Neutralizing Antibody Response in Vax004 Trial
• JID 2010:202 (15 August) • 595
M A J O R A R T I C L E
Magnitude and Breadth of a Nonprotective
Neutralizing Antibody Response in an Efficacy Trial
of a Candidate HIV-1 gp120 Vaccine
Peter Gilbert,1Maggie Wang,1Terri Wrin,2Chris Petropoulos,2Marc Gurwith,4Faruk Sinangil,3Patricia D’Souza,6
Isaac R. Rodriguez-Chavez,6,aAllan DeCamp,1Mike Giganti,1Phillip W. Berman,5Steve G. Self,1
and David C. Montefiori7
1Vaccine Infectious Disease Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington;
for Infectious Diseases, South San Francisco,
Cruz, Santa Cruz, California;
4PaxVax, Incorporated, San Diego,
5Baskin School of Engineering, University of California Santa
6Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
7Department of Surgery, Duke University Medical Center, Durham, North Carolina
protein was found previously to be nonprotective in an efficacy trial (Vax004) despite strong antibody responses
against the vaccine antigens. Here we assessed the magnitude and breadth of neutralizing antibody responses in
Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier
2) strains of HIV-1 subtype B in 2 independent assays. Vaccine recipients were stratified by sex, race, and high
versus low behavioral risk of HIV-1 acquisition.
Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1MNand other
tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier
1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition
had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response
that arose after infection.
Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of
protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying
A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120
Efforts to develop an effective human immunodefi-
ciency virus type 1 (HIV-1) vaccine have emphasized
an ability to elicit virus-specific CD8+T cells and neu-
tralizing antibodies (NAbs) [1–3]. Genetic variability
Received 18 November 2009; accepted 8 March 2010; electronically published
7 July 2010.
Potential conflicts of interest: M.G., F.S., and P.W.B. are former employees of
VaxGen; P.G. and S.G.S. received consulting fees from VaxGen in the past.
Presented in part: AIDS Vaccine 2005, Montreal, Quebec, Canada, 6–9 Sep-
Financial support: HIV Vaccine Trials Network and the National Institutes of
Health (grant AI46705).
aPresent affiliations: NIDCR AIDS and Immunosuppression Program, National
Institutes of Health, Bethesda, Maryland.
Reprints or Correspondence: Dr David C. Montefiori, Department of Surgery,
Box 2926, Duke University Medical Center, Durham, NC 27710 (email@example.com).
The Journal of Infectious Diseases
? 2010 by the Infectious Diseases Society of America. All rights reserved.
has given rise to multiple genetic subtypes of HIV-1
that exhibit a wide spectrum of antigenic diversity
within and between subtypes [4–8] and pose major
obstacles for vaccine development. Most variability oc-
curs in the surface gp120 and transmembrane gp41
envelope (Env) glycoproteins that mediate virus entry
and serve as the sole targets for NAbs [9–13]. HIV-1
evades many NAbs by altering primary recognition se-
quences and by masking epitopes with N-linkedglycans
and other conformational and steric constraints that
limit antibody access [10, 14, 15]. An ideal vaccine may
need to overcome these evasion strategies and elicit
NAbs against a wide range of circulating variants. Al-
though it is not clear how to achieve this goal, evidence
suggests that the virus has vulnerabilities, and that
broadly cross-reactive NAb induction is indeedpossible
Various Env-containing vaccine candidates haveelic-
by guest on February 2, 2011
596 • JID 2010:202 (15 August) • Gilbert et al
ited NAbs in phase 1 and phase 2 human clinical trials .
The antibodies often neutralize T cell line adapted strains and
other strains highly sensitive to neutralization, but they do not
neutralize most circulating strains of HIV-1 [18–20]. T cell line
adapted strains and a subset of circulating strains that exhibit
a high level of neutralization susceptibility are classified as tier
1 viruses . The tier 1 phenotype is associated with spon-
taneous epitope exposure in the sequence-variable cysteine-
cysteine loops and in the conserved coreceptor binding domain
of gp120 [22–24]. Most circulating strains have evolved under
immune pressure to conceal these epitopes, resulting in an
overall lower level of neutralization susceptibility that is clas-
sified as a tier 2 phenotype . Whether a certain level of
neutralizing activity against tier 1 and tier 2 viruses will predict
protection against HIV-1 is not known; however, it is generally
agreed that neutralization of tier 2 viruses should be a priority
for vaccines [25–28].
Many previous evaluations of vaccine-elicitedNAbresponses
against tier 2 viruses used poorly defined virologic reagents and
substandard assay methodologies. New high throughput assay
technologies are now available that use engineered cell lines
and reporter genes for highly sensitive, quantitative, and re-
producible results [14, 29]. These new assays have been opti-
mized and validated and use well-characterized Env-pseudo-
typed viruses, including transmitted and/or founder viruses
from sexually acquired infections that are thought to be im-
portant targets for vaccination [30–35].
Here we assessed the NAb response in the Vax004 efficacy
trial of a candidate HIV-1 gp120 vaccine (AIDSVAX B/B;
VaxGen) that was evaluated on the basis of eliciting NAbs [36,
37]. Strong antibody responses were detected byenzyme-linked
immunosorbent assay and by neutralization of HIV-1MN;
however, the vaccine did not prevent the acquisition of infec-
tion, nor did it impact viral loads in participants who acquired
infection after vaccination [39, 40]. A similar bivalent gp120
vaccine (AIDSVAX B/E; VaxGen)  was ineffective in an
efficacy trial in Bangkok intravenous drug users despite com-
parable antibody responses . Lack of efficacy in both trials
precluded an assessment of NAbs as a correlate of protection.
