This study investigated the epitope recognized by the broadly neutralizing antibody PG16 against HIV-1. PG16 was found to bind soluble gp140 proteins from clades B and C, but binding was lost for the clade C protein after incubation with a CD4-mimicking protein. Further experiments revealed a spatial component to PG16's neutralization mechanism, as binding of a V2-specific antibody reduced PG16 binding, suggesting they recognize overlapping regions. Additionally, western blot analysis showed PG16 had higher affinity for the clade B protein compared to the clade C protein. Overall, the results suggest PG16 may not recognize a quaternary epitope as previously thought.

1. Biochemical Characterization of Antibody PG16 and Evidence Against the Quaternary
Epitope Hypothesis with HIV-1 Clades B and C Soluble gp140 Trimers
By Lauren Drayer
ABSTRACT
The human immunodeficiency virus (HIV) type 1 is the most widespread around the
world, causing up to 3.1% of deaths worldwide. Efforts to develop an HIV vaccine are
thus important to control and prevent this devastating infection. PG16 is a monoclonal
antibody isolated from the pooled sera of about 1,800 HIV-1 clade A infected
individuals, and it neutralizes about 80% of HIV-1 isolates across all clades. Low serum
titers of PG16 can confer adequate HIV immunity, therefore making PG16 relevant in
studies aimed at uncovering the structural requirements of an HIV vaccine. The
incubations of HIV-1 clades B and C with PG16 and a CD4-mimicking miniprotein
produce complex neutralization patterns. These are modulated by loop deletions and
amino acid substitutions in the HIV surface proteins. Binding is confirmed for clade C
strain TV1 (C1) and, under certain conditions, clade B SF162. However, after incubation
with CD4-mimicking miniproteins, gp140 TV1 loses sensitivity to PG16. Further epitope
probing via a V2 specific antibody uncovers a strong spatial component in the PG16
neutralization mechanism. Specifically, V2 specific antibodies bind V2 in a manner that
must largely occlude region 156-162 from PG16. In addition, western blots confirm that
V1 and V2 are necessary for sensitivity to PG16 while, contrary to expectation, gp140

2. constructs lacking V3 show very strong PG16 binding responses. Further analyses of the
western blots reveal a significant difference in PG16 affinity for clades B and C.
Generally, these results suggest that PG16 may not primarily recognize a quaternary
epitope.
INTRODUCTION
The human immunodeficiency virus (HIV) is responsible for 7.8% of deaths in
low-income countries, and 3.1% of deaths worldwide (1). Therefore, efforts to sustain
novel HIV drug and vaccine design remain of great importance in the hopes of eventually
controlling this devastating infection. Two types of human immunodeficiency viruses are
currently reported to exist: HIV-1 and HIV-2. HIV-1 is the most widespread around the
world, and is divided into subgroups that are further divided into clades A, B, C, D, F, G,
H, J, K and circular recombinant forms (CRFs). CRFs occur when two viruses from
different clades combine to form a hybrid. In Europe and the Americas, clade B HIV is
ubiquitous while clade C HIV is found predominantly in parts of Asia and Africa (2).
Clades B and C are fairly abundant worldwide (2), making them of particular importance
in HIV studies focused both on antibodies and vaccine design. However the design of a
potent vaccine remains uniquely challenging because HIV can undergo many rapid
mutations, allowing it to evade the host immune system. Still, the key to such a vaccine
may be closer than anticipated: recently a study involving 16,000 participants in Thailand
demonstrated that a prime-boost vaccine regimen was safe and 31% effective in
preventing an HIV infection (3). Accordingly, an in-depth structural and biochemical

3. understanding of HIV envelope proteins and the antibodies that recognize them should
allow us to design even more effective vaccines in the future.
Specifically, vaccine design is best informed by the mechanisms that underlie
viral entry. The first step in HIV infection is initiated when an HIV envelope (Env)
glycoprotein trimer interacts with two receptors on the surface of the host cell: receptor
CD4 and CC-chemokine receptor 5 (CCR5) (4). The Env trimer is composed of three
gp120 monomers, each of which are non-covalently associated with a gp41 stem. Env is
thus a heterodimer of homotrimers, and is originally synthesized as a 160kDa precursor
protein (gp160) in the endoplasmic reticulum (ER), where it is also extensively
glycosylated (5). Glycosylation is a crucial feature in immune system evasion, since it
protects HIV from neutralizing antibodies (6). Then, the 160kDa precursor travels to the
Golgi apparatus where furin, a host provided protease, cleaves it the into the gp120 and
gp41 subunits. The two subunits continue to interact via non-covalent interactions to
form a native Env monomer, which further non-covalently associates with two other
monomers to form the full, native Env trimer. Each trimer’s gp120 subunits have
conserved cores and hypervariable loops. The conservation of the core is essential for
controlling the correct folding of gp120, and thus affects the infectivity of HIV (5). On
the other hand, the surface-exposed variable loops award HIV virions the advantage of
host immune system evasion (5), partly due to differential types of post-translational
glycosylation and the propensity of variable loops residues to undergo mutations.
With a few exceptions, such as HIV-2 strains (7), clades of Env generally utilize
the host receptor CD4 for viral entry. CD4 receptors are membrane glycoproteins
expressed on T lymphocytes, and they normally interact with major histocompatibility

