JOURNAL OF VIROLOGY, Dec. 2008, p. 12094–12103
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 24
Human Immunodeficiency Virus Type 1 Superinfection Occurs despite
Relatively Robust Neutralizing Antibody Responses?†
Catherine A. Blish,1,2Ozge C. Dogan,1Nina R. Derby,1Minh-An Nguyen,1Bhavna Chohan,1,3
Barbra A. Richardson,4and Julie Overbaugh1*
Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 981091;
Department of Medicine, University of Washington School of Medicine, Seattle, Washington 981952; and Department of
Pathobiology3and Department of Biostatistics,4University of Washington, Seattle, Washington 98195
Received 14 August 2008/Accepted 29 September 2008
Superinfection by a second human immunodeficiency virus type 1 (HIV-1) strain indicates that gaps in
protective immunity occur during natural infection. To define the role of HIV-1-specific neutralizing antibodies
(NAbs) in this setting, we examined NAb responses in 6 women who became superinfected between ?1 to 5
years following initial infection compared to 18 women with similar risk factors who did not. Although
superinfected individuals had less NAb breadth than matched controls at ?1 year postinfection, no significant
differences in the breadth or potency of NAb responses were observed just prior to the second infection. In fact,
four of the six subjects had relatively broad and potent NAb responses prior to infection by the second strain.
To more specifically examine the specificity of the NAbs against the superinfecting virus, these variants were
cloned from five of the six individuals. The superinfecting variants did not appear to be inherently neutral-
ization resistant, as measured against a pool of plasma from unrelated HIV-infected individuals. Moreover, the
superinfected individuals were able to mount autologous NAb responses to these variants following reinfection.
In addition, most superinfected individuals had NAbs that could neutralize their second viral strains prior to
their reinfection, suggesting that the level of NAbs elicited during natural infection was not sufficient to block
infection. These data indicate that preventing infection by vaccination will likely require broader and more
potent NAb responses than those found in HIV-1-infected individuals.
Human immunodeficiency virus type 1 (HIV-1) superinfection
occurs when an individual chronically infected with one strain of
HIV-1 becomes infected with a second strain, indicating that
natural immune responses to HIV-1 are not always protective.
Since superinfection occurs despite ongoing immune responses to
the first HIV-1 strain, it provides an avenue to explore how spe-
cific immune deficits allow HIV-1 infection to become estab-
lished. To date, approximately 30 well-characterized cases of
HIV-1 superinfection have been described based on longitudinal
follow-up (1, 7, 10, 13, 14, 28, 37, 38, 41, 43, 45, 51, 56); many
other presumed cases have been defined in cross-sectional stud-
ies, where there is evidence of dual infection at the time when
viral sequences were examined (reviewed in reference 43). Many
of the cases of superinfection identified in longitudinal studies
occurred within the first year following initial infection, when
immune responses to HIV-1 are often not fully mature. However,
HIV-1 superinfections have also been found frequently during
chronic infection (38), when the immune response to HIV-1
should be fully developed.
The frequency of superinfection likely depends on a variety
of factors, including the nature of the superinfecting strains,
the use of antiretroviral medications, and the immune status of
the individual. Several studies, which screened more than 3,000
individuals, found no evidence of HIV-1 superinfection,
though many of these individuals were receiving antiretroviral
therapy (6, 9, 50). In contrast, a study of Thai intravenous drug
users found two cases of HIV-1 superinfection among 130
chronically infected individuals (41). More recently, three pop-
ulation-based studies found that HIV-1 superinfection oc-
curred at a rate close to that of initial infection. In a study of
high-risk women in Kenya, the incidence of superinfection was
approximately 4% per year (7, 38), approximately half the
incidence of primary infection in the same cohort of 8% per
year (15). Among a cohort of men in southern California, the
incidence of superinfection was 5% (45), which was equal to
the initial infection rate of 5% per year in a similar cohort (12).
The frequent detection of superinfection in these more recent
studies calls into question what role, if any, immunity to the
first strain has in protection from the second strain.
The relatively small number of well-characterized cases of
superinfection has limited analysis of the role of the immune
response in superinfection. Thus, it remains unclear whether
only a subset of individuals with particularly poor immune
responses succumb to superinfection or whether immune re-
sponses during HIV-1 infection are in general inadequate to
prevent infection. All six superinfected subjects in whom cel-
lular immune responses have been assessed had cytotoxic T
lymphocytes (CTL) directed toward their initial strain, as mea-
sured by gamma interferon enzyme-linked immunospot assay
(1, 13, 41, 47, 55). While there were differences between the
studies in the number of potential epitopes evaluated, the
breadth of the immune responses to the initial HIV-1 strain
varied in these superinfected individuals, with four individuals
* Corresponding author. Mailing address: Division of Human Biol-
ogy, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N,
Mailstop C3-168, Seattle, WA 98109-1024. Phone: (206) 667-3524.
Fax: (206) 667-1525. E-mail: email@example.com.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 8 October 2008.
having very broad responses to multiple epitopes (1, 41, 55)
and two individuals having relatively narrow responses pre-
dominantly directed to a single epitope (13, 47). In four of
these cases, at least some of the CTL present were cross-
reactive with the superinfecting strain prior to reinfection (1,
41, 47, 55). However, in all six cases at least some of the
targeted CTL epitopes were altered in the superinfecting
strains, which could have contributed to the ability of these
strains to establish infection (1, 13, 41, 47, 55). Furthermore, as
CTL play a critical role in controlling an established infection
but are ineffective in preventing initial infection (reviewed in
references 5 and 35), it is perhaps not surprising that these
cellular responses were insufficient to prevent reinfection.
Unlike CTL, neutralizing antibodies (NAbs) to HIV-1 can
prevent infection in animal models (reviewed in reference 46);
NAbs, therefore, might be able to prevent superinfection in
humans if sufficiently broad and potent. Among two cases of
superinfection described by Ramos et al., binding antibodies to
the V3 region were generated to the initial strain though these
did not appear to cross-react with the superinfection strain
(41). Moreover, in this study, neutralization was not assessed
(41). In a third case, only weak NAbs were present to the initial
virus, and these antibodies did not neutralize the superinfect-
ing strain (1). In the only study to compare superinfected
individuals to those with similar risk factors who did not be-
come superinfected, three individuals who became superin-
fected had weak NAb responses to their initial infection (44).
These three superinfections all occurred relatively early, within
6 months of initial infection. In addition, this study examined
the ability of plasma from the superinfected individuals to
neutralize just three viruses, and responses to the superinfect-
ing virus itself were not examined. In order to more rigorously
evaluate the potential role of NAb responses in protection
from superinfection, we assessed the NAb responses in six
cases of superinfection that occurred throughout the course of
chronic HIV-1 infection using a larger panel of viruses, includ-
ing the superinfecting strains.
MATERIALS AND METHODS
Study population. The individuals in this study were part of a prospective
cohort study of high-risk women from Mombasa, Kenya, in which timing of the
first infection is defined by both HIV-1 serology and HIV RNA testing (23–25).
All of the women were HIV-1 infected through heterosexual contact, and none
reported antiretroviral therapy during follow-up for this study although some
have since started therapy due to CD4 counts of ?200/?l. From this cohort, a
total of 56 individuals were screened for superinfection by analysis of env se-
quences (7) or both env and gag sequences (38) in peripheral blood mononuclear
cells (PBMCs) within the first year of infection and again ?5 years later. Pre-
sumed cases of superinfection were confirmed using phylogenetic analyses, and
the time of superinfection was determined by examining the HIV sequences in
intervening PBMC samples, typically collected at 3-month intervals, using both
phylogenetic analysis and subtype-specific PCR (7, 38). The superinfection cases
and controls for the present study were identified from among these 56 women.