However, a recent trial of a prime-boost regimen that included
AIDSVAX B/E provided modest evidence for a reduced rate of
HIV-1 infection , which in the future may afford such
the magnitude and breadth of a nonprotective NAb response
in human efficacy trials of HIV-1 vaccines, providing a useful
reference for future vaccine evaluations.
VOLUNTEERS, MATERIALS, AND METHODS
Clinical trial design.
elsewhere [38–40]. The vaccine consisted of 2 gp120 proteins
derived from HIV-1 subtype B strains MN and GNE8. Vax004
The Vax004 trial design was described
and the present study were conducted in accordance with the
Declaration of Helsinki and local institutional review board
requirements. Written informed consent was obtained from all
Serologic specimens and stratification.
for plasma was collected in Vacutainer CPT tubes containing
sodium citrate as anticoagulant (Becton-Dickinson). Peripheral
blood for serum was collected withoutananticoagulant.Plasma
and serum samples were stored at ?80?C, thawed, and heat-
inactivated at 56?C for 1 h prior to assay. Vaccine recipients
were stratified by sex, race, and high versus lowriskofacquiring
HIV-1 infection, selected randomly within each group. Low
and higher risk groups were defined on the basis of a behavioral
risk score variable constructed from baseline questionnaire
data, which was used in the primary analyses of Vax004 [38,
39]. The low-risk group consists of participants with lowest
risk score 0, and the higher risk group were those with risk
score ?4. For the nonwhite and female strata, there were not
enough available participants with risk score ?4, and in these
cases the higher risk group includes some participants with risk
HIV-1 subtype B reference strains 6535.3,
RHPA4259.7, THRO4156.18, REJO4541.67, TRJO4551.58,
WITO4160.33, and CAAN5342.A2 closely approximate trans-
mitted/founder viruses from sexually acquired infections .
Additional subtype B viruses from sexually acquired in-
fections included WEAU-d15.410.787, BB1006–11.C3.1601,
6240.08.TA.4622, 6244.13.B5.4576, and 62357.14.D3.4589,
which are considered authentic transmitted/early founder vi-
ruses . Tier 1 viruses included HIV-1MN, SF162.LS, Bal.26,
BZ167.12, Bx08.16, SS1196.1, MW965.26, and 92BR025.9. All
tier 1 viruses are subtype B except MW965.26 and 92BR025.9,
which are subtype C. HIV-1MNwas used as an uncloned stock.
All other viruses were used as Env-pseudotyped viruses con-
taining a single full-length gp160 clone of the designated strain.
Additional viruses were derived by random sampling from
13 vaccine recipients and 14 placebo recipients within6months
of infection from Vax004 subjects who received at least 4 in-
oculations before infection. These viruses were used as cloned
quasispecies of plasma-derived Env-pseudotyped viruses .
Neutralization was measured with
blinded samples in 96-well culture plates by using firefly lu-
ciferase (Luc) reporter gene expression to quantify infection.
One assay [30, 31] was performed in a HeLa cell line (TZM-
bl, also known as JC53-BL) that expresses CD4, CCR5, and
CXCR4  and contains a Luc reporter gene . Unless
otherwise specified, plasma samples were assayed at 8 dilutions
starting at 1:10. Nab titers were calculated as the sample di-
lution conferring a 50% reduction in relative luminescence
by guest on February 2, 2011
Neutralizing Antibody Response in Vax004 Trial • JID 2010:202 (15 August) • 597
units (RLU) relative to virus control wells after subtraction of
background RLU in cell control wells. An additional set of
assays tested a 1:10 dilution of serum rather than plasma to
avoid the mild toxicity of anticoagulant. Results in these latter
assays were calculated as the percentage of reduction in RLU
in wells containing postimmunization serum relative to the
RLU in wellscontainingcorrespondingpreimmuneserumfrom
the same subject. HIV-1MNwas prepared in H9 cells. Env-pseu-
dotyped viruses were prepared by cotransfecting 293T/17 cells
(American Type Culture Collection) with an Env-expressing
plasmid plus an Env-defective backbone plasmid (pSG3Denv)
as described elsewhere [30, 31].
A second assay [29, 35] used an astroglioma celllineengineered
samples were assayed at 8 dilutions starting at 1:10. Nab titers
were calculated as the sample dilution conferring a 50% re-
duction in RLU relative to virus control wells after subtraction
of background RLU in cell control wells. Env plasmid libraries
were cloned from either infected cell cultures, env expression
vectors (tier 1 and 2 reference panels), or plasma from HIV-
infected trial participants. Viral stocks were prepared by co-
transfecting HEK293 cells with env plasmid libraries along with
an HIV genomic vector containing a Luc indicator gene in
place of env.
Box plots were used to graphically dis-
play distributions of log10 NAb titers to individual isolates.
