml).
The sequence data (Fig. 8A) indicate the minimum require-
ments for 4E10 recognition. Previously, 4E10 was mapped to
an epitope in gp41, NWFDIT (Env amino acid positions 671 to
676), with some sequence promiscuity at positions 671, 674,
and 676 (134, 153). Given the present data, we can now further
define the WFXI motif as the minimum core requirement for
4E10 neutralization. This motif is preserved in 2,828 of 2,894
(98%) M group HIV-1 sequences that span this portion of
gp41 presently available in the Los Alamos HIV database
(http://www.hiv.lanl.gov) and is highly conserved in all pure-
clade and recombinant forms. A variant with an F3L substi-
tution at position 673 is the most common variant; it is pre-
dominant in the O (outlier) virus group and was found in 31 of
2,894 (1%) M group sequences. However, the precise se-
quence of the 4E10 epitope in each virus is not the only factor
that determines the strength of neutralization. For example,
while BUSxxxMNc is neutralized with an IC50of 0.17 ?g/ml,
BUS93073, bearing an identical 4E10 epitope sequence, is
neutralized with an IC50of 11.7 ?g/ml (i.e., ?67-fold weaker
neutralization). This suggests that expression of the 4E10
epitope is dependent on structural context.
(b) 2F5. MAb 2F5 is also broadly neutralizing, although not
to the extent of 4E10. Our data confirm previous studies that
showed that 2F5 can neutralize viruses from clades A, B, D,
and E (35, 36, 114, 134, 138). However, 2F5 had no activity
against any of the clade C viruses tested. Scant activity was
found against clade D viruses. Sequence information (Fig. 8B),
together with previous peptide-mapping studies, defines the
minimal requirement for 2F5 recognition as DKW (Env posi-
tions 664 to 666 in gp41). Many viruses resistant to 2F5 neu-
tralization had a substitution in the DKW motif. For example,
most clade C viruses had DSW in place of the DKW motif. The
lack of clade C virus neutralization in our panel does not rule
out the possibility that 2F5 may occasionally neutralize viruses
from this clade. For example, it was previously shown that 2F5
neutralized a Zambian isolate, ZAM20 (138). However,
ZAM20 has an unusual 2F5 epitope for a clade C virus
(ALDKWN). Similarly, 2F5 was able to neutralize the AC
hybrid ACUG93086. This is consistent with the entire gp41
segment of this hybrid originating from a clade A isolate (2F5
epitope sequence, ALDKWA). In the Los Alamos HIV data-
base, the motif DKW is preserved in only 54 of 212 (25%)
clade C gp41 sequences sampled from 19 nations. The 2F5-
reactive motif, DKW, is enriched in certain regional clade C
samples; for example, it was found in 15 of 17 (88%) clade C
sequences from Burundi, 6 of 6 (100%) sequences from Brazil,
and 8 of 10 (80%) sequences from Ethiopia, while it is very
rare in other countries (for example, it was found in only 1 of
71 [1%] clade C sequences from Botswana). It is very likely
FIG. 5. Summary of the total number of primary viruses neutralized with an IC50, of ?50 ?g/ml. We excluded the data from T-cell line-adapted
isolates BUSxxxMNc, BFRxNL43c, and BFRxIIIBc. The data have been color coded as follows: a white box indicates that no viruses were
neutralized, a green box indicates that 1 to 30% of viruses were neutralized, a yellow box indicates that 31 to 60% of viruses were neutralized, an
orange box indicates that 61 to 90% of viruses were neutralized, and a red box indicates that ?90% of viruses were neutralized.
FIG. 4. IC50titers in the pseudovirus neutralization assay, using eight MAbs, the five-MAb cocktail, and the clade B HIV?plasma against 93
HIV-1 isolates. The MAbs are organized horizontally, and the viruses are organized from top to bottom. In the leftmost columns, viruses have been
grouped into clades A through J and are also color coded to reflect their country of origin where known. The viruses are also categorized as
molecular clones (C), cultured quasispecies (Q), or uncloned quasispecies made directly from plasma samples (UCQ). The MAb neutralization
titers have been color coded in exactly the same way as in Fig. 2. For the plasma, the color codes are as follows: a white box indicates an IC50of
?1:50 dilution, a green box indicates 1:50 ? IC50? 1:100, a yellow box indicates 1:100 ? IC50? 1:500, an orange box indicates 1:500 ? IC50?
1:1,000, and a red box indicates an IC50of ?1:1,000. Based on overall neutralizing potency, the viruses in each clade have been organized from
the most resistant at the top to the most sensitive at the bottom.
VOL. 78, 2004 CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs13239
Page 9
FIG. 6. IC90titers against the panel of 93 viruses. The data are organized as in Fig. 4.
Page 10
that there is a geographic lineage association with 2F5 suscep-
2F5 neutralized only 5 of 11 (45%) clade D viruses, while the
motif DKW is present in 64 of 91 (70%) clade D virus gp41
sequences available in the HIV sequence database. The clade
D viruses available for this study were all derived from Ugan-
dan samples, and those that were neutralized by 2F5 (5 of 11)
all exhibited the DKW motif, whereas the remaining 6 exhib-
ited a DQW motif. In the sequences from the database, 41 of
67 (61%) Ugandan samples carried DKW, while outside of
Uganda, almost all available clade D viruses—22 of 23 (96%)
clade D gp41 sequences from nine nations—carried the DKW
motif and so had the potential to react with 2F5. Thus, our
relatively low clade D susceptibility may be a reflection of
sampling from Uganda; clade D viruses from other regions are
likely to be more susceptible. Overall, as with clade C viruses,
there may be a geographic lineage association with the ob-
served 2F5 susceptibility among clade D viruses.
As with all the MAbs to linear epitopes, the presence of the
target sequence did not always predict 2F5 neutralization: the
exceptions here were the Brazilian isolates BFBR93019,
BFR93020, and CBR98004; the Rwandan isolate ARW93029;
and BITxPAVO. The DKW epitope motif may be conforma-
tionally masked in these viruses, so that neutralization was not
detectable at ?50 ?g/ml. Overall, widely different neutraliza-
tion potencies against viruses with the same 2F5 epitope se-
quence were observed (Fig. 8).
(c) b12. b12 was also broadly and potently neutralizing (Fig.
4 and 5) but not quite as broadly neutralizing as 2F5, and the
distribution of neutralized viruses was clearly quite different.
b12's activity was distributed more evenly across clades.