Verbal or written informed consent was obtained from all patients. The ethical
review committees of the University of Nairobi, the University of Washington,
and the Fred Hutchinson Cancer Research Center approved this study.
Cloning of full-length, functional env genes. In most cases, full-length env
genes for the virus panel and from superinfection cases were cloned directly from
PBMC DNA by limiting dilution nested PCR as described previously (21, 54).
Because insufficient PBMC DNA was available, env clones from subjects QD022
and QB008 were cloned from DNA from cultured PBMCs. The virus culture was
maintained for a maximum of 21 days, during which time there is limited impact
on the representation of the major viral species (53). Because there is variability
in the sequence targets for amplification, conditions were optimized for each
subject; primers and PCR conditions for each case are listed in Table S1 in the
supplemental material. The full-length sequences obtained were highly repre-
sentative of the sequences obtained previously by amplification of the V1-V5
region (7, 38). The PCR products were digested with MluI and NotI restriction
enzymes (Invitrogen) and cloned into the pCIneo vector (Promega). In the one
case where the restriction sites were not compatible (the env genes from subject
QA013), PCR products were cloned into pcDNA3.1/V5-His-TOPO vector (In-
vitrogen). All env sequences were evaluated to determine if recombination had
occurred between the initial and the superinfecting strains, as defined previously
(7, 38). Only one sequence, from subject QB008, was a recombinant sequence.
As it could not be conclusively established whether this recombination event
occurred in the subject or during the PCR amplification, this variant was not
studied further. To generate pseudotyped viral particles, plasmid DNA was
transfected into 293T cells along with an envelope-deficient HIV-1 subtype A
proviral plasmid, Q23?env, as described previously (21). Pseudotyped viruses
were screened for infectivity by a single-round infection of TZM-bl cells (29)
(AIDS Research and Reference Reagent Program, National Institutes of
Health). Approximately 50% of cloned env genes were capable of mediating
infection; the complete sequences of these functional clones, each from an
independent PCR, were determined using a BigDye Terminator, version 3.1,
cycle sequencing kit (Applied Biosystems) according to the manufacturer’s in-
Phylogenetic analyses. Full-length env sequences were examined using the
BLAST search tool from the National Center for Biotechnology Information
(NCBI; at http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to rule out contamination
from other laboratory strains or sample mix-up. Full-length env sequences from
the novel clones as well as sequences from the original V1-V5 sequences used to
define the superinfection cases (7, 38) were assembled using Sequencher soft-
ware (Gene Codes). The V2-V5 regions were then aligned using Clustal X (49),
and manually edited using MacClade, version 4.01 (22), to remove regions that
could not be unambiguously aligned, as described previously (7, 38). Phyloge-
netic trees were then constructed by neighbor-joining using an HKY85 model in
PAUP* (version 4.01b10) (48). Viral subtype was defined using the NCBI geno-
typing database (http://www.ncbi.nlm.nih.gov/) and by phylogenetic analyses with
reference sequences from the Los Alamos National Laboratory HIV database
Neutralization assays. Neutralization was assessed in triplicate with at least
two independent preparations of pseudoviral stock using the TZM-bl neutral-
ization assay as described previously (3, 29, 54). Briefly, 500 infectious particles
of pseudovirus, as determined by infection of TZM-bl cells, were incubated with
serial dilutions of plasma for 1 h; then TZM-bl indicator cells were added, and
infection levels were determined by assessing ?-galactosidase activity after 48 h.
Median inhibitory concentrations (IC50s) were defined as the reciprocal dilution
of plasma that resulted in 50% inhibition, calculated as described previously
using the linear portion of the neutralization curve (3, 54). The standard plasma
pool collected from 30 HIV-1-infected individuals in Kenya between 1998 and
2000 has been previously described (3).
Calculation of breadth and potency scores. Breadth and potency scores were
calculated for each plasma sample to compare neutralization between cases and
controls. A median IC50value was assigned to each panel virus, based on the
median of all the IC50values from every plasma sample tested against that virus.
To define a breadth score for an individual plasma sample, the plasma/virus
combinations in which the IC50was above the median IC50defined for that virus
with all plasma samples were given a score of 1, and those below were scored as
0. The overall breadth score was determined by summing these numbers for all
16 viruses tested. The potency score was derived by dividing the IC50value of a
given plasma/virus combination by the median virus IC50value. As with the
breadth score, the potency scores against all 16 panel viruses were added to
obtain an overall potency score. Thus, if a plasma sample had an IC50of 250 for
a virus whose median score was 50, this plasma would receive 1 breadth point but
5 potency points toward its total score.
Statistical analyses. All statistical analyses were performed using Intercooled
Stata, version 10.0 (College Station, TX). In order to compare median IC50,
mean IC50, breadth score, and potency score between superinfection cases and
controls, the values for the three controls for each case were averaged. The
matched values for superinfection cases and controls were compared by a Wil-
coxon signed ranks test. In order to determine whether the timing of the super-
infection sample influenced the neutralization scores, the number of years
postinfection (ypi) was compared with the mean, median, breadth, or potency
scores using a Spearman rank correlation. A two-sample Wilcoxon rank sum test
was used to compare the plasma pool neutralization sensitivity of the superin-
fecting variants to that of the initial variants. The same test was used to compare
neutralization sensitivity of superinfecting and early variants.
VOL. 82, 2008 ANTIBODY RESPONSES DURING HIV-1 SUPERINFECTION 12095
Nucleotide sequence accession numbers. The novel sequences from the su-
perinfected subjects have been deposited in the GenBank database under acces-
sion numbers FJ396012 through FJ396031.
Breadth and potency of NAb responses in superinfection
cases and controls. Six cases of superinfection that occurred
between ?1 to 5 years following initial infection were previ-
ously identified within a female sex worker cohort study in
Kenya (Fig. 1 and Table 1) (7, 38). Viral loads were approxi-
mately 40,000 copies/ml in most individuals near the time of
superinfection (7, 38). Absolute CD4 counts were available
prior to superinfection in only two subjects, and both had CD4
counts exceeding 500 cells/?l (38). The remaining four subjects
had CD4 counts exceeding 200 cells/?l at the first available
time point following superinfection, which ranged from 1 to 4
years following superinfection (7, 38). Thus, none of these
individuals appeared to be in an advanced state of immuno-
suppression at the time of reinfection. In order to determine
whether deficits in the breadth and potency of their NAb
responses were a correlate of superinfection, we examined
three controls in which we could not detect superinfection for
each case. Controls were matched to cases according to the
initial HIV subtype (A, C, or D), the timing of plasma samples
in relation to initial infection, and the viral load (Table 2). NAb
responses in cases and controls were examined at approxi-
mately 1 year following initial infection in all subjects to allow
us to compare all individuals at the same time point (Fig. 1 and
Table 2), as well as just prior to documented superinfection
(Fig. 1 and Table 2). In order to assess the breadth and potency
of the plasma from the superinfection cases and controls, we
tested a panel of 16 HIV-1 pseudoviruses representing trans-
mitted variants from various HIV-1 subtypes including A, A/D
recombinant, C, D, and B. The majority of variants were
cloned from individuals from Kenya (3, 54; also unpublished
data), except for 6535.3 (B5), THRO4156.18 (B15), and
Du156.12 (C1), which are part of the previously described
standard subtype B and C virus panels (17, 18). These variants
were selected for the panel on the basis of their neutralization
sensitivity to pooled plasma from HIV-1-infected individuals in
Kenya. Selecting variants that showed detectable neutraliza-
tion with pooled plasma increased the chances of observing,
and thus differentiating, neutralizing activity between individ-
ual plasma samples.