NAb responses to an individual isolate were summarized by
the percentage of subjects who had a positive response (“pos-
itive response rate”), and by the geometric mean titer (GMT)
of NAbs (“titers of NAbs”) within the subgroup of subjects
with a positive response (responders). Positive response rates
were compared between groups by 95% confidence intervals
(CIs) about the difference in positive response rates, and by a
Fisher exact test for different rates. Titers of NAbs among re-
sponders were compared between groups by 95% CIs about
the ratio of GMTs. Equality of the overall distribution of log10
NAb titers between 2 groups was tested as described elsewhere
, using 10,000 permutated data sets to compute a P value.
The false discovery rate (FDR) was used to determine tests that
remained statistically significant after adjustment for the mul-
tiple hypothesis tests. The FDR method was performed at level
Assessment of magnitude and breadth of neutralization of
a panel of isolates.
A magnitude-breadth (M-B) curve was
used to describe the magnitude (NAb titer) and breadth (num-
ber of isolates neutralized) of an individual plasma sample as-
sayed against a panel of tier 2 HIV-1 isolates . On the basis
of NAb titers to m isolates, the x-axis of an M-B plot is the
threshold of neutralization that is considered positive, whereas
the y-axis is the percentage of the m targets neutralized. The
area under the curve (AUC) of a M-B curve provides an overall
summary of the M-B profile and equals the average log10 NAb
titer over the m targets. The Mann-Whitney test was used to
compare the AUC of M-B curve between groups, which pro-
vides an overall test for different aggregate NAb responses.
Wilcoxon signed rank tests were used to compare within-sub-
ject differences in the AUC of M-B plots between 2 distinct
panels of HIV-1 isolates, which determined whether one panel
was more easily neutralized than the other. All P values are 2-
Preinfection NAb responses.
weeks after fourth inoculation (90 vaccine recipients and 30
placebo recipients who were uninfected at the time of blood
draw) were assessed in 2 independent assays; this time point
corresponds to peak vaccine-elicited antibody responses .
High-titer NAbs were detected against HIV-1MNand SF162.LS
in most vaccine recipients in both assays (Figure 1A and 1B).
Sporadic weak neutralizing activity was detected against tier 2
reference strains in both assays (Figure 1A and 1B). Positive
response rates (frequency of results ?1:10 plasma dilution)
and titers of NAbs against the tier 2 reference viruses were
significantly higher for vaccine than placebo recipients for 9 of
12 viruses in the TZM-bl assay and for 6 of 12 viruses in the
U87.CD4.CCR5.CXCR4 assay. False positive results (ie, higher
responses in placebo recipients than in vaccine recipients) were
obtained with RHPA4259.7 in the TZM-bl assay and with
PVO.4 in the U87.CD4.CCR5.CXCR4 assay. Because of the low
plasma dilutions tested, occasional false positive neutralization
was not unexpected. Overall positive response rates against any
tier 2 viruses were 47% (range, 17%–92%) and 23% (range,
0–57%) for vaccine and placebo recipients, respectively, in the
TZM-bl assay. Corresponding positive response rates in the
U87.CD4.CCR5.CXCR4 assay were 44% (range, 12%–72%)
and 32% (range, 0–60%), respectively. Therefore, net positive
response rates for vaccine recipients (subtracting positive re-
sponse rates for placebo recipients) were 24% in the TZM-bl
assay and 12% in the U87.CD4.CCR5.CXCR4 assay. Neutral-
ization of tier 2 reference strains was significantly greater for
vaccine recipients than for placebo recipients in both assays
when magnitude and breadth of neutralization were consid-
ered in aggregate.
Preinfection plasma from vaccine recipients exhibited weak
neutralizing activity against early viruses from 13 vaccine and
14 placebo recipients (Figure 2). Pooling over the 27 isolates,
overall positive response rates were 5% for vaccine and 0% for
placebo recipients (Mann-Whitney test,
curves were compared, vaccine-elicited antibodies were more
likely to neutralize viruses from placebo recipients than viruses
from vaccine recipients (P p .004
of this latter difference was small, with 54 vaccine recipients
Plasma samples obtained 2
). When M-BP p .05
; Figure 2B). The magnitude
by guest on February 2, 2011
598 • JID 2010:202 (15 August) • Gilbert et al
2 reference strains. NAbs in plasma samples from 90 randomly selected vaccine recipients and 30 randomly selected placebo recipients, all of whom
who were uninfected at the time of blood draw (2 weeks after the fourth inoculation), were assessed against HIV-1MN, SF162.LS and a panel of 12
subtype B tier 2 reference strains. Positive response rates (frequency of positive results at ?1:10 plasma dilution), titers of NAbs and magnitude-
breadth (M-B) curves were derived from results obtained in the TZM-bl (A) and U87.CD4.CCR5.CXCR4 (B) assays. For the box plots of NAb titers
(middle panel), 25% of values lie below the box, 25% lie above the box, and 50% lie below the horizontal line (the median) inside the box. Vertical
lines above the box extend to a distance 50% greater than the height of the box; points beyond this are unusually high values (outliers). Subject-
specific and group averages in M-B plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.
Comparison of preinfection neutralizing antibody (NAb) responses among vaccine and placebo recipients as measured with tier 1 and tier
having equal AUC for the 2 sets of viruses, 23 having greater
AUC for placebo viruses, and 8 having smallerAUC forplacebo
viruses; thus, the result may be of little biological importance.