4. complex class II antigens (MHC II). Furthermore, MHC II is also preferentially
expressed on T-lymphocytes, causing the CD4-MHC II interaction to have an important
part in the T-cell activation process (8). In addition to surface-exposed variable loops,
post-translational glycosylation of the HIV Env trimer causes it to be largely
immunologically silent. Furthermore, trimerization itself may be responsible for a non-
negligible degree of shielding of the conserved epitopes (9). As a result, recently isolated
antibodies like PG9 and PG16 (10) are important in overcoming the vaccine design
challenge because they target the “silent” variable loops that are thus far understood to
conceal HIV from the immune system (11). Their characterization has the potential to
bring novel HIV vaccine design one step closer to success for the following reasons: (1)
Understanding the unique features that make PG9 and PG16 potent antibodies should
help improve the rational design of vaccines. (2) Better vaccines should successfully
elicit protective antibodies against HIV.
PG16, the focus of this review, is a monoclonal antibody (MAb) isolated from the
pooled sera of about 1,800 HIV-1 clade A infected individuals (12) that neutralizes about
80% of HIV-1 isolates across all clades (13). Its half maximal inhibitory concentration
(IC50) is several orders of magnitude lower than other tested broadly neutralizing
antibodies (bNAbs), meaning low concentrations of PG16 can confer adequate protection
from HIV viral particles in the bloodstream. Consequently, PG16 is highly relevant in
studies toward uncovering the structural requirements for an efficient HIV vaccine. Its
salient feature is an antigen-binding fragment for which a crystal structure at 2.5Å reveals
an unusually long 28-residue (Kabat numbering) (14) heavy chain third complementarity-
determining region (CDR H3). While long CDRs do exist in nature, PG16’s is still one of

5. the longest ever recorded. It is said to resemble an “axe,” with the N-terminal residues
(AGGP99) and C-terminal residues (YYNY100Q) coming together to form the handle of
the axe. Hydrogen bonding stabilizes the structure, though the N-terminal amino acids are
further steadied by the side-chains of Tyr100N and Tyr100Q, which interact with amino
acids Gly98 and Pro99 of the C-terminal of the CDR H3 (14). Moreover, data collected
from monoclinic crystals suggest that in addition to the stable stalk at the base of the
CDR H3 structure, two intertwined loops made of an antiparallel β-sheet and a 310-helical
turn (both constituting the H3 “hammerhead”) are important in PG16 reactivity.
Additionally, alanine-scanning mutagenesis shows that mutation of AspH100I in
particular abrogates neutralization by PG16, thus emphasizing the importance of the
stalk’s sequence and structure in antigen binding. Finally, insertions of polyglycine
linkers demonstrate that the specificity loop and the topmost β-strand of the H3
subdomain make crucial contacts with the Env trimer. Studies have found that mutations
in those domains change the conformation of the CDR H3, thus causing a drastic
reduction in PG16 neutralization (15).
While structural information about the shape and function of the CDR H3 is
important for the development of novel, structural vaccine designs, structure only
represents a portion of PG16’s neutralization mechanism. Successful vaccine design must
also consider the key biochemical features of PG16’s epitope. Reportedly, the target of
PG16 is expressed on the surface of the native Env trimer, and includes conserved
residues in V2, V3, the V1/V2 stem, and possibly parts of the coreceptor binding site
(16). A particularly critical residue in the V2 loop is the asparagine at position 160,
without which PG16 cannot recognize Env (10). Interestingly, the location of the V1/V2