We assessed the ability of plasma to neutralize the panel
viruses by determining the median IC50(defined as the recip-
rocal dilution of plasma that resulted in 50% inhibition) for
each plasma/virus pair at ?1 ypi (Fig. 1 and Fig. 2a). Plasma
from both cases and controls exhibited a broad range of neu-
tralizing potency against these transmitted variants (Fig. 2a).
To quantitate potential differences between superinfection
cases and matched controls, each case was compared to the
average of the three controls per case. The mean IC50value
was lower in the superinfecting plasma than in the controls
(P ? 0.05) while there was a trend for a lower median IC50
value in superinfection cases than in controls (P ? 0.07). Be-
cause each panel virus had a different overall neutralization
sensitivity, we normalized the IC50value of each virus/plasma
pair to the median IC50for each virus with all plasma to
calculate breadth and potency scores. At ?1 year after initial
FIG. 1. Timing of analyzed samples for the six superinfection cases.
Each of the six superinfection cases is identified on the left, with a
horizontal line indicating the time since the initial infection, according
to the scale at the bottom. The interval during which superinfection
occurred is indicated by the gray bar. The ?1-year time point from
which plasma samples were evaluated is indicated by the open squares,
and the presuperinfection time point is indicated by the black circles.
For subjects QA013 and QB008, the ?1-year time point and the
presuperinfection time point were the same since superinfection oc-
curred at approximately 1 year. The gray circles indicate the first time
point at which the superinfecting variants were identified; all env vari-
ants were cloned from this time point. SI, superinfection.
TABLE 1. Characteristics of superinfection casesa
aReferences 7 and 38.
TABLE 2. Selection of cases and controls for assessment of
breadth and potency of NAb responses
Group and case
Cases at ?1 year
aFor each case, 3 controls per case were matched according to HIV-1 subtype,
timing of plasma samples tested, and VLs. Insufficient data were available to
compare CD4 counts between cases and controls.
bND, not done.
12096BLISH ET AL. J. VIROL.
infection, the superinfection cases had significantly lower
breadth scores than the matched controls (Wilcoxon signed
ranks test, P ? 0.046) (Fig. 2b). In addition, there was a trend
toward less potency in the NAb responses at ?1 year in su-
perinfection cases than in controls (P ? 0.075) (Fig. 2c). To
ensure that variability in the sample timing was not altering
results, we compared the breadth or potency scores with the
number of years postinfection by Spearman rank correlation.
There was no significant relationship between the timing of the
plasma samples and the breadth (P ? 0.36) or the potency
(P ? 0.51) scores. As much of the neutralizing activity ob-
served was relatively weak, we also calculated 70 and 80%
inhibitory concentration (IC70and IC80, respectively) values
(see Fig. S1 and S2 in the supplemental material). On the basis
of IC70values, we observed less potency and a trend for less
breadth among superinfection cases than in controls (see Fig.
S1 in the supplemental material). IC80values were generally
below the limit of detection, and no significant differences in
neutralizing activity were observed between superinfection
cases and controls (see Fig. S2 in the supplemental material).
Plasma samples from the superinfection cases and controls
were then compared at the time point immediately prior to
superinfection, as the breadth and the potency of the NAbs at
this time should reflect those faced by the entering superin-
fecting virus (Fig. 1 and 3a and Table 2). NAbs present in four
of the six superinfection cases (QA013, QA413, QB045, and
QB726) could neutralize the majority of the heterologous
panel viruses at this time (Fig. 3a). There was no detectable
difference between the superinfection cases and controls in the
breadth (P ? 0.35) (Fig. 3b) or the potency (P ? 0.92) (Fig. 3c)
FIG. 2. NAb responses at approximately 1 ypi. (a) The plasma samples tested are displayed along the left, with a subject identification code
followed by the number of years postinfection at which the plasma sample was obtained. Each superinfection case is displayed in larger font at the
top of a group, with the three matched controls in the box beneath. The 16 viruses tested are shown at the top, followed by a column for the mean,
median, breadth score, and potency scores for each plasma sample (four rightmost columns). The 16 panel viruses are abbreviated by subtype along
the top. The virus variants are: A1, Q461d1; A2, Q168b23; A3, Q842d16; A4, BJ613.E1; A5, BS208.B1; A6,Q769b9; A/D1, BF535.A1; A/D2,
QA790.204I.ENV.C1; D1, QD435.100 M.ENV.A4; D2, QA465.59 M.ENV.D1; A/D3, QZ100.ENV.D83; C1, Du156.12; C2,QB099.391
M.ENV.C8; C3, QC406.70 M.ENV.F3; B5, 6535.3; B15, THRO4156.18. IC50values are shown as numerical values in the table. The data are color
coded, with darker blue boxes denoting more potent neutralization. A gray bar indicates that ?50% neutralization was observed at a plasma
dilution of 1:50, which was the highest dilution tested. For the purposes of statistical analyses, these IC50values were assigned a level of 25. The
two values marked “*auto” in red indicate that the plasma sample was autologous to the panel virus and was therefore not included in calculation
of the breadth and potency scores. (b) Comparison of breadth scores between superinfection cases and the average value from the three matched
controls for each case. Breadth scores are shown along the y axis, and the superinfecting and control groups are compared along the x axis. The
lines between the data points denote comparison between the superinfection cases and the matched controls. P values for the superinfection cases
compared to controls were obtained by comparing scores with the Wilcoxon signed ranks test. (c) Comparison of potency scores, displayed as per
breadth scores in panel b.
VOL. 82, 2008ANTIBODY RESPONSES DURING HIV-1 SUPERINFECTION 12097
of their NAb responses just prior to superinfection. Similarly,
there were no significant differences in either mean (P ? 0.92)
or median (P ? 0.92) IC50values between superinfection cases
and controls. Furthermore, as with the ?1-year time point,
there was no significant correlation between the timing of the
samples and the breadth (P ? 0.31) or potency (P ? 0.65)
scores. Finally, comparison of IC70and IC80values also did not
reveal any significant differences between cases and controls
(see Fig. S3 and S4 in the supplemental material).
Characterization of superinfecting envelope variants. In or-
der to evaluate the neutralization profile of the superinfecting
variants, we cloned full-length, functional HIV-1 envelope
variants from the first time point in which superinfecting vari-
ants were detected. Clones representing the envelope from the
superinfecting variants were obtained for five of the six cases;
in subject QB045 the superinfecting variant was always a mi-
nority variant (38), and despite cloning ?10 functional vari-
ants, we were unable to obtain a superinfecting variant and
could not evaluate this case further (data not shown). In three
cases (QB008, QA413, and QB726), we cloned variants repre-
sentative of both the initial and superinfecting variants, as
defined by sequence analyses in previous studies (Fig. 4) (7,
38). In two cases, only clones representing the superinfecting
variant were obtained (Fig. 4), presumably because the super-
infecting strain became dominant (QA013) or replaced the
initial strain (QD022). The V1-V5 sequences within the full-
length env variants were representative of the previously de-
scribed sequences (Fig. 4). The diversity observed at this early
time point could reflect the transmission of multiple variants,
as has been previously observed among women infected het-
erosexually (20). The diversity and evolution of the superin-
fecting variants following reinfection have been discussed in
more detail previously (7, 38).