Whereas b12 neutralized a fraction of viruses from almost
every clade and geographic location, consistent with previous
findings (138), 2F5 neutralized most viruses from some clades
but was very poor against others. b12 was also significantly
more potent than 2F5 and 4E10 against many virus isolates,
especially those from clade B (as noted above).
(d) b6. b6 is a prototype "nonneutralizing" CD4bs-specific
MAb derived from the same HIV?human donor as b12 (5, 19,
121). Inclusion of this MAb provides a benchmark for the
activity of "garden variety" CD4bs MAbs. In the single-round
assay, b6 did not neutralize any isolate that was not neutralized
by b12, and it was never as potent. Here, we found that b6
neutralizes only a small number of highly neutralization-
sensitive clade B primary viruses and T-cell line-adapted virus
strains (BUSx1196, BUSxxBaL, BFRxBX08, BTTQZ458,
BUSSF162, BFRxIIIBc, BFRxNL43c, and BUSxxxMNc). It is
likely that steric restrictions limit the access of this MAb to its
epitope on the vast majority of primary viruses (13, 63, 121,
(e) 2G12. MAb 2G12 neutralized a proportion of isolates in
our panel similar to that neutralized by b12 but with a pattern
that was more restricted by clade (Fig. 4 and 5). It had no
activity against clade C or E isolates in our panel. This is
consistent with, and expands, previous findings (17, 35, 134,
138). 2G12 is thought to recognize glycans at residues 332 and
392, with some dependence on glycans at positions 295, 339,
and 386 (23, 126, 129). The lack of clade C neutralization may
in some cases be due to a loss of the glycan at position 295 at
the C-terminal base of the V3 loop. In our group of 12 clade C
viruses, the only one that retains this site is CBR98004 (8%); in
the Los Alamos database, a somewhat higher fraction (474 of
2,042 [24%]) of C clade viruses retain this glycosylation site.
The lack of clade C neutralization may also be due to the loss
of the N-linked glycan at position 339 in viruses CIN98022,
CZA97012, and CBR98004 (25% of our viruses). In the data-
base (and in our group of clade C viruses), the glycan at
position 339 is fairly well conserved among clade C Env se-
quences (664 of 891 [75%]), comparable to its level of reten-
tion in most other clades (149). Since CBR98004 retains the
glycan at position 295, it is likely that the loss of one at position
339 leads to loss of 2G12 reactivity. Overall, the lack of a
glycan at position 295 appears to be most important in explain-
ing the lack of clade C neutralization. In contrast to its lack of
neutralization of pure clade C viruses, 2G12 does neutralize
the clade AC hybrids ACKE94105 and ACUG93086 in which
the glycosylation sites at 295 and 339 are retained.
2G12 does not neutralize CRF01 clade AE viruses, perhaps
due to an N-to-E mutation at position 332 at the C-terminal
side of the V3 loop that abolishes a critical glycan in the
epitope. The lack of clade AE neutralization may also be partly
explained by the presence of an additional disulfide bond in the
FIG. 7. Summary of the total number of primary viruses neutralized with an IC90of ?50 ?g/ml, regardless of potency. The data are organized
as in Fig. 5.
VOL. 78, 2004CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs13241
Page 11
Page 12
V4 region (129). Overall, with very few exceptions, it appears
that when the residues important for 2G12 are present, neu-
tralization is predictable. This may be because the 2G12
epitope is fully exposed on the outer domain of gp120 and not
subject to conformational masking as MAbs to other epitopes
appear to be.
(f) 447-52D. The V3 loop MAb 447-52D neutralizes 45% of
the clade B viruses tested at IC50?50 ?g/ml (Fig. 4 and 5) but
only 14% at IC90?50 ?g/ml (Fig. 6 and 7). It is particularly
effective against the globally most sensitive viruses (35, 91).
MAb 447-52D has been reported to occasionally neutralize
some viruses from non-B clades, including clades A, D, and F
(45, 48, 49, 101). However, in our panel, neutralization outside
clade B was rare (4 of 61 [?7%]). In one case (isolate
BFITVLGC2), 447-52D neutralized when no other anti-gp120
MAbs neutralized. Alignment of the V3 loop sequences con-
firms that the 447-52D core epitope is GPGR (Fig. 8C) (28,
45). While the dominant V3 sequence motif in clade B viruses
is GPGR (appearing in 1,272 of 1,911 clade B V3 sequences
[67%]), it appears in only 140 of 1,753 (8%) non-clade B M
group viruses, where the dominant V3 sequence motif is
GPGQ (42, 43). Therefore, 447-52D activity against non-B
viruses depends on the unusual presence of a GPGR V3 se-
quence (45, 49).
BUSJRCSFc virus was found to be resistant to 447-52D
here, but in other studies it was sensitive to this MAb (47). This
might be explained by two amino acid differences (503 R/A and
674 G/D) in distal locations between the BUSJRCSFc clone
used here and the previously published sequence. However, it
is not immediately clear how such mutations in the gp120 C5
domain and the C terminus of the gp41 ectodomain might
influence neutralization by V3 loop MAbs. As an alternative
explanation, it is possible that the particular BUSJRCSFc virus
stock used in previous studies underwent serial PBMC pas-
sages that rendered it more sensitive to V3 MAb neutralization
(8, 113).
(g) 58.2. Like 447-52D, MAb 58.2 neutralized a significant
proportion of clade B primary isolates but did not neutralize
any viruses outside clade B (Fig. 4 and 5). It is narrower in
breadth than 447-52D but not remarkably so. V3 loop sequenc-
ing suggested that its minimal epitope is (H/T)IGPGR(A/T)
(F/L) (Fig. 8D).
(h) X5. X5 is a MAb whose epitope on gp120 is preferen-
tially exposed by engagement of CD4; in other words, its
epitope is CD4 induced (CD4i). Like b6, X5 neutralized only
highly sensitive clade B isolates, as described recently (66). The
CD4i epitope therefore appears to be accessible in neutraliza-
tion-sensitive isolates, but it appears to be sterically occluded
in most primary isolates. However, smaller fragments of CD4i
MAbs are known to be able to neutralize primary isolates by
overcoming the steric restriction (66). Unlike the V3 loop, the
CD4i epitope is well conserved, since it overlaps the coreceptor
binding site. X5 was able to neutralize 2 of 25 non-B isolates in
the PBMC assay (Fig. 2). It was not able to neutralize these
viruses in the pseudovirus assay, perhaps due to differential
coreceptor expression, as discussed above.