6. and V3 loop regions is still a point of contention. According to some, these regions are
located at the apex of the Env trimer, where they are solvent accessible and able to
function as shields against the human immune system (16). However, other groups have
determined that, in the native Env trimer, the V3 variable region is located on the outer
edge of the monomers, while the V2 variable region is proximal to the viral membrane
(17). Other uncertainties—about PG16’s epitope—are analyzed in this review: while the
quaternary epitope of PG16 is reportedly expressed on membrane-bound Env trimers,
whether soluble trimers display the quaternary epitope is mostly unknown (15). This
information remains critical for the practical aspects of vaccine design; it dictates whether
vaccinologists must keep relying on whole virion vaccines instead of using viral proteins.
What is known is that PG16’s epitope appears on the gp140 monomers in the absence of
the correct quaternary structure (10), so it may not be entirely correct to refer to PG16 as
a “quaternary-structure-specific” monoclonal antibody. For instance, it has been observed
that the IgG form of PG16 recognizes gp140 monomers better than gp140 trimers, while
Fab forms of PG16 recognize trimers and monomers with similar binding kinetics (10).
While these results are partly justified by the fact that soluble trimers may not display the
quaternary epitope at all, further testing is absolutely needed to uncover and characterize
the true PG16 epitope.

7. MATERIALS AND METHODS
Antibody PG16
Human FAb PG16 was provided by the International AIDS Vaccine Initiative (IAVI). It
was obtained as described in Walker (18): the breadth of neutralization in the sera of
about 1,800 HIV-1 infected individuals was analyzed, and donors were selected for
monoclonal antibody (mAb) generation. High-throughput neutralization screens were
used to identify PG16.
Soluble Env gp140
Neutralization by PG16 can only occur if Env displays an asparagine at position 160 in
gp120, which is located in the V2 loop. Most HIV strains do have an asparagine at this
position, though this is not true of clade B gp140 SF162 in particular (9). The point
mutation K160N renders gp140 SF162 vulnerable to PG16 (9), and as such gp140 SF162
and gp140 SF162K160N are used here as negative and positive controls, respectively.
Also, for experimental purposes gp140 can be engineered such that it will be cleavage-
defective. In those cleavage-defective constructs the wild type cleavage sequence REKR
is replaced with a non-scissile IEGR motif (19), and this ensures the integrity of the Env
monomers throughout the various experimental procedures. Clade B gp140 SF162 and its
point mutants SF162 K160ND368R, SF162 K160ND368R ΔV1 and SF162
K160ND368R ΔV2 were provided by Noah Sather (Seattle BioMed, Seattle, WA). Clade
C gp140 TV1, full length, was provided by Novartis, Inc. (Cambridge, MA).

8. Recombinant gp140 CN54 (C4) was provided by Jonathan Heeney, (Cambridge
University, UK).
CD4 mimetic
CD4m was provided by Loic Martin, CEA, France. It was constructed the following way:
The CD4 binding site of gp120 was transplanted onto a scorpion-toxin scaffold. The
resulting miniprotein was optimized by combinatorial chemistry and one of the final
optimized miniproteins, M48, was used for our experiments (20).
Coomassie staining
4 microliters of protein samples were loaded onto 10% Native PAGE and 10% SDS
PAGE gels and stained with Coomassie blue.
Western Blotting of gp140 Env
4 microliters of protein samples were loaded onto the 10% Native PAGE gels. The
proteins were then transferred to a PVDF membrane following a non-reducing wet
transfer protocol. The transfer was performed at 100 volts and 400 amps for 60 minutes
in 1X transfer buffer comprised of glycine, Tris base, methanol and water. After
completion of the transfer the membranes were rinsed in 1X TBS, then blocked with 5%
milk in TBST overnight at 4°C with agitation. After blocking the membranes were
washed 2 times in TBST. Primary antibody Fab PG16 was added in TBST and left to
incubate for 2 hours at room temperature with agitation. The membranes were then
washed 3 times in TBST while agitating (5 minutes per wash) to remove excess primary

9. antibody. Secondary antibody Anti-Human IgG (Fab specific)-Alkaline Phosphatase
(Sigma-Aldrich, St. Louis, MO) was added in TBST and left to incubate for 60 minutes at
room temperature, with agitation. The membranes were washed again, 3 times for 5
minutes, and were left to soak in TBS for 20 minutes at room temperature. Lastly the
membranes were developed with Sigmafast (Sigma-Aldrich, St. Louis, MO) according to
the manufacturer’s protocol.
Western Blot Quantification
Three western blots with bands for both gp140 SF162K160N and gp140 TV1 were
chosen for quantification. The images were captured with an iPhone 4 and analyzed in
ImageJ (21). The images were converted to 8-bit grayscale and inverted. A line was
drawn through the bands for which the intensity values were needed, and a plot profile
was obtained (Fig.4). In Excel, the intensity values from the profile were divided by 255
to generate a plot with a maximum value of 1 on the y-axis (Fig.5). Visual analysis of the
profile identified the peak intensity values for gp140 TV1 and gp140 SF162K160N.
These were tabulated in Excel and a 2-tailed t-test was applied to the data set using
StatPlus (22).
RESULTS
Western blots
PG16 binding to cross-clade gp140 without prior CD4m incubation
PG16 is shown to bind gp140 SF162 K160N (positive control), while it does not bind
gp140 SF162 WT (negative control). Binding is also confirmed for gp140 TV1, and