In order to determine whether the superinfecting variants
were particularly neutralization resistant, the sensitivity of
these variants to a plasma pool derived from 30 HIV-positive
individuals in Kenya (3) was examined. No significant differ-
ences in the neutralization sensitivity of the superinfecting
variants was observed compared to either the initial viruses
from the superinfection cases or a collection of viruses
pseudotyped with envelope variants of HIV-1 derived from
other infected individuals early in their infections (Table 3).
Thus, the superinfecting variants did not appear to be inher-
ently neutralization resistant.
FIG. 3. NAb responses immediately prior to superinfection. (a) IC50values for plasma/virus combinations are presented as described in the
legend to Fig. 2a. Here, the viruses tested are the same as in the experiment shown in Fig. 2, but the plasma tested was from a different time point,
i.e., the time point immediately prior to documented superinfection. (b) Comparison of breadth scores between superinfection cases and matched
controls prior to superinfection as described in the legend of Fig. 2b. (c) Comparison of potency scores between superinfection cases and matched
controls prior to superinfection as described in the legend of Fig. 2c.
12098BLISH ET AL.J. VIROL.
Neutralization of superinfecting variants by plasma from
the superinfected individuals. To more specifically examine the
NAb sensitivity of the envelope of variants from superinfected
individuals near the predicted time of exposure, each variant was
tested against plasma from the time point just prior to docu-
mented superinfection (Fig. 5). In addition, variants were tested
against plasma samples from later in infection in order to deter-
mine whether these individuals were able to mount an autologous
response to these variants (Fig. 5). In subject QA013, superinfec-
tion occurred between 0.72 and 1.1 ypi (7). Three of the four
superinfecting variants (denoted by an asterisk) from 1.1 ypi were
neutralized by plasma from 0.72 ypi, with IC50values of 106 for
QA013.H12*, 113 for QA013.Q4*, and 94 for QA013.R3* (Fig.
5a). Variant QA013.J36* was remarkably neutralization sensitive,
with an IC50value of 1,343 with the presuperinfection plasma and
IC50values exceeding 25,000 with later plasma samples (Fig. 5a).
This subject mounted a potent autologous NAb response, with
IC50values of ?1,800 to all of the superinfecting variants at 6.3
ypi (Fig. 5a).
In subject QB008, superinfection occurred between 0.83 and
1.6 ypi (7), and two initial and two superinfecting variants were
cloned at 1.6 ypi (Fig. 4). One of the two superinfecting vari-
ants (QB008.C9*) was weakly neutralized by the plasma from
0.83 ypi, with an IC50of 66 (Fig. 5b). The other superinfecting
variant, QB008.E2*, failed to reach 50% neutralization at a
1:50 dilution of plasma, which was the highest concentration
tested, making it resistant in this assay (Fig. 5b). All of the
variants were neutralized by autologous plasma at the contem-
poraneous time point with IC50values of 56 to 202, and this
subject went on to develop a preferential NAb response to the
initial virus variants (Fig. 5b).
We first detected superinfection in subject QA413 at 2.8 ypi
(38) and cloned six viral variants from that time point: two rep-
resenting the initial strain and four representing the superinfect-
ing virus. Plasma from 2.0 ypi was able to weakly neutralize three
of the four superinfecting variants, with IC50values of 53 for
QA413.G3*, 72 for QA413.H6*, and 72 for QA413.K3* (Fig. 5c).
The fourth superinfecting variant, QA413.E15*, was resistant to
neutralization by plasma from 2.0 ypi. One of the initial viral
variants (denoted by the caret), QA413.C3ˆ, was resistant to
neutralization at all but the latest time point evaluated, while the
tralization, with IC50values from 210 to 285 throughout the
course of infection. This subject developed only moderate autol-
ogous NAb responses, with IC50values from 77 to 193 to all the
variants tested (Fig. 5c), despite good breadth in the heterologous
NAb responses (Fig. 3a).
Subject QB726 was superinfected between 2.8 and 3.2 ypi
(38). The superinfecting variant cloned at 3.2 ypi was neutral-
ized by the plasma from 2.8 ypi with a IC50of 59, while the
initial variant was slightly more susceptible to this plasma sam-
TABLE 3. Neutralization sensitivity of superinfecting, initial, and
early variants of HIV-1 to a pool of plasma from HIV-1
aInitial variants from the superinfection cases were cloned at the time point
when the superinfecting variants were first detected. Early variants represent
full-length functional HIV-1 variants from within 1 year of infection that were
cloned from individuals infected with subtypes A, A/D recombinant, C, and D in
Kenya. The plasma pool was collected from 30 HIV-1-infected individuals in
Kenya between 1998 and 2000 and has been previously described (13).
bSince more than one variant was examined from each subject and these
variants might be more similar to each other than those from other subjects, a
median IC50value of all the variants per person was determined. These per
person IC50values were then used to compare between superinfecting, initial,
and early variants.
cP values were determined by two-sample Wilcoxon rank sum testing for
superinfecting variants compared to either initial or early variants.
FIG. 4. Neighbor-joining phylogenetic tree of V2-V5 sequences
from superinfection cases. Reference sequences for subtype G, D, C,
and A from the Los Alamos HIV database (http://www.hiv.lanl.gov
/content/index) are displayed in black. Sequences from each subject
are denoted in a separate color. For reference, sequences that were
originally obtained from these cases using primers that amplify a sub-
genomic portion of envelope (V1-V5) (7, 38) are shown in italics. The
case reference sequences were obtained from the first time point at
which the superinfecting variants were detected and contain represen-
tatives of both the initial and superinfecting strains, except for case
QD022, in which the superinfecting variants completely replaced the
initial variants. Thus, the QD022.A and QD022.B initial sequences in
bold italics were from a presuperinfection time point (0.14 ypi). Se-
quences from the full-length env clones from these same individuals
isolated as part of this study are shown in bold print, with an asterisk
denoting a superinfecting sequence and a caret denoting a sequence
from the initial virus population, as defined in the previous study.
Full-length envelope sequences were cloned either directly from
PBMC DNA (subjects QA013, QB726, and QA413) or from PBMC
DNA following short-term coculture with uninfected PBMCs (subjects
QB008 and QD022) from the first time point at which superinfecting
sequences were detected.
VOL. 82, 2008 ANTIBODY RESPONSES DURING HIV-1 SUPERINFECTION12099
ple with a IC50value of 132 (Fig. 5d). This subject, despite a
broad and potent heterologous NAb response prior to super-
infection (Fig. 3a), developed only moderate NAb responses to
the variants cloned at 3.2 ypi, with IC50values of 50 to 170 to
these autologous variants (Fig. 5d).
Subject QD022 was superinfected between 5.0 and 5.4 ypi
(38). In this subject, all of the superinfecting variants cloned
from 5.4 ypi were resistant to the 5.0 ypi plasma sample (Fig.
5e). These variants were weakly neutralized by the contempo-
raneous plasma sample, with IC50values of 56 to 104, but
subsequent plasma samples neutralized these variants more
potently, with IC50values of 170 to 726 (Fig. 5e).
Overall, one of the two subjects with relatively narrow NAb
responses prior to superinfection (Fig. 3a, QB008) was still
able to neutralize one of her superinfecting strains, while the
other subject with relatively narrow responses (QD022) was
reinfected with viral strains that were resistant to host NAbs.