Results with postinfection plasma from placebo recipients (ie,
natural NAb response to infection) showed that viruses from
infected placebo recipients were intrinsically slightly more sen-
sitive to neutralization (data not shown;
Plasma from a subset of vaccine and placebo recipients in
Figure 1 were assessed for neutralizationbreadthagainstalarger
panel of tier 1 viruses and one additional prototypic tier 2 virus
).P p .013
by guest on February 2, 2011
Neutralizing Antibody Response in Vax004 Trial • JID 2010:202 (15 August) • 599
trial participants. Plasma samples in Figure 1 were assessed for neutralizing activity against viruses from 27 trial participants obtained at the earliest
available postinfection time point. A, Neutralization response rates and the titers of NAbs. The first 13 viruses from the left are from vaccine recipients
and the second 14 viruses are from placebo recipients. B, Magnitude-breadth (M-B) curves to the vaccine recipient isolate panel and to the placebo
recipient isolate panel (top) and differences in AUC of M-B curves for the placebo and vaccine isolate panels (bottom). Subject-specific and group
averages in M-B plots are shown as light and heavy lines, respectively. All results in A and B were obtained in the U87.CD4.CCR5.CXCR4 assay.
Parallel assessments in the TZM-bl assay were not performed.
Comparison of preinfection neutralizing antibody (NAb) responses among vaccine and placebo recipients as measured with viruses from
(JR-FL) in the TZM-bl assay (Figure 3A). Plasma from placebo
recipients were mostly negative. Plasma obtained from all vac-
cine recipients neutralized HIV-1MNand SF162.LS, with GMTs
of 4931 and 1431, respectively. Moderate to low levels of NAbs
were detected against tier 1 viruses MW965.26, SS1196.1,
Bal.26, Bx08.16, 92BR025.9 and BZ167.12, with GMTs of 263,
134, 48, 44, 34 and 17, respectively. Plasma obtained from a
single vaccine recipient neutralized JR-FL (titer, 24).
Additional assays were performed with serum rather than
with plasma and compared a 1:10 dilution of postimmune
serum (2 weeks after 4th inoculation) to a 1:10 dilution of
corresponding preimmune serum from additional randomly
sampled vaccine and placebo recipients who were uninfected
at the time of blood draw. This method automatically adjusts
for nonspecific activity in corresponding preimmune serum
and thus may be a more stringent measure of true neutrali-
zation. Serum samples were assayed against the 12 tier 2 ref-
erence strains and 8 authentic tier 2 transmitted/founder vi-
ruses. Subjects were randomly sampled to comprise an equal
distribution of blacks and whites of both sexes; this number
was not adequate for statistical comparisons between races and
sexes. Vaccine recipients exhibited weak but statistically signif-
icant neutralizing activity against both sets of viruses (Figure
3B). Transmitted and early founder viruses were slightly less
sensitive to neutralization than the tier 2 reference viruses, but
this difference was not significant (
ients andfor placebo recipients). Overall positive re-P p .53
sponse rates (?50% neutralization) against the tier 2 reference
viruses were 8.3% for vaccine recipients and 0% for placebo
recipients. The positive response rate against transmitted/foun-
der viruses was 0% for both the vaccine and placebo groups.
Association between neutralization of HIV-1MNand of tier
Titers of NAbs against HIV-1MNin vaccine recip-
ients were positively correlated with titers against 4 tier 2strains
in the U87.CD4.CCR5.CXCR4 assay (6535.3, THRO4165.18,
REJO4541.67, PVO.4; Spearman rank test,
which was significant after FDR adjustment (6535.3;
HIV-1MNNAb titers were weakly positivelycorrelatedwithAUC
for vaccine recip-P p .09
), one of
r p 0.40
r 1 0.20
by guest on February 2, 2011
600 • JID 2010:202 (15 August) • Gilbert et al
samples from 24 randomly selected vaccine recipients and 5 placebo recipients (2 weeks after the fourth immunization, before infection) among the
same 120 trial participants in Figure 1 were assayed against HIV-1MN, SF162.LS, 6 additional tier 1 viruses and 1 prototypic tier 2 virus (JR-FL) in
the TZM-bl assay. Plasma samples were assayed at 8 dilutions starting at 1:20. NAb titers !20 were assigned a value of 10. Results are shown for
vaccine recipients only. Results with placebo recipient plasma were low (SS1196.1, 4 samples with NAb titers of 29–59; MW965.26, 1 sample with
a NAb titer of 31) or negative (all remaining tests). Positive response rate (% of values ?50 neutralization) is shown above each scatter plot. B,
Serum samples from additional vaccine and placebo recipients ( each) were tested for neutralizing activity at a 1:10 dilution in the TZM-bln p 20
assay against the 12 subtype B tier 2 reference strains (same as Figure 1A, excluding tier 1 viruses MN and SF162.LS). Many of these same samples
(16 vaccine and 17 placebo recipients) were also assayed against 8 tier 2 transmitted/founder clade B strains (WEAU-d15.410.787, BB1006–11.C3.1601,
BB1054–07.TC4.1499, BB1056–10.TA11.1826, BB1012–11.TC21, 6240.08.TA.4622, 6244.13.B5.4576, 62357.14.D3.4589); sufficient quantities were not
available for all samples to be assayed against this latter panel of viruses. Serum samples before the first inoculation (preimmune) and 2 weeks after
fourth inoculation (before infection) were assayed in triplicate on the same assay plate. Percentage of neutralization was calculated by dividing the
average RLU of preimmune serum by the average RLU of postimmune serum, subtracting this result from 1 and multiplying by 100. For each subject
and each tier 2 panel (12 reference viruses and 8 transmitted/founder viruses), the average of the percent neutralization values across the isolates
in the panel was computed. These averages were compared between the vaccine and placebo groups for each panel with Mann-Whitney tests, and
were compared between the 2 panels with a paired data Wilcoxon signed-rank test. Filled symbols, vaccine recipients; open symbols,placeborecipients.