10. gp140 SF162 K160ND368R ΔV3. There was no binding for gp140 SF162
K160ND368R, SF162 K160ND368R ΔV1, SF162 K160ND368R ΔV2 or CN54
(Fig. 1 and 3).
PG16 binding to cross-clade gp140 after CD4m incubation
With one exception, incubation with CD4m did not affect binding to constructs for which
binding was previously observed: gp140 SF162 K160N and gp140 SF162 K160ND368R
ΔV3. Differential binding was observed for gp140 TV1, for which CD4m incubation
abrogated PG16 binding. CD4m did not enhance binding to constructs for which there
had been no previous binding: gp140 SF162 WT, and gp140 SF162 K160ND368R
(Fig. 1).
PG16 and 697-30D binding TV1 after prior incubation with various antibodies
PG16 binds gp140 TV1 after its prior incubation with antibodies b12, 17b, and 2F5 (Fig.
9). It was observed that PG16 binds TV1 8.23% less efficiently after prior gp140 TV1
incubation with 697-30D, an anti-V2 antibody (Fig. 7). PG16 and PG9 do not impede the
binding of 697-30D (Fig. 8).
Quantification of Western Blots
There is a significant difference in band intensities between gp140 SF162 K160N and
gp140 TV1 (p=0.00009), which suggests PG16 has different affinities for the two strains:
PG16 has significantly more affinity for gp140 SF162 K160N than for gp140 TV1.

11. Measurements of PG16 Fab
Measurements of the Fab in Chimera showed that the Fab, at its widest point, is about
51Å. Measurements also showed that the Fab is about 87Å long, from the tip of the CDR
H3 to the bottom of the stalk (Fig. 3a, 3b). Measurements of the Fab showed that the
CDR H3 “hammerhead” is about 26.5Å (not shown).
DISCUSSION
PG16 binds preferentially in the concurrent presence of variable loops 1 and 2
PG16 is a MAb that neutralizes about 80% of HIV-1 isolates across all clades
(13), and results from recent studies (10) show that it is capable of binding soluble HIV
Envs. Clade B SF162 is a known PG16-resistant isolate that becomes PG16-sensitive
upon introducing a K160N mutation in the V2 loop (23). In the wild-type strain, the
lysine at position 160 prevents recognition by PG16, while replacement with an
asparagine at that position results in recognition by PG16. Hence, the asparagine is
posited to be an N-linked glycosylation site that is simply absent in SF162 WT (10),
which further suggests that PG16’s epitope is largely dependent on glycosylation (23).
Here, binding patterns of PG16 to clade B SF162, its point mutant and clade C strains are
confirmed with western blots.
In accordance with previously published data, these western blot results confirm
that variable loops 1 and 2 are necessary for sensitivity to PG16. When V1 and V2 are
absent binding is never observed. Doores et al. hypothesize that the variable loops
themselves do not confer sensitivity to PG16; rather, critical glycosylation sites on the
variable loops are responsible for the biochemistry of the epitope. However, our results

12. do not directly confirm the importance of these critical glycosylation sites. Still, since our
gp140 constructs are glycosylated, it appears our results do agree with Doores’
conclusions to some extent. Furthermore, we observed that the third variable loop (V3)
acts as a critical component of the PG16 epitope, such that when the critical glycosylation
sites are removed from the V1 and V2 loops, the correct conformation of the V3 loop is
not maintained and optimal recognition by PG16 is not achieved (23). Accordingly,
western blot results confirm this relationship because the deletion of V1 and V2 cancel
PG16’s ability to recognize V3 and bind to it. On the other hand, western blots of ΔV3
constructs show very strong PG16 binding responses in the presence of V1 and V2. This
implies that while V3 may enhance the recognition of PG16’s epitope, its presence is not
required.
Probing the boundaries of PG16’s epitope via other antibodies that have known
footprints also provides important mechanistic insights. Probing with 697-30D mAb in
particular is informative because it is V2 specific (24), requiring the amino acid region
164-194 in the V2 loop to neutralize HIV isolates (25). Since PG16 requires the nearly
overlapping region 156-162 (6), it was hypothesized that sequential binding of 697-30D
and PG16 in either order would lead to steric hindrance, and thus less efficient binding.
Evidence of the less efficient binding would subsequently be observed in western blots as
light or even absent bands. However, results show that while 697-30D lessens PG16’s
ability to bind gp140 TV1, PG16 does not hinder 697-30D at all (Fig. 7, 8). This
phenomenon implies a possible dependency on the spatial orientation of the binding
antibodies. Specifically, 697-30D may bind V2 in a manner that largely occludes region
156-162 from being accessed by PG16. On the other hand, PG16 binds V2 in such a way