(i) The five-MAb cocktail. It has been questioned whether
MAbs to different epitope clusters can synergize in achieving
greater neutralization potency or breadth. We found that a
cocktail of five MAbs (b12, 2G12, 447-52D, 2F5, and 4E10) at
FIG. 8. Mapping the sequence requirements for neutralization by MAbs to linear epitopes. Env sequences from each virus are depicted to show the epitopes of gp41 MAbs 4E10 (amino
acids 667 to 677) (A) and 2F5 (amino acids 658 to 667) (B) and V3 MAbs 447-52D (C) and 58.2 (amino acids 306 to 320 are shown) (D). For each MAb, to determine which amino acid
substitutions are permissible, viruses that were most potently neutralized were organized at the top of each column and those that were not neutralized were placed at the bottom. Those residues
that appear to be important for MAb binding are highlighted in red. A dash indicates consistency with the sequence at the top of each figure. A dot indicates a deletion. An X represents a
residue where there is variation at an amino acid position. Definitive sequencing of ARW92021 was not possible due to extensive sequence heterogeneity in the quasispecies.
VOL. 78, 2004 CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs13243
Page 13
FIG. 9. Two-dimensional hierarchical clustering of viruses and MAbs based on IC50neutralization profiles. Viruses were clustered according
to their neutralization profiles along the vertical axis. Simultaneously, the MAbs were arranged according to their abilities to neutralize the panel
of viruses. Dendrogram patterns are shown to the right (for viruses) and bottom (for MAbs). The same color scheme for neutralization data points
as in Fig. 2 was used. The most common clades in this study, A to F, were assigned a color, indicated on the end of the branches in the dendrograms.
No color was assigned for viruses from clades that were not represented by many samples in this study or for interclade recombinants.
Page 14
an equimolar ratio neutralized all viruses with an IC50?50
?g/ml. This was not surprising, considering that the cocktail
includes 4E10, which neutralizes every virus. The IC50of the
IgG cocktail in each case was similar to the average IC50of its
component MAbs (adjusting for the fact that in the five-MAb
cocktail, any one MAb concentration is reduced to 20%), in-
dicating a lack of obvious synergy. The IC90data (Fig. 6 and 7)
allow examination of the activity of the cocktail at a level of
stringency where 4E10 is no longer panneutralizing. Here
again, however, there is no evidence for strong synergistic
neutralization, nor was there a significant improvement in
breadth. Although the effects of the cocktail appear to be near
additive, our analysis is not sophisticated enough to rule out
the possibility of subtle synergistic effects.
(j) N16 HIV?plasma. Plasma from an HIV-1-positive donor
infected with a clade B virus exhibited weak neutralization of
a significant fraction of viruses from all clades (IC50s were
generally below 1:100 plasma dilution). As reported previously
by other groups (6, 10, 43, 58, 59, 71, 75, 90, 93, 102, 145), there
is a great breadth of response in interclade (non-B) reactions.
However, there was a modest preference for clade B neutral-
ization: excluding the sensitive viruses HXB2r, MN, SF162,
and NL4-3, 22 of 28 (79%) clade B viruses were neutralized,
while 40 of 61 (66%) non-clade B viruses were neutralized. The
neutralization potency of the plasma within clade B was also
statistically higher. If we exclude the four sensitive clade B
isolates, using a conservative nonparametric Wilcoxon test, the
clade B data have a distribution of IC50s that is higher, with a
median IC50titer of 72 and an interquartile range of 50 to 120
compared to the non-clade B median IC50of 60 with an inter-
quartile range of 43 to 89 (P ? 0.042). Therefore, there is an
indication of somewhat higher intraclade neutralization.
Neutralization of viruses bearing interclade recombinant
Envs. Our virus panel included several viruses with interclade
recombinant Envs (Fig. 1). We found that the neutralization
profiles of these viruses were largely predictable from the com-
positions of the hybrid Envs. The BF and BG recombinants were
entirely composed of Env ectodomains of clade B, the junction
with another Env clade arising either in the gp41 transmembrane
domain or the cytoplasmic tail (Fig. 1). Accordingly, their neu-
tralization profiles were similar to those of clade B viruses. Sim-
ilarly, the two AG recombinants behaved like clade A viruses,
with the possible exception that AGxVLGC1 is not susceptible to
2F5. Since the breakpoint between clade A and G Env proteins is
close to this epitope, clade G sequence could explain this 2F5
resistance, a point that could not be verified in the sequence due
to some heterogeneity in the DKW epitope (Fig. 8B). The AC
recombinants ACUG93086 and ACKE94105 are true Env hy-
brids and appear to have unique neutralization profiles. Unlike
2G12. Also, despite having an entirely clade A gp41, ACKE94105
still resists 2F5, due to a mutation in its core epitope (Fig. 8). It is
difficult to comment on the behavior of AE recombinants in the
absence of any pure clade E viruses to compare. However, these
that most of their Env sequence derives from clade E parents.
Two-dimensional hierarchical clustering of viruses and an-
tibodies based on IC50neutralization data. Dendrograms were
constructed (Fig. 9) in which viruses were clustered according
to their profiles of neutralization susceptibility (IC50data) to
the panel of antibodies, and antibodies were clustered accord-
ing to their patterns of reactivity with the panel of viruses,
using a two-dimensional clustering strategy (51). To our knowl-
edge, this represents the first attempt to classify viruses based
on their levels of neutralization sensitivity to MAbs. The vi-
ruses segregated into relatively clear clusters, 1 to 5 (Fig. 3).
The most common clades in this study, A to F, were assigned
colors, indicated on the end of each branch in the dendrogram.
When there is no colored box on the end of a branch, this is
because the virus was either from one of the clades not repre-
sented by many samples in this study or was a recombinant.
The dendrogram revealed that MAb neutralization patterns
are somewhat associated with clades, although imperfectly.
Statistical analysis confirmed that the clade association with
IC50neutralization clusters is highly significant (using a chi-
square test, P ? 10?15). This test was used to test the null
hypothesis that viruses were randomly distributed among the
different clusters defined on the basis of neutralization suscep-
tibility. Only clade B is found in cluster 1, the cluster that
defines the group of viruses that are most susceptible to neu-
tralization. This may be in part because clade B infections gave
rise to all the antibodies under investigation. Considering the
fundamental tenets of immune reactivity, it might be expected
that these MAbs would be most reactive with clade B viruses.