Three subjects with relatively broad NAb responses prior to
superinfection (QA013, QA413, and QB726) (Fig. 3a) were
able to neutralize the majority of their superinfecting viruses
prior to their reinfection with these strains. All individuals
were able to mount autologous NAb responses to their super-
infecting variants following reinfection (Fig. 5), indicating that
these variants were not inherently neutralization resistant, con-
sistent with the findings using pooled plasma (Table 3).
We comprehensively evaluated the role of the breadth and the
potency of the HIV-1 NAb response in protection from infection
using six cases of superinfection that were identified among a
cohort of female sex workers with extensive long-term follow-up
(7, 38). At the time of superinfection, no significant deficits in
NAb responses were observed in the superinfected individuals
compared to matched controls. Thus, even NAb levels typically
found during chronic infection can fail to protect from reinfection
with circulating strains of HIV-1. Furthermore, in four of five
cases evaluated, superinfection occurred despite preexisting
plasma NAbs capable of neutralizing the strains that established
the second infection.
The breadth and potency of the NAb responses were hetero-
geneous among these individuals at the time of exposure to the
FIG. 5. Neutralization of superinfecting and initial variants by plasma from the superinfected individuals. Each panel represents the evolving
NAb response in the superinfected subject indicated in the upper left. The autologous plasma IC50value against the various initial and
superinfecting env variants is plotted over time. Superinfecting variants are denoted with blue symbols, and initial variants are indicated with orange
symbols. All the env variants were cloned from the time point immediately after superinfection, and the light-blue bars denote the interval in which
superinfection occurred. The gray bar indicates the limit of detection of our assay. When a virus was neutralized ?50% at a plasma dilution of
1:50, the highest dilution tested, the IC50value was assigned a level of 25 and is within the gray area. PI, postinfection.
12100BLISH ET AL. J. VIROL.
superinfecting virus. As the variants within the virus panel were
chosen on the basis of their neutralization sensitivity to pooled
plasma, most individuals were able to neutralize at least one of
these heterologous variants. Overall, the breadth and potency
of the NAb responses were similar to those found in the
matched controls and other chronically infected individuals. In
particular, four subjects had relatively robust NAb responses,
while two others had comparatively narrow responses. In fact,
the four superinfected subjects with the broadest NAb re-
sponses could neutralize the ?70% of the viruses within the
panel with average IC50s of ?110, while the matched controls
neutralized ?40% of the panel viruses with average IC50s of
?90. Admittedly, limitations in the numbers of cases and con-
trols limited the robustness of the statistical analyses. We
therefore compared the NAb responses of the superinfected
individuals to those of 72 individuals from the same cohort
whose NAb responses were assessed at 5 ypi (K. Bosch, D.
Panteleeff, and J. Overbaugh, unpublished data). Three super-
infected subjects (QA413, QB045, and QB726) had breadth
and potency scores within the upper quartile of these 5-year
responses. Subject QA013 had breadth and potency scores at
the median of the 5-year NAb responses despite having been
superinfected relatively early after the first infection (?1 year),
at a time when the breadth and potency of the NAb response
has not yet peaked (42). These 72 women were not selected
based on any clinical or immunological findings; thus, they
represent typical NAb responses in high-risk African women
during chronic infection. Four of the six superinfections there-
fore occurred in individuals with relatively broad and potent
NAb responses at the time of exposure. It is difficult to draw
precise comparisons with our data and NAb responses ob-
served in other cohorts because of differences in assays and test
strains used. In addition, most of the published studies have
focused on selected individuals, particularly long-term nonpro-
gressors, which is not an ideal comparison group (2, 8, 19, 30,
32, 39, 57). While rare individuals in these populations exhib-
ited apparently broader and more potent NAb responses (e.g.,
neutralization of ?90% of viruses tested at average IC50s of
?230) (8) than the women in our study, many other individuals
exhibited less breadth and potency in NAb responses against
the test strains used. On the basis of all these comparisons—
including to matched controls, to chronically infected women
in the same population, and to published studies—we conclude
that the women who became superinfected did not have defi-
cits in the breadth or potency of their NAb responses relative
to other HIV-infected groups when they became superin-
fected. Overall, NAb responses in superinfected individuals
were typical of HIV-1 infection, ranging from narrow to rela-
At ?1 ypi, we observed relatively narrow NAb responses in
superinfected individuals compared to controls. This early lack
of breadth in the superinfected women mirrors the findings in
three superinfected men who acquired HIV-1 through sex with
men (44). Interestingly, this same association was observed
despite the fact that the study of Smith et al. used a small
number of primarily laboratory-adapted viruses whereas ours
employed a large collection of variants cloned directly from
infected individuals near the time of transmission. This early
association between breadth and risk of superinfection in both
studies suggests that some association between HIV-1-specific
antibodies and risk of superinfection may exist early in infec-
tion. However, we did not observe a correlation between NAb
breadth at the time of superinfection and whether a woman
became superinfected. At these later times, which seem to be
a more relevant measure of the role of NAbs in protection
from infection, there was a broadening of the NAb responses
in most individuals prior to their documented superinfection.
Perhaps a more important measure of the role of NAbs in
protection from superinfection is the study of the antigenicity
of the specific viruses that established the second infection. In
this study, we examined such viruses derived from the first time
point following documented superinfection. We found that
these viruses were not unusually neutralization resistant as
there were no differences in the neutralization sensitivities of
these variants and other circulating variants to pooled plasma
from HIV-1-infected individuals. Moreover, the infected indi-
viduals were able to mount autologous NAb responses to the
superinfecting variants, suggesting that the strains could them-
selves elicit NAbs. Importantly, the superinfecting variants
were often susceptible to neutralization by plasma from the
person who became infected by these strains: in four of five
subjects, at least one superinfecting variant was susceptible to
the host plasma from the time prior to superinfection. Even if
a “sieve” effect weeded out the majority of the neutralization-
sensitive viral variants from the donor virus, at least some of
these variants apparently established infection despite encoun-
tering NAbs capable of neutralizing them. While we cannot
rule out that these variants evolved within a narrow window
after transmission, it is not clear what would drive them to
become more susceptible to neutralization. Overall, these data
suggest that the levels of NAbs found in these individuals were
insufficient to prevent infection even by variants that showed
Several caveats to these data need to be considered. First, as
with all cases of superinfection, it remains possible that the
superinfecting viruses were present at low levels and/or com-
partmentalized prior to their first detection. We have reason-
able confidence that the superinfecting strains were detected
soon after they became established in these cases because we
used a sensitive subtype-specific PCR assay that gives ?92%
probability of detecting a strain present with a prevalence of
5% to define the time of superinfection (38). Moreover, the
women in this cohort have relatively few partners (on average
1 to 2 per week) (16), making superinfection in a short time
frame less likely than reported in women who have many more
partners (11). Secondly, we cannot conclude that control sub-
jects were protected from superinfection because superinfec-
tions could be missed if the second virus did not persist (56) or
recombined with the initial virus within the regions of the
genome analyzed (38). If such cases were missed within the
control group, this would decrease our ability to detect differ-
ences between cases and controls. Third, new infections are
generally established at mucosal sites, where NAb levels could
be lower than those assessed in the plasma, possibly allowing
local establishment and spread of infection before immune
control could be attained. It is therefore possible that differ-
ences could be observed in NAb levels at the mucosal sites, in
particular, against the superinfecting strains near the time of
While this study suggests that the presence of any detectable
VOL. 82, 2008 ANTIBODY RESPONSES DURING HIV-1 SUPERINFECTION12101
NAbs of the proper specificity may not protect against HIV-1
infection, it does not rule out the possibility that such antibod-
ies would be effective if present at higher levels. These data are
consistent with the nonhuman primate (NHP) model, where
sterile protection from infection generally required very high
NAb levels that produced ?99% in vitro neutralization of the
challenge simian-human immunodeficiency virus with between
1:8 and 1:200 dilutions of plasma (26, 27, 31, 34, 36). While
differences in the assays used to assess neutralization between
the NHP studies and our human study make direct compari-
sons difficult, the NAb levels in the superinfected individuals
were probably not at this potency. Even the unusually neutral-
ization-sensitive variant QA013.J36* was neutralized at 90%
by host plasma at a 1:78 dilution but did not achieve 99%
neutralization at the lowest plasma dilution tested (1:50). The
remaining superinfecting variants were neutralized at levels of
?90% with a 1:50 dilution of plasma and were therefore not at
the levels required for sterilizing immunity in the NHP model.