13. that region 164-194 always remains accessible for neutralization. In addition, PG16 is
shown not to hinder neutralization by antibodies 2F5, b12, 17b and 4E10 (data not
shown). Those results make sense because these antibodies do not compete for the same
epitope. Furthermore, those results indicate that binding of PG16 does not alter the
conformation of gp140 TV1 enough to misshape 2F5, b12, 17b, or 4E10’s respective
neutralizing regions.
In summary, PG16 cannot recognize V3 in the absence of V1 and V2, but absence
of V3 has no effect on recognition as long as V1 and V2 are present. Interactions between
V1 and V2 are thus critical, because sensitivity to PG16 is only conferred when these
variable loops are present concurrently. While current research still considers PG16 a
quaternary specific antibody (26), results here suggest that it is probably not PG16’s
primary neutralizing mechanism. We hypothesize that PG16 may sometimes neutralize
gp140s via a quaternary epitope composed of the V1, V2 and V3 loops, but that the major
mechanism of recognition is only concerned with V1 and V2. This is supported by the
western blot results that show enhanced binding when V3 is absent, and by the Davenport
et al. results that show that Fab versions of PG16 recognize monomeric gp140 just as
well as trimeric gp140. Indeed, the epitope on the monomer should still be accessible in
the trimer, provided there are no unfavorable steric interactions and considering the
position of the variable loops in the unliganded Env. Generally, recognition of the
trimeric form cannot prove the presence of a crucial quaternary epitope, since the
antibody could be recognizing the monomers only. Furthermore, if the variable loops are
assumed to be located near the apex of the monomers, then the CDR H3 is not wide
enough to reach the span of two gp140 monomers: the head of the CDR H3 spans about

14. 26.5Å (Fig. 3 a, b), while the distance between variable loops on adjacent monomers
would be at least 30-40Å (17). Besides, fluctuations in the distance between trimer
components across a population of trimers would most likely be detrimental to efficient
recognition by PG16, although fluctuations in a small subset of trimers could conceivably
accommodate a fleeting quaternary epitope. To confirm these hypotheses more
investigating needs to focus on the binding interactions between PG16 and the variable
loops. Hence, the crystallization of PG16 in complex with gp140 would allow us to
access the physical characteristics of this particular antigen-antibody interaction.
Further research could also look into clade C gp140 sensitivity to PG16, and the
details of the interactions between gp140, 697-30D and PG16. Results from the
quantification of a subset of western blots confirm that the affinity for clade C is
markedly different than the affinity for clade B. Results from the westerns also show that
clade C strains sometimes respond to conformational changes induced by CD4m, as was
observed with C1. Additionally, it was shown that 697-30D hinders PG16 in binding its
region in the V2 loop. This hindrance may indicate important spatial elements that guide
the neutralizing mechanism of PG16. It would thus be informative to understand the
nuances in the sensitivity of clade C gp140 to PG16, as well as elucidating the interaction
between 697-30D and PG16. Any biochemical detail gleaned from PG16-antigen
contacts will help in the development of a successful vaccine.
CONCLUSION
The mode of neutralization of PG16 seems to rely heavily on V1 and V2. This
should simplify future designs for antigens that will need to elicit PG16-like antibodies,
because efforts can focus on these two variable loops which are in the same vicinity in

15. both the native and CD4-induced trimeric forms (27). Furthermore, V1 and V2 are the
only variable loops required for binding, while V3 never impacts affinity for PG16, thus
confirming results previous reported in Davenport et al. These results generally suggest
that V3 does not carry much weight in epitope recognition. Furthermore it appears that a
strong spatial component exists as part of PG16’s neutralizing mechanism.
Understanding the details of how PG16 approaches its target may be very helpful in
painting a clearer picture of neutralization at V2 in particular.
On the other hand, the rational design of antigens for HIV vaccine has
consistently failed in eliciting broadly neutralizing antibodies, perhaps as a result of an
overly reductionist approach (28). Thus it could be valuable to supplement structural and
biochemical studies of PG16 with extensive studies in affinity maturation. These studies
could expose the physical challenges that exist in the elicitation of PG16-like antibodies,
and provide alternative solutions to novel HIV vaccine design (14).

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