Breadth of preinfection neutralizing antibodies (NAbs) against tier 1 and tier 2 viruses among vaccine and placebo recipients. A, Plasma
of M-B curves against the 12 tier 2 reference viruses (r p
andTZM-bl assay;0.24P p .025
U87.CD4.CCR5.CXCR4 assay). No significant correlation was
seen between HIV-1MNNAb titers and neutralization of viruses
from trial participants.
Comparison of postinfection NAb responses among vaccine
and placebo recipients.
NAbs were assessed in plasma from
14 vaccine recipients and 14 placebo recipients 12–24 months
after diagnosis of infection (prior to antiretroviral therapy). All
subjects received 4 inoculations of either the vaccine or placebo
prior to diagnosis. Results in the TZM-bl assay were published
elsewhere . Results in the U87.CD4.CCR5.CXCR4 assay are
shown in Figure 4. Titers of postinfection NAbs against HIV-
1MNwere significantly higher for vaccine recipients than place-
bo recipients in both assays, suggesting the vaccine augmented
the response to HIV-1MN. No significant difference was seen
between vaccine and placebo recipients for NAbs against
SF162.LS, the 12 tier 2 reference strains and the 27 viruses from
trial subjects. Assays with virusesfrominfectedtrialparticipants
included autologous plasma and virus combinations from 2
andr p 0.15P p .16
vaccine and 8 placebo recipients that yielded considerably
stronger neutralization than heterologous combinations. Dif-
ferences among vaccine and placebo recipients were nonsig-
nificant regardless of whether autologous combinations were
included in the statistical analysis.
Comparison of NAb responses among preinfection vaccine
elicited NAb responses in 90 trial participants (2 weeks after
fourth inoculation) were compared with the early response that
arose after infection in 14 placebo recipients (1–2 years after
diagnosis). Results are shown in Figure5. TitersofNAbsagainst
HIV-1MNwere similar in both cases, whereas titers against
SF162.LS were significantly elevated in infected placebo recip-
ients (GMT 2451 vs 288,P ! .001
vs 184, in U87.CD4.CCR5.CXCR4 assay). M-B curvesP ! .001
in the TZM-bl assay showed that responses against the tier 2
reference strains were similar among the 2 groups (
whereas a small but significantly elevated response was seen in
infected placebo recipients using the U87.CD4.CCR5.CXCR4
assay: for all 39 tier 2 viruses (data not shown);P ! .001
in TZM-bl assay; GMT 1006
),P p .24
by guest on February 2, 2011
Neutralizing Antibody Response in Vax004 Trial • JID 2010:202 (15 August) • 601
samples from 14 vaccine recipients and 14 placebo recipients 12–24 months after diagnosis of infection. All subjects were antiretroviral therapy naı ¨ve
at the time of plasma collection. A, Assays with MN, SF162.LS and the subtype B reference panel of tier 2 viruses. B, Assays with viruses from trial
participants. In the top 2 diagrams, the first 13 viruses from the left are from vaccine recipients and the second 14 viruses are from placebo recipients.
Autologous virus/plasma combinations in the middle diagram (neutralization response levels) are indicated by an asterisk. All results in panels A and
B were obtained in the U87.CD4.CCR5.CXCR4 assay. Subject-specific and group averages in magnitude-breadth plots are shown as light and heavy
lines, respectively, and are for the tier 2 viruses only.
Comparison of postinfection neutralizing antibody (NAb) responses among vaccine and placebo recipients. NAbs were assessed in plasma
for the tier 2 reference strains shown in Figure 5. Thus,
the vaccine-elicited response did not exceed the response that
arose after 1–2 years of infection in the absence of vaccination.
Influence of demographic factors on the preinfection neu-
tralizing antibody response in vaccine recipients.
the 90 vaccine recipients (2 weeks after fourth inoculation,
before infection) were compared among sex, race (blacks and
whites), and low versus high risk behavior groups. Results in
both assays demonstrated higher titers of NAb against HIV-
1MNand SF162.LS in women than in men (∼2-fold increase
in GMT; ). Additionally, M-B curves showed high-P ! .008
er aggregate responses to the 12 tier 2 reference viruses in
by guest on February 2, 2011
602 • JID 2010:202 (15 August) • Gilbert et al
Plasma obtained from 90 vaccine recipients (2 weeks after the fourth inoculation) and 14 placebo recipients (1–2 years after diagnosis) were assayed
against MN, SF162.LS, and the subtype B reference panel of tier 2 viruses. A, TZM-bl assay. B, U87.CD4.CCR5.CXCR4 assay. Subject-specific and
group averages in magnitude-breadth plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.