The presence of clade B Envs in cluster I may also be partly
attributed to the fact that some of the clade B isolates are, in
general, particularly neutralization sensitive, perhaps because
some of them have been cultured more extensively in vitro or
selected and cloned, a process that tends to render viruses
more neutralization sensitive.
It could be argued that the association between clades and
neutralization susceptibility profiles is solely driven by clade B
viruses in cluster 1, especially since clusters 2 to 5 contain
viruses from more than one clade. Therefore, we performed a
chi-square test on the distribution of non-clade B viruses in
clusters 2 to 5. Here, the association remained highly signifi-
cant (P ? 0.00002). In Fig. 10, the clades and neutralizing
clusters have been plotted histogramatically using the informa-
tion from Fig. 9. It can be seen more clearly that there is an
association of clade with cluster. Cluster 2 is formed mostly
from clade C and D viruses and is characterized by resistance
FIG. 10. Neutralization patterns are associated with clades. The
total number of viruses of each clade in each neutralization immuno-
type cluster are shown.
VOL. 78, 2004 CROSS-CLADE NEUTRALIZATION ANALYSIS OF ANTI-HIV-1 MAbs13245
Page 15
to 2G12 and 2F5. All CRF01 AE viruses (10 of 10) are found
in cluster 3, with some contributions from A and B viruses.
Resistance to 2G12 but sensitivity to 2F5 and the clade B
plasma characterize this cluster. Cluster 4 contains viruses
from several clades. Cluster 5 includes clade A, C, and F
viruses, and five of seven of the clade F viruses are in this
cluster. Cluster 5 generally shows resistance to b12 and 2G12.
The only geographic trend readily apparent in the clustering
data was that no clade B viruses from Brazil appeared in the
highly reactive cluster 1; four of five of the Brazilian B viruses
were located in cluster 3 and were distinguishable from other
B viruses in being quantitatively less susceptible to 2F5, 4E10,
and b12 and resistant to 2G12.
Clustering based on IC90neutralization profiles (data not
shown) was less informative than the IC50profile, in part be-
cause there were so many reactions that had undetectable
neutralization. Still, some patterns are clearly evident: clade C
viruses tended to be sensitive to 4E10, and clade E (CRF01)
tended to be sensitive to both 4E10 and 2F5. Many clade A, D,
and F viruses could not be neutralized by any MAbs, even at
the highest concentration tested. Among these three clades,
those that could be neutralized by one or two of the MAbs
showed sporadic, clade-independent sensitivity to particular
Comparison of the PBMC and pseudovirus neutralization as-
says. We observed reasonable agreement between the
pseudovirus and PBMC neutralization assays reported here
(Fig. 2) and elsewhere (17, 48, 49, 134, 153). Our results lend
support to the pseudovirus assay as a useful and relevant ap-
proach for measuring neutralization. In an unpublished mul-
tilaboratory blinded comparison of the neutralization activities
of several MAbs and HIV?sera in various neutralization as-
says (D. Montefiori, personal communication), the pseudovi-
rus assay also showed good concordance with PBMC neutral-
ization assays, although there were some differences in assay
sensitivities. We found the greatest discrepancies between the
two assays for the MAbs 4E10 and X5. As discussed below, it
is possible that these inconsistencies are related to different
virus infection kinetics with the target cells used in each assay,
as reported by Reeves et al. (118), or perhaps to differences in
the nature of the viruses used.
(i) Discrepancy in 4E10 neutralization. A significant obser-
vation was that 4E10 neutralized all the viruses in our panel in
the pseudovirus assay (Fig. 4). However, in PBMC assays here
(Fig. 2) and elsewhere, 4E10 did not neutralize all viruses (134,
153). To explain this discrepancy, it could be argued that the
PBMC assay is not sensitive enough to detect 4E10 neutral-
ization, especially given that PBMC assay neutralization is
usually reported at IC90. Alternatively, it could be argued that
the ability of 4E10 to neutralize after CD4 and CCR5 binding
(postattachment) (11) may make it particularly sensitive to the
receptor expression levels or the nature of the target cell types.
Notably, MAb 2F5, which has also been suggested to be capa-
ble of some virus neutralization after CD4 and CCR5 engage-
ment, neutralized equivalently in both neutralization formats.
Overall, the basis of the differential activity of 4E10 in these
neutralization assays remains unresolved. The panneutralizing
activity of 4E10 in the pseudovirus assay suggests that it rec-
ognizes a very highly conserved site on the viral Env protein
that may be essential for virus replication; this is consistent
with the preservation of the core epitope for 4E10 in 98% of
the sequences in the HIV database (however, 4E10 does not
neutralize SIVmac239 and SIVmac316 [J. Binley, unpublished
observations]). For this reason, it would be of interest to de-
termine if escape from 4E10 neutralization is possible, and if
so, how this affects the resistant virus.
(ii) Discrepancy in X5 neutralization. In contrast to 4E10,
MAb X5 exhibited neutralization in the PBMC assay but not in
the pseudovirus assay (Fig. 2). X5 recognizes Env that has
engaged CD4 but not coreceptor (66). Differing coreceptor
densities on the target cells could affect the window of time
during which this epitope is available. X5 neutralization in the
pseudovirus assay was limited, perhaps because of the rela-
tively high levels of coreceptor on the U87 target cells (C. J.
Petropoulos and T. Wrin, preliminary data) that increase in-
fection kinetics and decrease the opportunity for X5 neutral-
(iii) Other discrepancies. Other discrepancies occurred in
which there was neutralization in the pseudovirus assay but not
in the PBMC assay (Fig. 2). These might be explained by
differences in Env sequence in the pseudovirion and PBMC-
derived virus populations, which might be related to the pas-
sage history of the viruses and Envs used. It is known that
prolonged PBMC culturing renders primary viruses progres-
sively more neutralization sensitive (8, 113). The Env on the
surfaces of the pseudovirions may be derived from a molecular
or biological clone, low- or high-passage PBMC stock, or cir-
culating virus that may differ somewhat from the PBMC-de-
rived live-virus stock to which it is compared in the other
neutralization assay format. It is also possible to explain some
discrepancies by the fact that some viruses have relatively rapid
replication kinetics in the PBMC assay, and in these cases, the
nonneutralized virus subpopulation may rebound significantly
by the time of harvesting on day 7.