Thus, it remains unclear whether the higher levels of NAbs
achieved by passive transfer within the NHP model would be
protective in humans.
These findings in exposed humans, where there is extensive
diversity in potential infecting strains, unfortunately suggest a
high bar for the levels of antibodies required for eliciting pro-
tective immunity. While weak, narrow NAb responses could
have contributed to superinfection in a subset of individuals,
others became reinfected despite relatively robust NAb re-
sponses to their first strain. Since NAbs can clear virus without
dependence on the cellular immune system, the levels of an-
tibodies required for protection in these previously infected
individuals are likely similar to those required in uninfected,
vaccinated individuals. Furthermore, most effective vaccines
are thought to provide protection primarily by stimulating neu-
tralizing antibodies to clear cell-free virus (35, 40); thus, the
assays used here should provide a valid measure of viral pro-
tection by this mechanism. An effective HIV-1 vaccine will
therefore need to elicit more robust NAb responses than found
during natural infection. Indeed, this is the case for some other
viral vaccines, such as those for hepatitis B and human papil-
lomavirus, which elicit equivalent or higher levels of NAbs than
natural infection (4, 33, 52). HIV-1 presents additional chal-
lenges because of the extreme genetic diversity of the virus.
Our results suggesting that reinfection occurs even in individ-
uals who have antibodies capable of neutralizing diverse strains
further underscore this challenge.
We thank like the Mombasa research team for their support and
efforts, Stephanie Rainwater for assistance with phylogenetic analyses,
and Anne Piantadosi for helpful comments on the manuscript. We also
gratefully acknowledge the women who participated in the study.
This study was supported by NIH grant AI38518 to J.O. and grant
K08 AI068424-01 to C.A.B. C.A.B. was also supported in part by NIH
training grant T32AI07140.
1. Altfeld, M., T. M. Allen, X. G. Yu, M. N. Johnston, D. Agrawal, B. T. Korber,
D. C. Montefiori, D. H. O’Connor, B. T. Davis, P. K. Lee, E. L. Maier, J.
Harlow, P. J. Goulder, C. Brander, E. S. Rosenberg, and B. D. Walker. 2002.
HIV-1 superinfection despite broad CD8?T-cell responses containing rep-
lication of the primary virus. Nature 420:434–439.
2. Beirnaert, E., P. Nyambi, B. Willems, L. Heyndrickx, R. Colebunders, W.
Janssens, and G. van der Groen. 2000. Identification and characterization of
sera from HIV-infected individuals with broad cross-neutralizing activity
against group M (Env clade A-H) and group O primary HIV-1 isolates.
J. Med. Virol. 62:14–24.
3. Blish, C., R. Nedellec, K. Mandaliya, D. Mosier, and J. Overbaugh. 2007.
HIV-1 subtype A envelope variants from early in infection have variable
sensitivity to neutralization and to inhibitors of viral entry. AIDS 6:693–702.
4. Bocher, W. O., S. Herzog-Hauff, W. Herr, K. Heermann, G. Gerken, K. H.
Meyer Zum Buschenfelde, and H. F. Lohr. 1996. Regulation of the neutral-
izing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vac-
cine recipients and patients with acute or chronic hepatitis B virus (HBV)
infection. Clin. Exp. Immunol. 105:52–58.
5. Brown, S. A., J. L. Hurwitz, X. Zhan, P. C. Doherty, and K. S. Slobod. 2005.
CD8?T-cells: are they sufficient to prevent, contain or eradicate HIV-1
infection? Curr. Drug Targets Infect. Disord. 5:113–119.
6. Chakraborty, B., L. Valer, C. De Mendoza, V. Soriano, and M. E. Quinones-
Mateu. 2004. Failure to detect human immunodeficiency virus type 1 super-
infection in 28 HIV-seroconcordant individuals with high risk of reexposure
to the virus. AIDS Res. Hum. Retrovir. 20:1026–1031.
7. Chohan, B., L. Lavreys, S. M. J. Rainwater, and J. Overbaugh. 2005. Evi-
dence for frequent reinfection with human immunodeficiency virus type 1 of
a different subtype. J. Virol. 79:10701–10708.
8. Dhillon, A. K., H. Donners, R. Pantophlet, W. E. Johnson, J. M. Decker,
G. M. Shaw, F. H. Lee, D. D. Richman, R. W. Doms, G. Vanham, and D. R.
Burton. 2007. Dissecting the neutralizing antibody specificities of broadly
neutralizing sera from human immunodeficiency virus type 1-infected do-
nors. J. Virol. 81:6548–6562.
9. Gonzales, M. J., E. Delwart, S. Y. Rhee, R. Tsui, A. R. Zolopa, J. Taylor, and
R. W. Shafer. 2003. Lack of detectable human immunodeficiency virus type
1 superinfection during 1072 person-years of observation. J. Infect. Dis.
10. Gottlieb, G. S., D. C. Nickle, M. A. Jensen, K. G. Wong, J. Grobler, F. Li,
S. L. Liu, C. Rademeyer, G. H. Learn, S. S. Karim, C. Williamson, L. Corey,
J. B. Margolick, and J. I. Mullins. 2004. Dual HIV-1 infection associated
with rapid disease progression. Lancet 363:619–622.
11. Grobler, J., C. M. Gray, C. Rademeyer, C. Seoighe, G. Ramjee, S. A. Karim,
L. Morris, and C. Williamson. 2004. Incidence of HIV-1 dual infection and
its association with increased viral load set point in a cohort of HIV-1
subtype C-infected female sex workers. J. Infect. Dis. 190:1355–1359.
12. Harro, C. D., F. N. Judson, G. J. Gorse, K. H. Mayer, J. R. Kostman, S. J.
Brown, B. Koblin, M. Marmor, B. N. Bartholow, and V. Popovic. 2004.
Recruitment and baseline epidemiologic profile of participants in the first
phase 3 HIV vaccine efficacy trial. J. Acquir. Immune Defic. Syndr. 37:1385–
13. Jost, S., M. C. Bernard, L. Kaiser, S. Yerly, B. Hirschel, A. Samri, B. Autran,
L. E. Goh, and L. Perrin. 2002. A patient with HIV-1 superinfection.
N. Engl. J. Med. 347:731–736.
14. Koelsch, K. K., D. M. Smith, S. J. Little, C. C. Ignacio, T. R. Macaranas,
A. J. Brown, C. J. Petropoulos, D. D. Richman, and J. K. Wong. 2003. Clade
B HIV-1 superinfection with wild-type virus after primary infection with
drug-resistant clade B virus. AIDS 17:F11–F16.