Comparison of preinfection neutralizing antibody (NAb) responses in vaccine recipients to postinfection NAbresponsesinplaceborecipients.
women than in men (
U87.CD4.CCR5.CXCR4 assay) (Figure 6). A nonsignificant
trend toward higher M-B curves was also seen for women
when all 39 tier 2 viruses were considered in aggregate
( ; U87.CD4.CCR5.CXCR4 assay). Race and risk be-P p .073
havior level had no significant effect.
, TZM-bl assay;,P ! .001P p .034
Comparison of tier 2 reference strains and viruses fromtrial
The tier 2 reference strains were more suscep-
tible to nonspecific neutralization (
placebo samples) and to specific neutralization (
preinfection vaccine samples) (Figure 2B) than viruses from
trial participants. Having a positive response to the reference
P ! .001
P p .004
by guest on February 2, 2011
Neutralizing Antibody Response in Vax004 Trial • JID 2010:202 (15 August) • 603
1), as measured with the tier 1 and tier 2 reference strains evaluated in Figure 1. Positive response rates (frequency of positive results at ?1:10
plasma dilution), titers of NAbs and magnitude-breadth (M-B) curves were derived from results obtained in the TZM-bl (A) and U87.CD4.CCR5.CXCR4
(B) assays. Subject-specific and group averages in M-B plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.
Comparison of preinfection neutralizing antibody (NAb) responses among men and women vaccine recipients (, evaluated in Figuren p 90
panel was predictive of having a positive response to the trial
participant panel for postinfection vaccine and placebo samples
(odds ratio, 8.17; ) but not for preinfection vaccineP p .019
samples. Because viruses from trial participants were only as-
sayed in U87.CD4.CCR5.CXCR4 cells, where slightly elevated
NAb responses were detected after infection, the magnitude of
vaccine-elicited NAb response against tier 2 viruses might bor-
der the magnitude required to achieve reproducible results in
the 2 independent assays.
We confirm that most vaccine recipients in Vax004 possessed
moderate to high titers of NAbs against HIV-1MN. Moderate
by guest on February 2, 2011
604 • JID 2010:202 (15 August) • Gilbert et al
neutralizing activity was often detected against other tier 1
strains, but only occasional weak neutralizing activity was de-
tected against tier 2 strains. Prior vaccination augmented the
NAb response against HIV-1MNafter infection but had little
measurable effect on the postinfection NAb response against
tier 2 viruses. Overall, the vaccine-elicited NAb response was
no better than the relatively weak response that arose after 1–
2 years of infection in the absence of vaccination. Relatively
weak NAb responses against tier 2 strains is consistent with the
lack of protection in this trial.
Vaccine-elicited NAb responses against tier 2 viruses, albeit
weak, were statistically significant (compared to placebo)
against tier 2 Env-pseudotyped reference strains and against
pseudoviruses containing a more recent set of authentic trans-
mitted/founder Envs, suggestingthatthereferencepaneldetects
NAbs of interest for vaccines. Both sets of pseudoviruses con-
tained single Env clones, whereas pseudovirusescontainingEnv
from trial participantswereaquasispecies.Greatergeneticcom-
plexity of the Env quasispecies might account for observed
differences in nonspecific activity and neutralization-sensitivity
when assayed in U87.CD4.CCR5.CXCR4 cells. In both cases,
neutralization of tier 2 viruses was poorly predicted by NAbs
against the HIV-1MN, thus reinforcing the importance of in-
cluding tier 2 viruses when assessing vaccine-elicited NAbs.
Additionally, vaccine recipient plasma appeared more likely to
neutralize Env quasispecies from infected placebo recipients
than from infected vaccine recipients (
small magnitude of this possible effect suggests little if any
biological significance. Beyond Vax004, for efficacy trials with
evidence for positive vaccine efficacy, a larger effect of this kind
could indicate that some circulating viruses are more sensitive
to vaccine-elicited NAbs that blocked their transmission to ex-
posed vaccine recipients. We encourage similar assessments of
NAbs, combined with complementary genetic analyses of the
viruses , in RV144 and future trials where measurable pro-
tection is achieved.
Our results are consistent with a previous report  show-
ing significantly more elevated titers of NAbs against HIV-1MN
in women than in men (2 times higher GMT in both assays)
and no significant difference in the response between high and
low behavioral risk groups in Vax004. We also observed sig-
nificantly stronger responses in women than in men for NAbs
against SF162.LS and tier 2 reference strains. Contrary to pre-
vious reports [38, 49, 50], we found no significant difference
in NAb responses between blacks and whites. Our results lend
support to a possible effect of sex on the NAb response to
certain HIV-1 vaccines. Additional studies are needed to de-
lineate the nature of this effect.
Modest protection in the recent efficacy trial in Thailand
(RV144) will provide additional opportunities to learn more
about the requirements for effective vaccination against HIV-
), although the
P p .004
1. One way to improve the efficacy of current HIV-1 vaccines
may be to elicit stronger NAb responses against tier 2 strains
of the virus. The magnitude and breadth of neutralization re-
ported here for a nonprotective vaccine should serve as a useful
reference to identify improved vaccine designs.