There are also some differences between our PBMC assay
results and PBMC assay results reported previously. In an
earlier report, 4E10 neutralized isolate ARW92021 potently
(134), as it did in the pseudovirus assay here. However, we did
not observe neutralization in our PBMC assays (Fig. 2). This
may be due to differences in the assay sensitivity or passage
It has been reported that b12 can, at certain concentrations,
lead to enhancement of virus infection (usually ?40%) (104).
However, we did not observe any infectivity enhancement in
pseudovirus neutralization assays. Although enhancement
might arise with particular clade A Envs (104), it is more likely
to be a phenomenon associated with the conditions of neutral-
ization assays, for example, the receptor density on the target
Comparison of neutralization against gp120 and gp41
epitopes. Our results highlight a dichotomy in the behavior of
the anti-gp120 and anti-gp41 MAbs. Compared to the anti-
gp120 MAbs, the anti-gp41 MAbs neutralize more evenly, with
relatively greater breadth but with a somewhat lower potency.
The greater breadth of the gp41 MAbs might be anticipated,
given the conservation of their epitopes. Their relatively mod-
est potencies, even against neutralization-sensitive clade B vi-


text:
Perhaps it is just someone sick of conspiracy sites polluting the minds of the easily swayed.
quote:but if someone cares enough to single them out, maybe there really is something out there someone doesn't want seen.
quote:Here is a recording of the hacker calling one forum co-owner on her private phone.Kinda scary....
Mention "TAVISTOCK" on that site and see what they do!
Exactly.....GLP is said to be a front for govt.... Not a good site to be on! (They randomly block people for no reason,etc....
I gotta admit, I lol'd.A conspiracy theory about a conspiracy theory site. Regressive conspiracy theories!
1 recommendation
Aren't you at all bothered by the rampant anti-semitism and homophobia at the other end of your link?
Is there such thing as a "mainstream conspiracy website"?
If I try to connect to »www.godlikeproductions.com/ I get OUTGOING dropped packets from their IP.Outgoing from their IP?
From all I have heard about that site i wouldnt wanna be there!
3 recommendations
There's a guy locally offering to remove people's shrubs so they can see the feds hiding behind them. He says business is good.

subset: commoncrawl


text:
I bought the S5 on Craiglist and feel like I got rip off. The phone is the store display version. I can't do anything on it. I try Odin and nothing happens. Is there any other solution to remove store display version?
XDA Developers was founded by developers, for developers. It is now a valuable resource for people who want to make the most of their mobile devices, from customizing the look and feel to adding new functionality.
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text: Repositório Comunidade: http://hdl.handle.net/10451/4457 2015-03-29T16:33:07Z A systematic approach for designing a HBM Pilot Study for Europe http://hdl.handle.net/10451/17770 Título: A systematic approach for designing a HBM Pilot Study for Europe Autor: Becker, Kerstin; Seiwert, Margarete; Casteleyn, Ludwine; Joas, Reinhard; Joas, Anke; Biot, Pierre; Aerts, Dominique; Castano, Argelia; Esteban, Marta; Angerer, Jürgen; Koch, Holger M.; Schoeters, Greet; Den Hond, Elly; Sepai, Ovnair; Exley, Karen; Knudsen, Lisbeth E.; Horvat, Milena; Bloemen, Louis; Kolossa-Gehring, Marike; DEMOCOPHES consortium Resumo: The objective of COPHES (Consortium to Perform Human biomonitoring on a European Scale) was to develop a harmonised approach to conduct human biomonitoring on a Europeanscale. COPHES developed a systematic approach for designing and conducting a pilot study for an EU-wide cross-sectional human biomonitoring (HBM) study and for the implementation of the fieldwork procedures. The approach gave the basis for discussion of the main aspects of study design and conduct, and provided a decision making tool which can be applied to many other studies. Each decision that had to be taken was listed in a table of options with their advantages and disadvantages. Based on this the rationale of the decisions could be explained and be transparent. This was important because an EU-wide HBM study demands openness of all decisions taken to encourage as many countries as possible to participate and accept the initiative undertaken. Based on this approach the following study design was suggested: a cross-sectional study including 120 children aged 6–11 years and their mothers aged up to 45 years from each participating country. For the pilot study the children should be sampled in equal shares in an urban and a rural location. Only healthy children and mothers (no metabolic disturbances) should be included, who have a sufficient knowledge of the local language and have been living at least for 5 years at the sampling location. Occupational exposure should not be an exclusion criterion. Recruitment should be performed via inhabitant registries or schools as an alternative option. Measures suitable to increase the response rate should be applied. Preferably, the families should be visited at home and interviewed face-to-face. Various quality control measures to guarantee a good fieldwork performance were recommended. Descrição: © 2013 Elsevier GmbH. All rights reserved. 2014-01-01T00:00:00Z Economic benefits of methylmercury exposure control in Europe: monetary value of neurotoxicity prevention http://hdl.handle.net/10451/17690 Título: Economic benefits of methylmercury exposure control in Europe: monetary value of neurotoxicity prevention Autor: Bellanger, Martine; Pichery, Céline; Aerts, Dominique; Berglund, Marika; Castaño, Argelia; Čejchanová, Mája; Crettaz, Pierre; Davidson, Fred; Esteban, Marta; Fischer, Marc E.; Gurzau, Anca Elena; Halzlova, Katarina; Katsonouri, Andromachi; Knudsen, Lisbeth E.; Kolossa-Gehring, Marike; Koppen, Gudrun; Ligocka, Danuta; Miklavčič, Ana; Reis, M. Fátima; Rudnai, Peter; Tratnik, Janja Snoj; Weihe, Pál; Budtz-Jørgensen, Esben; Grandjean, Philippe Resumo: Background: Due to global mercury pollution and the adverse health effects of prenatal exposure to methylmercury (MeHg), an assessment of the economic benefits of prevented developmental neurotoxicity is necessary for any cost-benefit analysis. Methods: Distributions of hair-Hg concentrations among women of reproductive age were obtained from the DEMOCOPHES project (1,875 subjects in 17 countries) and literature data (6,820 subjects from 8 countries). The exposures were assumed to comply with log-normal distributions. Neurotoxicity effects were estimated from a linear dose-response function with a slope of 0.465 Intelligence Quotient (IQ) point reduction per μg/g increase in the maternal hair-Hg concentration during pregnancy, assuming no deficits below a hair-Hg limit of 0.58 μg/g thought to be safe. A logarithmic IQ response