15. Lavreys, L., J. M. Baeten, V. Chohan, R. S. McClelland, W. M. Hassan, B. A.
Richardson, K. Mandaliya, J. O. Ndinya-Achola, and J. Overbaugh. 2006.
Higher set point plasma viral load and more-severe acute HIV type 1
(HIV-1) illness predict mortality among high-risk HIV-1-infected African
women. Clin. Infect. Dis. 42:1333–1339.
16. Lavreys, L., J. M. Baeten, J. Overbaugh, D. D. Panteleeff, B. H. Chohan,
B. A. Richardson, K. Mandaliya, J. O. Ndinya-Achola, and J. K. Kreiss.
2002. Virus load during primary human immunodeficiency virus (HIV) type
1 infection is related to the severity of acute HIV illness in Kenyan women.
Clin. Infect. Dis. 35:77–81.
17. Li, M., F. Gao, J. R. Mascola, L. Stamatatos, V. R. Polonis, M. Koutsoukos,
G. Voss, P. Goepfert, P. Gilbert, K. M. Greene, M. Bilska, D. L. Kothe, J. F.
Salazar-Gonzalez, X. Wei, J. M. Decker, B. H. Hahn, and D. C. Montefiori.
2005. Human immunodeficiency virus type 1 env clones from acute and early
subtype B infections for standardized assessments of vaccine-elicited neu-
tralizing antibodies. J. Virol. 79:10108–10125.
18. Li, M., J. F. Salazar-Gonzalez, C. A. Derdeyn, L. Morris, C. Williamson, J. E.
Robinson, J. M. Decker, Y. Li, M. G. Salazar, V. R. Polonis, K. Mlisana, S. A.
Karim, K. Hong, K. M. Greene, M. Bilska, J. Zhou, S. Allen, E. Chomba, J.
Mulenga, C. Vwalika, F. Gao, M. Zhang, B. T. Korber, E. Hunter, B. H.
Hahn, and D. C. Montefiori. 2006. Genetic and neutralization properties of
subtype C human immunodeficiency virus type 1 molecular env clones from
acute and early heterosexually acquired infections in Southern Africa. J. Vi-
19. Li, Y., S. A. Migueles, B. Welcher, K. Svehla, A. Phogat, M. K. Louder, X.
Wu, G. M. Shaw, M. Connors, R. T. Wyatt, and J. R. Mascola. 2007. Broad
HIV-1 neutralization mediated by CD4-binding site antibodies. Nat. Med.
20. Long, E. M., H. L. Martin, Jr., J. K. Kreiss, S. M. Rainwater, L. Lavreys,
D. J. Jackson, J. Rakwar, K. Mandaliya, and J. Overbaugh. 2000. Gender
differences in HIV-1 diversity at time of infection. Nat. Med. 6:71–75.
21. Long, E. M., S. M. J. Rainwater, L. Lavreys, K. Mandaliya, and J. Over-
12102 BLISH ET AL.J. VIROL.
baugh. 2002. HIV type 1 variants transmitted to women in Kenya require the
CCR5 coreceptor for entry, regardless of the genetic complexity of the
infecting virus. AIDS Res. Hum. Retrovir. 18:567–576.
22. Maddison, D., and W. Maddison. 2005. MacClade 4: analysis of phylogeny
and character evolution. Sinauer, Sunderland, MA.
23. Martin, H. L., D. J. Jackson, K. Mandaliya, J. Bwayo, J. P. Rakwar, P.
Nyange, S. Moses, J. O. Ndinya-Achola, K. Holmes, F. Plummer, E. Ngugi,
and J. Kreiss. 1994. Preparation for AIDS vaccine evaluation in Mombasa,
Kenya: establishment of seronegative cohorts of commercial sex workers and
trucking company employees. AIDS Res. Hum. Retrovir. 10:S235–S237.
24. Martin, H. L., P. M. Nyange, B. A. Richardson, L. Lavreys, K. Mandaliya,
D. J. Jackson, J. O. Ndinya-Achola, and J. Kreiss. 1998. Hormonal contra-
ception, sexually transmitted diseases, and the risk of heterosexual transmis-
sion of HIV-1. J. Infect. Dis. 178:1053–1059.
25. Martin, H. L., B. A. Richardson, P. M. Nyange, L. Lavreys, S. L. Hillier, B.
Chohan, K. Mandaliya, J. O. Ndinya-Achola, J. Bwayo, and J. Kreiss. 1999.
Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency
virus type 1 and sexually transmitted disease acquisition. J. Infect. Dis.
26. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes,
M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb,
H. Katinger, and D. L. Birx. 1999. Protection of macaques against patho-
genic simian/human immunodeficiency virus 89.6PD by passive transfer of
neutralizing antibodies. J. Virol. 73:4009–4018.
27. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter,
C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, and M. G. Lewis.
2000. Protection of macaques against vaginal transmission of a pathogenic
HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat.
28. McCutchan, F. E., M. Hoelscher, S. Tovanabutra, S. Piyasirisilp, E. Sanders-
Buell, G. Ramos, L. Jagodzinski, V. Polonis, L. Maboko, D. Mmbando, O.
Hoffmann, G. Riedner, F. von Sonnenburg, M. Robb, and D. L. Birx. 2005.
In-depth analysis of a heterosexually acquired human immunodeficiency virus
type 1 superinfection: evolution, temporal fluctuation, and intercompartment
dynamics from the seronegative window period through 30 months postinfec-
tion. J. Virol. 79:11693–11704.
29. Montefiori, D. 2004. Evaluating neutralizing antibodies against HIV, SIV, and
SHIV in luciferase reporter gene assays, p. 1–15. In J. E. Coligan, A. M.
Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, and R. Coico (ed.),
Current protocols in immunology, vol. 12. John Wiley and Sons, New York, NY.
30. Montefiori, D. C., G. Pantaleo, L. M. Fink, J. T. Zhou, J. Y. Zhou, M. Bilska,
G. D. Miralles, and A. S. Fauci. 1996. Neutralizing and infection-enhancing
antibody responses to human immunodeficiency virus type 1 in long-term
nonprogressors. J. Infect. Dis. 173:60–67.
31. Nishimura, Y., T. Igarashi, N. Haigwood, R. Sadjadpour, R. J. Plishka, A.
Buckler-White, R. Shibata, and M. A. Martin. 2002. Determination of a
statistically valid neutralization titer in plasma that confers protection
against simian-human immunodeficiency virus challenge following passive
transfer of high-titered neutralizing antibodies. J. Virol. 76:2123–2130.
32. Nyambi, P. N., J. Nkengasong, P. Lewi, K. Andries, W. Janssens, K. Fransen,
L. Heyndrickx, P. Piot, and G. van-der-Groen. 1996. Multivariate analysis of
human immunodeficiency virus type 1 neutralization data. J. Virol. 70:6235–
33. Olsson, S. E., L. L. Villa, R. L. Costa, C. A. Petta, R. P. Andrade, C. Malm,
O. E. Iversen, J. Hoye, M. Steinwall, G. Riis-Johannessen, A. Andersson-
Ellstrom, K. Elfgren, G. von Krogh, M. Lehtinen, J. Paavonen, G. M.
Tamms, K. Giacoletti, L. Lupinacci, M. T. Esser, S. C. Vuocolo, A. J. Saah,
and E. Barr. 2007. Induction of immune memory following administration of
a prophylactic quadrivalent human papillomavirus (HPV) types 6/11/16/18
L1 virus-like particle (VLP) vaccine. Vaccine 25:4931–4939.