We thank George Shaw, Beatrice Hahn, and the Center for HIV/AIDS
Vaccine Immunology (CHAVI) for contributing the transmitted/founder
HIV-1 Envs. We also thank Michael Peterson and David Jobesforassistance
in coordinating the study. In addition, we acknowledge the excellent tech-
nical assistance of Alicia Gaitan, Barbara Sokolik-Wolak, Hongmei Gao,
and Kelli Greene.
1. Letvin NL. Progress and obstacles in the development of an AIDS
vaccine. Nat Rev Immunol 2006;6:930–9.
2. McMichael AJ. HIV vaccines. Annu Rev Immunol 2006;24:227–55.
3. Mascola JR, Montefiori DC. The role of antibodies in HIV vaccines.
Annu Rev Immunol 2010;28:413–444.
4. Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C, Detours
V. Evolutionary and immunological implications of contemporary
HIV-1 variation. Br Med Bull 2001;58:19-42.
5. McCutchan FE. Understanding the genetic diversity of HIV-1. AIDS
6. Moore JP, Cao Y, Leu J, Qin L, Korber B, Ho DD. Inter- and intraclade
neutralization of human immunodeficiency virus type 1: genetic clades
do not correspond to neutralization serotypes but partially correspond
to gp120 antigenic serotypes. J Virol 1996;70:427–44.
7. Mascola JR, Louwagie J, McCutchan FE,etal. Twoantigenicallydistinct
subtypes of human immunodeficiency virus type 1: viral genotype
predicts neutralization serotype. J Infect Dis 1994;169:48–54.
8. Binley JM, Wrin T, Korber B, et al. Comprehensive crossclade neu-
tralization analysis of a panel of anti-human immunodeficiency virus
type 1 monoclonal antibodies. J Virol 2004;78:13232–52.
9. Gaschen B, Taylor J, Yusim K, et al. Diversity considerations in HIV-
1 vaccine selection. Science 2002;296:2354–2360.
10. Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: fusogens, an-
tigens, and immunogens. Science 1998;280:1884–8.
11. Yang X, Lipchina I, Cocklin S, Chaiken I, Sodroski J. Antibody binding
is a dominant determinant of the efficiency of human immunodefi-
ciency virus type 1 neutralization. J Virol 2006;80:11404–11408.
12. Crooks ET, Moore PL, Richman D, et al. Characterizing anti-HIV
monoclonal antibodies and immune sera by defining the mechanism
of neutralization. Human Antibodies 2005;14:101–113.
13. Moore PL, Crooks ET, Porter L, et al. The nature of nonfunctional
envelope proteins on the surface of human immunodeficiency virus
type 1. J Virol 2006;80:2515–2528.
14. Wei X, Decker JM, Wang S, et al. Antibody neutralization and escape.
15. Kwong PD, Doyle ML, Casper DJ, et al. HIV-1 evades antibody-me-
diated neutralization through conformational masking of receptor-
binding sites. Nature 2002;420:678–682.
16. Stamatatos L, Morris L, Burton DR, Mascola JR. Neutralizing anti-
bodies generated during natural HIV-1 infection: good news for an
HIV-1 vaccine? Nat Med 2009;15:866–870.
17. Walker LM, Phogat SK, Chan-Hui P-Y, et al. Broad and potent neu-
tralizing antibodies from an African donor reveal new HIV-1 vaccine
target. Science 2009;326:285–289.
18. Mascola JR, SnyderSW,WeislowOS,etal.Immunizationwithenvelope
subunit vaccine products elicits neutralizing antibodies against labo-
by guest on February 2, 2011
Neutralizing Antibody Response in Vax004 Trial • JID 2010:202 (15 August) • 605 Download full-text
ratory-adapted but not primary isolates of human immunodeficiency
virus type 1. J Infect Dis 1996;173:340–348.
19. Bures R, Gaitan A, Zhu T, et al. Immunization with recombinant
canarypox vectors expressing membrane-anchored gp120 followed by
gp160 protein boosting fails to generate antibodies that neutralize R5
primary isolates of human immunodeficiency virus type 1. AIDS Res
Hum Retroviruses 2000;16:2019–2035.
20. Belshe RB, Gorse GJ, Mulligan MJ, et al. Induction of immune re-
sponses to HIV-1 canarypox virus (ALVAC) HIV-1 and gp120 SF-2
recombinant vaccines in uninfected volunteers. AIDS 1998;12:2407–
21. Seaman MS, Janes H, Hawkins N, et al. Tiered categorization of a
diverse panel of HIV-1 Env pseudoviruses for assessments of neutral-
izing antibodies. J Virol 2010;84:1439–1452.
22. Bou-Habib DC, Roderiquez G, Oravecz T, Berman PW, Lusso P, Nor-
cross MA. Cryptic nature of envelope V3 region epitopes protects
primary monocytotropic human immunodeficiency virus type 1 from
antibody neutralization. J Virol 1994;68:6006-6013.
23. Davis KL, Gray ES, Moore PL, et al. High titer HIV-1 V3-specific
antibodies with broad reactivity but low neutralizing potency in acute
infection and following vaccination. Virology 2009;387:414–426.
24. Decker JM, Bibollet-Ruche F, Wei X, et al. Antigenic conservation and
immunogenicity of the HIV coreceptor binding site. J Exp Med 2005;
25. Matthews TJ. Dilemma of neutralization resistance of HIV-1 filed iso-
lates and vaccine development. AIDS Res Hum Retroviruses 1994;10:
26. Moore JP, Burton DR. Urgently needed: a filter for the HIV-1 vaccine
pipeline. Nat Med 2004;10:769–771.