was used in sensitivity analyses. The estimated IQ benefit cost was based on lifetime income, adjusted for purchasing power parity. Results: The hair-mercury concentrations were the highest in Southern Europe and lowest in Eastern Europe. The results suggest that, within the EU, more than 1.8 million children are born every year with MeHg exposures above the limit of 0.58 μg/g, and about 200,000 births exceed a higher limit of 2.5 μg/g proposed by the World Health Organization (WHO). The total annual benefits of exposure prevention within the EU were estimated at more than 600,000 IQ points per year, corresponding to a total economic benefit between €8,000 million and €9,000 million per year. About four-fold higher values were obtained when using the logarithmic response function, while adjustment for productivity resulted in slightly lower total benefits. These calculations do not include the less tangible advantages of protecting brain development against neurotoxicity or any other adverse effects. Conclusions: These estimates document that efforts to combat mercury pollution and to reduce MeHg exposures will have very substantial economic benefits in Europe, mainly in southern countries. Some data may not be entirely representative, some countries were not covered, and anticipated changes in mercury pollution all suggest a need for extended biomonitoring of human MeHg exposure. Descrição: © 2013 Bellanger et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2013-01-01T00:00:00Z A pilot study on the feasibility of European harmonized Human Biomonitoring: Strategies towards a common approach, challenges and opportunities http://hdl.handle.net/10451/17689 Título: A pilot study on the feasibility of European harmonized Human Biomonitoring: Strategies towards a common approach, challenges and opportunities Autor: Casteleyn, L.; Dumez, B.; Becker, K.; Kolossa-Gehring, M.; Den Hond, E.; Schoeters, G.; Castaño, A.; Koch, H. M.; Angerer, J.; Esteban, M.; Exley, K.; Sepai, O.; Bloemen, L.; Horvat, M.; Knudsen, L. E.; Joas, A.; Joas, R.; Biot, P.; Koppen, G.; Dewolf, M-C.; Katsonouri, K.; Hadjipanayis, A.; Cerná, M.; Krsková, A.; Schwedler, G.; Fiddicke, U.; Nielsen, J. K. S.; Jensen, J. F.; Rudnai, P.; Közepésy, S.; Mulcahy, M.; Mannion, R.; Gutleb, A. C.; Fischer, M. E.; Ligocka, D.; Jakubowski, M.; Reis, M. F.; Namorado, S.; Lupsa, I-R.; Gurzau, A. E.; Halzlova, K.; Jajcaj, M.; Mazej, D.; Snoj, J. Tratnik; Posada, M.; López, E.; Berglund, M.; Larsson, K.; Lehmann, A.; Crettaz, P.; Aerts, D. Resumo: In 2004 the European Commission and Member States initiated activities towards a harmonized approach for Human Biomonitoring surveys throughout Europe. The main bjective was to sustain environmental health policy by building a coherent and sustainable framework and by increasing the comparability of data across countries. A pilot study to test common guidelines for setting up surveys was considered a key step in this process. Through a bottom-up approach that included all stakeholders, a joint study protocol was elaborated. From September 2011 till February 2012, 17 European countries collected data from 1844 mother–child pairs in the frame of DEMOnstration of a study to COordinate and Perform Human Biomonitoring on a European Scale (DEMOCOPHES).1 Mercury in hair and urinary cadmium and cotinine were selected as biomarkers of exposure covered by sufficient analytical experience. Phthalate metabolites and Bisphenol A in urine were added to take into account increasing public and political awareness for emerging types of contaminants and to test less advanced markers/markers covered by less analytical experience. Extensive efforts towards chemo-analytical comparability were included. The pilot study showed that common approaches can be found in a context of considerable differences with respect to experience and expertize, socio-cultural background, economic situation and national priorities. It also evidenced that comparable Human Biomonitoring results can be obtained in such context. A European network was built, exchanging information, expertize and experiences, and providing training on all aspects of a survey. A key challenge was finding the right balance between a rigid structure allowing maximal comparability and a flexible approach increasing feasibility and capacity building. Next steps in European harmonization in Human Biomonitoring surveys include the establishment of a joint process for prioritization of substances to cover and biomarkers to develop, linking biomonitoring surveys with health examination surveys and with research, and coping with the diverse implementations of EU regulations and international guidelines with respect to ethics and privacy. Descrição: © 2014 Elsevier Inc. All rights reserved. 2015-01-01T00:00:00Z Patterns of perceptions of workplace violence in the Portuguese health care sector http://hdl.handle.net/10451/17688 Título: Patterns of perceptions of workplace violence in the Portuguese health care sector Autor: Ferrinho, Paulo; Biscaia, André; Fronteira, Inês; Craveiro, Isabel; Antunes, Ana Rita; Conceição, Cláudia; Flores, Isabel; Santos, Osvaldo Resumo: This article characterizes the problem of violence against health professionals in the workplace (VAHPITWP) in selected settings in Portugal. It addresses the questions of what types of violence are most frequent and who are the most affected health professionals. Three methodological approaches were followed: (i) documentary studies, (ii) a questionnairebased hospital and health centre (HC) complex case study and (iii) semi-structured interviews with stakeholders. Of the different types of violence, all our study approaches confirm that verbal violence is the most frequent. Discrimination, not infrequent in the hospital, seems to be underestimated by the stakeholders interviewed. Violence seems much more frequent in the HC than in the hospital. In the HC, all types of violence are also most frequently directed against female health workers and, in the hospital, against male workers. These studies allow us to conclude that violence is frequent but underreported. Descrição: © 2003 Ferrinho et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 2003-01-01T00:00:00Z


text:
practical range hood question
To those of you with glass (or partially glass) range hoods, do you find them difficult to keep clean? I'd picked out an all stainless hood, but the one thing I'm not crazy about is that the lights in it are at the back.:
I found another for about the same price that has the lights at the front, but it's the style with a stainless center and chimney and a curved glass canopy.
I like both styles equally, and I definitely prefer the lights at the front, but between grease and dust it seems like a glass range hood would have to be wiped down pretty much every day to stay looking good. I keep up on cleaning, but I'm not sure I'd really keep up with that kind of maintenance.