34. Pal, R., V. S. Kalyanaraman, B. C. Nair, S. Whitney, T. Keen, L. Hocker, L.
Hudacik, N. Rose, I. Mboudjeka, S. Shen, T. H. Wu-Chou, D. Montefiori, J.
Mascola, P. Markham, and S. Lu. 2006. Immunization of rhesus macaques
with a polyvalent DNA prime/protein boost human immunodeficiency virus
type 1 vaccine elicits protective antibody response against simian human
immunodeficiency virus of R5 phenotype. Virology 348:341–353.
35. Pantaleo, G., and R. A. Koup. 2004. Correlates of immune protection in
HIV-1 infection: what we know, what we don’t know, what we should know.
Nat. Med. 10:806–810.
36. Parren, P. W., P. A. Marx, A. J. Hessell, A. Luckay, J. Harouse, C. Cheng-
Mayer, J. P. Moore, and D. R. Burton. 2001. Antibody protects macaques
against vaginal challenge with a pathogenic R5 simian/human immunodefi-
ciency virus at serum levels giving complete neutralization in vitro. J. Virol.
37. Pernas, M., C. Casado, R. Fuentes, M. J. Perez-Elias, and C. Lopez-Galindez.
after primoinfection. J. Acquir. Immune Defic. Syndr. 42:12–18.
38. Piantadosi, A., B. Chohan, V. Chohan, R. S. McClelland, and J. Overbaugh.
2007. Chronic HIV-1 infection frequently fails to protect against superinfec-
tion. PLoS Pathog. 3:e177.
39. Pilgrim, A. K., G. Pantaleo, O. J. Cohen, L. M. Fink, J. Y. Zhou, J. T. Zhou,
D. P. Bolognesi, A. S. Fauci, and D. C. Montefiori. 1997. Neutralizing anti-
body responses to human immunodeficiency virus type 1 in primary infection
and long-term-nonprogressive infection. J. Infect. Dis. 176:924–932.
40. Plotkin, S. A. 2001. Immunologic correlates of protection induced by vacci-
nation. Pediatr. Infect. Dis. J. 20:63–75.
41. Ramos, A., D. J. Hu, L. Nguyen, K. O. Phan, S. Vanichseni, N. Promadej, K.
Choopanya, M. Callahan, N. L. Young, J. McNicholl, T. D. Mastro, T. M.
Folks, and S. Subbarao. 2002. Intersubtype human immunodeficiency virus
type 1 superinfection following seroconversion to primary infection in two
injection drug users. J. Virol. 76:7444–7452.
42. Richman, D. D., T. Wrin, S. J. Little, and C. J. Petropoulos. 2003. Rapid
evolution of the neutralizing antibody response to HIV type 1 infection.
Proc. Natl. Acad. Sci. USA 100:4144–4149.
43. Smith, D. M., D. D. Richman, and S. J. Little. 2005. HIV superinfection.
J. Infect. Dis. 192:438–444.
44. Smith, D. M., M. C. Strain, S. D. W. Frost, S. K. Pillai, J. K. Wong, T. Wrin,
C. J. Petropolous, E. S. Daar, S. J. Little, and D. D. Richman. 2006. Lack of
neutralizing antibody response to HIV-1 predisposes to superinfection. Vi-
45. Smith, D. M., J. K. Wong, G. K. Hightower, C. C. Ignacio, K. K. Koelsch,
E. S. Daar, D. D. Richman, and S. J. Little. 2004. Incidence of HIV super-
infection following primary infection. JAMA 292:1177–1178.
46. Srivastava, I. K., J. B. Ulmer, and S. W. Barnett. 2005. Role of neutralizing
antibodies in protective immunity against HIV. Hum. Vaccine 1:45–60.
47. Streeck, H., B. Li, A. F. Poon, A. Schneidewind, A. D. Gladden, K. A. Power,
D. Daskalakis, S. Bazner, R. Zuniga, C. Brander, E. S. Rosenberg, S. D.
Frost, M. Altfeld, and T. M. Allen. 2008. Immune-driven recombination and
loss of control after HIV superinfection. J. Exp. Med. 205:1789–1796.
48. Swofford, D. 1991. PAUP: phylogenetic analysis using parsimony, 3rd ed.
Illinois Natural History Survey, University of Illinois, Champaign.
49. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G.
Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools. Nucleic Acids
50. Tsui, R., B. L. Herring, J. D. Barbour, R. M. Grant, P. Bacchetti, A. Kral,
B. R. Edlin, and E. L. Delwart. 2004. Human immunodeficiency virus type 1
superinfection was not detected following 215 years of injection drug user
exposure. J. Virol. 78:94–103.
51. van der Kuyl, A. C., K. Kozaczynska, R. van den Burg, F. Zorgdrager, N.
Back, S. Jurriaans, B. Berkhout, P. Reiss, and M. Cornelissen. 2005. Triple
HIV-1 infection. N. Engl. J. Med. 352:2557–2559.
52. Villa, L. L., K. A. Ault, A. R. Giuliano, R. L. Costa, C. A. Petta, R. P.
Andrade, D. R. Brown, A. Ferenczy, D. M. Harper, L. A. Koutsky, R. J.
Kurman, M. Lehtinen, C. Malm, S. E. Olsson, B. M. Ronnett, F. E. Skjeld-
estad, M. Steinwall, M. H. Stoler, C. M. Wheeler, F. J. Taddeo, J. Yu, L.
Lupinacci, R. Railkar, R. Marchese, M. T. Esser, J. Bryan, K. U. Jansen,
H. L. Sings, G. M. Tamms, A. J. Saah, and E. Barr. 2006. Immunologic
responses following administration of a vaccine targeting human papilloma-
virus types 6, 11, 16, and 18. Vaccine 24:5571–5583.
53. Voronin, Y., B. Chohan, M. Emerman, and J. Overbaugh. 2007. Primary
isolates of human immunodeficiency virus type 1 are usually dominated by
the major variants found in blood. J. Virol. 81:10232–10241.
54. Wu, X., A. B. Parast, B. A. Richardson, R. Nduati, G. John-Stewart, D.
Mbori-Ngacha, S. M. Rainwater, and J. Overbaugh. 2006. Neutralization
escape variants of human immunodeficiency virus type 1 are transmitted
from mother to infant. J. Virol. 80:835–844.
55. Yang, O. O., E. S. Daar, B. D. Jamieson, A. Balamurugan, D. M. Smith, J. A.
Pitt, C. J. Petropoulos, D. D. Richman, S. J. Little, and A. J. Brown. 2005.
Human immunodeficiency virus type 1 clade B superinfection: evidence for
differential immune containment of distinct clade B strains. J. Virol. 79:860–
56. Yerly, S., S. Jost, M. Monnat, A. Telenti, M. Cavassini, J. P. Chave, L.
Kaiser, P. Burgisser, and L. Perrin. 2004. HIV-1 co/super-infection in in-
travenous drug users. AIDS 18:1413–1421.
57. Zhang, Y. J., C. Fracasso, J. R. Fiore, A. Bjorndal, G. Angarano, A. Gringeri,
and E. M. Fenyo. 1997. Augmented serum neutralizing activity against pri-
mary human immunodeficiency virus type 1 (HIV-1) isolates in two groups
of HIV-1-infected long-term nonprogressors. J. Infect. Dis. 176:1180–1187.
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