27. Douek DC, Kwong PD, Nabel GJ. The rational design of an AIDS
vaccine. Cell 2006;124:677–681.
28. Burton DR, Desrosiers RC, Doms RW, et al. HIV vaccine design and
the neutralizing antibody problem. Nat Immunol 2004;5:233–236.
29. Richman DD, Wrin T, Little SJ, Petropoulos CJ. Rapid evolution of
the neutralizing antibody response to HIV type 1 infection. Proc Natl
Acad Sci U S A 2003;100:4144–4149.
30. Li M, Gao F, Mascola JR, et al. Human immunodeficiency virus type
1 env clones from acute and early subtype B infections forstandardized
assessments of vaccine-elicited neutralizing antibodies. J Virol 2005;
31. Li M, Salazar-Gonzalez JF, Derdeyn CA, et al. Genetic and neutrali-
zation properties of subtype C human immunodeficiency virus type 1
molecular env clones from acute and early heterosexually acquired
infections in southern Africa. J Virol 2006;80:11776–11790.
32. Kulkarni SS, Lapedes A, Tang H, et al. Highly complex neutralization
determinants on a monophyletic lineage of newly transmitted subtype
C human immunodeficiency virus type 1 env clones from India. Vi-
33. Blish CA, Nedellec R, Mandaliya K, Mosier DE, Overbaugh J. HIV-1
subtype A envelope variants from early in infection have variable sen-
sitivity to neutralization and to inhibitors of viral entry. AIDS 2007;
34. Keele BF, Giorgi EE, Salazar-Gonzalez JF, et al. Identification and char-
acterization of transmitted and early founder virus envelopes in pri-
mary HIV-1 infection. Proc Natl Acad Sci U S A 2008;105:7552–7557.
35. Schweighart B, Liu Y, Huang W, et al. Development of an HIV-1
reference panel of subtype B envelope clones isolated from the plasma
of recently infected individuals. J Acquir Immune Defic Syndr 2007;
36. Berman PW. Development of bivalent rgp120 vaccines to prevent HIV
type 1 infection. AIDS Res Hum Retroviruses 1998;14:S277-S289.
37. Francis DP, Gregory T, McElrath MJ, et al. Advancing AIDSVAX? to
phase 3. Safety, immunogenicity, and plans for phase 3. AIDS ResHum
38. Gilbert PB, Peterson ML, Follmann D, et al. Correlation between im-
munologic responses to a recombinant glycoprotein 120 vaccine and
incidence of HIV-1 infection in a phase 3 HIV-1 preventive vaccine
trial. J Infect Dis 2005;191:666–677.
39. Flynn NM, Forthal DN, Harro CD, et al. Placebo-controlled phase 3
trial of recombinant glycoprotein 120 vaccine to prevent HIV-1 infec-
tion. J Infect Dis 2005;191:654–665.
40. Gilbert PB, Ackers ML, Berman PW, et al. HIV-1 virologic and im-
munologic progression and initiation of antiretroviral therapy among
HIV-1-infected subjects in a trial of the efficacy of recombinant gly-
coprotein 120 vaccine. J Infect Dis 2005;192:974–983.
41. Berman PW, Huang W, Riddle L, et al. Development of bivalent (B/
E) vaccines able to neutralize CCR5-dependent virusesfromtheUnited
States and Thailand. Virology 1999;265:1–9.
42. Pitisuttithum P, Gilbert PB, Gurwith M, et al. Randomized, placebo-
controlled efficacy trial of a bivalent rgp120 HIV-1 vaccine among
injecting drug users in Bangkok, Thailand. J Infect Dis 2006;194:
43. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with
ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl
J Med 2009;361:2209–2220.
44. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D. Effects of
CCR5 and CD4 cell surface concentrations on infection by macro-
phage tropic isolates of human immunodeficiency virus type 1. J
45. Wei X, Decker JM, Liu H, et al. Emergence of resistant human im-
munodeficiency virus type 1 in patients receiving fusion inhibitor (T-
20) monotherapy. Antimicrob Agents Chemother 2002;46:1896–1905.
46. Lachenbruch PA. Comparison of two-part models with competitors.
Statistics in Medicine 2001;20:1215–1234.
47. Huang Y, Gilbert PB, Montefiori DC, Self SG. Simultaneous evaluation
of the magnitude and breadth of a left- and right-censored multivariate
response, with application to HIV vaccine development.StatBiopharm
48. Pe ´rez-Losada M, Jobes DV, Sinangil F, Crandall KA, Posada D, Berman
PW. Phylodynamics of HIV-1 from a phase III AIDS vaccine trial in
North America. Mol Biol Evol 2010;27(2):417–425.
49. Montefiori DC, Metch B, McElrath MJ, Self S, Weinhold KJ, Corey L.
Demographic factors that influence the neutralizing antibody response
in recipients of recombinant HIV-1 gp120 vaccines. J Infect Dis 2004;
50. Pe ´rez-Losada M, Posada D, Arenas M, et al. Ethnic differences in the
adaptation rate of HIVgp120fromavaccinetrial.Retrovirology2009;6:
by guest on February 2, 2011