Oh, and while I do want a ducted hood that actually moves some air and heat to the great outdoors, I don't have a pro-style range, so I'm not really concerned with the difference in the size of the uptake area between the two. (The ancient range hood we just took out when we demo'd the kitchen was a recirculating one and I couldn't even use it - the first time I turned it on after we moved into the house there was a loud crashing noise as the plastic blades on the fan all snapped off and went crashing around the housing!)
Don't have glass, but I have to dust my stainless hood a lot. Also, the way the switches are on mine, I get fingerprints every time I turn it on or off, so I have to wipe those off. It only takes me a minute to do it, but I'm not sure that glass would be any worse.
Audry, I have a hood with the curved glass canopy, but the lights are along the sides rather than just in front. I do wipe the glass portion fairly often to keep it clean - not every day; it depends on what I was cooking. It isn't hard to do. Mine is also ducted out.
Stainless or glass, it's pretty much the same. Either the shiny stainless will show the dust or the clear glass will.
It's a 30 second chore to dust the hood (ANY hood) in the morning while you are waiting on the coffee to perk. Or, it's about a 90 second chore to get out the spray cleaner, spritz, and wipe if you cooked the night before and have some grease on it.
If you spend less than 2 minutes a day doing those things, then you won't have to spend 30 minutes once a week waiting on the degreaser to cut through the gunk and scrubbing. You will still have to clean the filters once a week though. Pop them in the DW one night, and if you've got a spare pair, pop those in at the same time. It takes maybe 30 seconds with no downtime if you do that. Otherwise, you've got to wait for the overnight hours to put the filters back in after the DW has run it's load for the day.
Thanks for the replies everyone. It's good to know that the stainless-only one will probably require pretty regular cleaning/ dusting as well, but you've also made me think of something I hadn't before... if I "have" to clean it every day, it will mean I don't have to clean off built-up grease. Which, if I can't see it, I'm definitely more likely to let it build up to the point where it's more of a pain to clean.
We just installed a glass one and I love it! I can see if there is dust on it and then I wipe it. Not a big deal. Definitely love the look of the glass better than the all stainless one. Get the one you like better, don't worry about cleaning. They will both need the same amount.


text:
Official Free Agent Frenzy Thread (Deals from other teams)
View Single Post
Registered User
Join Date: Nov 2005
Location: Montreal
Posts: 25,142
vCash: 500
Originally Posted by
Not that for me though because Gainey acquired his rights doesn't mean that Sundin want to go there.
People are being frustrated 'cause they're beginning to realize that another one will look another way.....I don't think that they're offended 'cause he's not doing something right away but mostly 'cause we won't be doing it at all....or that's my belief.
Wondering if it would not have been possible for Gainey, before making that "deal" to just wonder via Fletcher if Sundin is actually considering retirement at this point. If so, don't you think that a big decision like that wouldn't take 1 week and it doesn't matter who has the exclusivity? Seems that Gainey speaks to him every day and every day we keep hearing that Sundin says his mind isn't made up....What's the point in talking to him then? Is he suddenly going to wake up June 31st and because it's crunch time he'll take a decision like that?.....if or course the whole "retirement" thing is true....which I don't believe but again it's just an opinion.
Sundin might hate the Leafs but I don't believe he'll sign with us after July 1st.....unless the deal stands even if he signs before. Maybe the deal is not if the signing is before July 1st....it could be the exclusive rights before July 1st and if you sign him, the deal stands no matter when you sign him.
Anyway, just have the feeling that he's thinking of retirement only because TO and Montreal are in the picture right now....I'm that pessimistic about that "deal". As soon as the Rangers starts dancing, Sundin will start realizing.......
Going to NYR might be his plan. But it's not a reason to be pissed off. You see these posts and threads pop up calling Sundin names just because it appears we aren't the center of the universe.
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text:
An Ode to the Ski BootMar 30th 2014
AKA Why I Ski.
- Views: 7722
- Comments: 38
AKA Why I Ski.
A journey back in time to when I got started skiing, and becoming a 'Freeskier'.
Style and innovation were the judging criteria for the Big Air contest that took place on the 29th of March 2014 in Livigno, Italy for the Suzuki Nine Queens presented by ONeill. After many days of shooting at the colossal castle the invited riders where amped to push the level of riding in the jam session. Spectators basked in the sun on Mottolino Fun Mountain cheering while the girls sent it. Lisa Zimmermann claimed the top spot out of the freeskiers with her two best tricks, a breezy 900 tail grab and by stomping her very first switch 1080 mute. Elena Könz (SUI) was the snowboard winner with her signature backside 540 mute and frontside 720 melon
Emma Dahlström is currently one of the world's top female park skiers. I caught up with her for a few minutes at Nine Queens.


text:
Lively Cinema 10
759 Highway 98 East,
759 Highway 98 East,Destin, FL 32541
No one has favorited this theater yet
The Destin Cinema 10 was opened in 1987. Owned by O'Neil Theatres, which owns 60 screens in seven theatres in Florida, Alabama, Mississippi and Louisiana.
It was last known as Lively Cinema 10 and closed August 17, 2014.
Contributed by Dave Bonan
Recent comments (view all 1 comments)
OCRon on March 18, 2015 at 10:46 pm
According to this story in the Destin Log, Cinema 10 closed on August 17, 2014. It was said to have opened in 1987 and was most recently called the Lively Cinema 10.
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text:
LJ is Hiring
Jan 03, 2008 By Carlie Fairchild
Linux Journal is looking to add a sales person to its team. Must be strong in sales, energetic, intelligent, interested in Linux (we'll train the right candidate) and fun. (Those are our only demands, we swear.) :-)
This person will be in charge of both print and on-line advertising sales for a selected territory. Interested or know someone who may be? Please drop me a line.
Carlie Fairchild is the publisher of Linux Journal.
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- Define service virtualization
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Webinar: 8 Signs You're Beyond Cron
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Join Linux Journal and Pat Cameron, Director of Automation Technology at HelpSystems, as they discuss the eight primary advantages of moving beyond cron job scheduling. In this webinar, you'll learn about integrating cron with an enterprise scheduler.Join us!
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- Tighten Up SSH
- diff -u: What's New in Kernel Development


text:
|Email firstname.lastname@example.com
|Follow on Twitter
Alan J. Heavens has been writing about real estate and home improvement for the last 14 years. A veteran of 35 years as a reporter, he holds B.A. and M.A. degrees in history.
Heavens cannot make recommendations about contractors, Realtors, mortgage services or neighborhoods.
We've barely gotten over winter's misery and already are being reminded about hurricane preparedness. Yet the season officially started June 1, so perhaps a few tips from the American Bankers Association are in order:
When we last met up with Jeffrey Tubbs, he was two years into his first building project and doing well, though it was 2010 and we were still mired in the real estate downturn.
House-hunting here? Act fast
- If 22 people are considering buying houses in Westtown right now, one of them could well end up elsewhere.
Warrington By the Numbers
- Population: 23,418 (2010). Median income: $84,633. Area: 13.8 square miles. Settlements in the last three months: 41. Homes for sale: 130.
Welcome to the 'year of the connected home'
- Ever since Sandy, or maybe it was Hurricane Irene, I've been thinking about buying a portable generator sufficient to run our sump pump, our freezer and refrigerator, and a few appliances in case of a lengthy power disruption.
Development on Route 611 helps sweeten market
- Ask about Warrington real estate, and agent Sharon Ermel Spadaccini mentions a huge fringe benefit of selling in the Bucks County township: The cream doughnuts they make at Warrington Pastry Shop.
Real estate study data leaves out details
- Research by a pair of professors at the University of Chicago Booth School of Business has found that "shabby urban neighborhoods are the wisest choice" for real estate investors.
Hire someone else to fix leaning garage
- Question: I have a 75-year-old, one-car, detached wooden garage that has started to lean. What do I need to do to straighten it up?
Upper Dublin schools boost housing, too
- The locales that make up Upper Dublin are often more familiar to outsiders than the township itself.
Why wood floors chip; worries over water
- A few weeks back, a reader asked for reasons why a wood floor was starting to extrude chips and slivers from between the boards.
By the Numbers
- Population: 2,794 (2010) Median income: $88,623 Area: 0.75 sq. miles Settlements in the last three months: 9 Homes for sale: 18
Past and present mingle along the Delaware
- One in a continuing series spotlighting real estate markets in the region's communities. It's Tuesday morning, nearly 20 minutes past 11, and the bells warning of the approach of the next River Line train to Trenton startle a pedestrian waiting to cross Broad Street at Main Street near the Riverton station.


text:
KDE Releases Frameworks 5 Tech Preview 51
from the when-kde-and-qt-become-one dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
"True innovation would be to provide a script-based approach to most of the GUI stuff, e.g. nodejs/browser API
We started working on exactly that ~5 years ago and the result is QML2 and Plasma (two separate things, but they work wonderfully together). As the node.js project founder said when he saw QML for the first time: "Wow, it's HTML5 done right."
So KDE is truly innovative, you're just too uninformed to have known and ~5 years too late to the suggestion table. (I'm not entirely sure how the
... is the idea of load as few as possible libraries to run a KDE application in a non KDE environment.
No, that's not it. The purpose is to break useful functionality out of KDE and make it available to QT developers in general without requiring KDE itself. The benefit to KDE is, it prepares the ground for migration to QT 5, cleans things up, and enables the possibility of contributions to the framework components by non-KDE developers. Or in other words, whatever is good for QT it good for KDE.
To err is human -- to blame it on a computer is even more so.


text:
Volatility has been creeping back into markets, creating headaches for investors as they try to navigate wild swings in currencies, oil and geopolitical uncertainty.» Read More
Analyst Hunter Keay expects an uptick in airline buybacks this year, funded by an inflow of money from cheaper oil.» Read More
There's a real sense that something foreboding may be lurking just around the corner, said Jim Cramer.
If the ruling on allowing Northwestern University football players to unionize stands all challenges, it may not be so easy to do.
A top U.S. regulator gave a spirited defense of new rules forcing foreign banks to hold more capital in their U.S. units.
BlackBerry reported a quarterly loss, as smartphone sales continued to slide across all regions.
Wal-Mart filed a $5 billion lawsuit against Visa, saying the company passed along unreasonable fees when shoppers used plastic at its stores.
Mark Zuckerberg said that Facebook will use "drones, satellites and lasers" to bring internet connectivity to the world. NBC News reports.
The number of shareholder security class action lawsuits soared 57% in 2013 as investors turned to the courts to be made whole from alleged wrongdoing.
Not all coffee is created equally, which means it matters where you go if you are looking for a pick-me-up.
There has been much chatter about fresh stimulus in China, but those who are hoping for an 'all-guns-blazing' announcement could be left disappointed.
CBS Outdoor Americas said it priced its initial public offering at $28 per share, valuing the outdoor advertising company at about $3.36 billion.
Coca-Cola survived U.S. soda volume declines for years by raising prices. But the recent decline in Diet Coke consumption looks tough.
Tech stocks have gotten hit by a one-two punch this week, Cowen's David Seaburg explains.
Connecticut hikes minimum wage to highest in US
The search area of the missing pane has shifted northeast of where a team of investigators had been looking for debris. NBC News reports.
Microsoft said on Thursday its Office suite of applications, including the core Word, Excel and PowerPoint programs, will be available as an iPad app.
The gadget will compete with Apple, Roku and Google.
Prominent value investor Bill Miller on Thursday said he is investing in bitcoin.
A US magistrate judge recommended dismissal of a lawsuit against Bank of America over $850 million in mortgage debt, according to a court filing.
Art Cashin of UBS Financial Services explains why investors need to watch some key support levels for the Nasdaq and S&P.
SweetWater is partnering with Delta to offer the brewery's 420 Extra Pale Ale on Delta's 34 daily flights between Atlanta New York.


text: go nogo - Sad (Woolfy Remix) 128bit MP3 - released by Emerald & Doreen Records published on 2013/02/11 16:11:41 +0000 With last year's album release "The Return Of Love" (Permanent Vacations), two well-known German magazines for electronic music perceived Woolfy's style as "disco for the hammock" (De:bug) and "analogue-synth-disco for people with slippers on" (Groove)… Both reviewers dwell on the irresistible coziness derived from a mix that sports Steely Dan's West Coast-flavour as well as modern folklore like the ones from singer/songwriter Bon Iver. Woolfy's Remix of go nogo's "Sad" simply sounds like a lost session from his own material. His version deservedly bears the new disco tag, for there's hardly been a track that combines a boogy-bounce with a taste of U.K. Dubstep-baseness. https://soundcloud.com/woolfys