Ancestral and consensus envelope immunogens for HIV-1 subtype C
Denise L. Kothea, Yingying Lib, Julie M. Deckerb, Frederic Bibollet-Rucheb, Kenneth P. Zammitb,
Maria G. Salazarb, Yalu Chenb, Zhiping Wengb, Eric A. Weaverd, Feng Gaod, Barton F. Haynesd,
George M. Shawa,b,c, Bette T.M. Korbere,f, Beatrice H. Hahna,b,⁎
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
bDepartment of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA
cHoward Hughes Medical Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
dDuke Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA
eLos Alamos National Laboratory, Los Alamos, NM 87545, USA
fSanta Fe Institute, Santa Fe, NM 87501, USA
Received 15 March 2006; returned to author for revision 19 April 2006; accepted 8 May 2006
Available online 14 June 2006
Immunogens based on “centralized” (ancestral or consensus) HIV-1 sequences minimize the genetic distance between vaccine strains and
contemporary viruses and should thus elicit immune responses that recognize a broader spectrum of viral variants. However, the biologic,
antigenic and immunogenic properties of such inferred gene products have to be validated experimentally. Here, we report the construction and
characterization of the first full-length ancestral (AncC) and consensus (ConC) env genes of HIV-1 (group M) subtype C. The codon-usage-
optimized genes expressed high levels of envelope glycoproteins that were incorporated into HIV-1 virions, mediated infection via the CCR5 co-
receptor and retained neutralizing epitopes as recognized by plasma from patients with chronic HIV-1 subtype C infection. Guinea pigs immunized
with AncC and ConC env DNA developed high titer binding, but no appreciable homologous or heterologous neutralizing antibodies. When tested
by immunoblot analysis, sera from AncC and ConC env immunized guinea pigs recognized a greater number of primary subtype C envelope
glycoproteins than sera from guinea pigs immunized with a contemporary subtype C env control. Mice immunized with AncC and ConC env
DNA developed gamma interferon T cell responses that recognized overlapping peptides from the cognate ConC and a heterologous subtype C
Env control. Thus, both AncC and ConC env genes expressed functional envelope glycoproteins that were immunogenic in laboratory animals and
elicited humoral and cellular immune responses of comparable breadth and magnitude. These results establish the utility of centralized HIV-1
subtype C Env immunogens and warrant their continued evaluation as potential components of future AIDS vaccines.
© 2006 Elsevier Inc. All rights reserved.
Keywords: HIV-1 genetic variation; Centralized HIV-1 immunogens; HIV-1 envelope glycoprotein; Subtype C
Globally circulating strains of HIV-1 exhibit an extraordi-
nary degree of genetic diversity, which poses a formidable
challenge for AIDS vaccine development (Douek et al., 2006;
Garber et al., 2004; Gaschen et al., 2002; Joseph et al., 2005).
Members of the pandemic-associated main group of HIV-1
(group M) can differ in up to 35% of envelope amino acid
residues, and even within individual group M subtypes,
envelope protein sequence variation can be as high as 20%
(Gaschen et al., 2002). Despite this extent of diversity, only few
AIDS vaccine strategies are addressing HIV-1 genetic variation
directly. The great majority of HIV-1 immunogens in preclinical
and clinical evaluation are derived from contemporary viruses,
frequently selected based on availability and geographic
representation, and in some instances envelope co-receptor
preference or preservation of cross-reactive epitopes (IAVI,
2006; Douek et al., 2006; HVTN, 2006; Nkolola and Essex,
2006). Several studies are also aiming to improve cross-
reactivity by including immunogens from more than one HIV-1
subtype (Chakrabarti et al., 2005; Kong et al., 2003; Seaman et
Virology 352 (2006) 438–449
⁎Corresponding author. Department of Medicine, University of Alabama at
Birmingham, 720 20th Street South, Kaul 816, Birmingham, AL 35294, USA.
Fax: +1 205 934 1580.
E-mail address: firstname.lastname@example.org (B.H. Hahn).
0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
al., 2005), and in one instance a vaccine strain was selected
based on its genetic relatedness to the consensus sequence of the
locally circulating subtype (Burgers et al., 2006). However, the
most direct approach to minimize HIV-1 genetic diversity is the
use of “centralized” immunogens (Gaschen et al., 2002;
Novitsky et al., 2002; Ellenberger et al., 2002; Nickle et al.,
2003; Mullins et al., 2004). This concept is based on the fact
that reconstructed ancestral or consensus sequences are only
about half as distant from contemporary HIV-1 strains as these
are to each other. Thus, centralized immunogens should elicit
more cross-reactive immune responses than immunogens
derived from any single contemporary HIV-1 strain. However,
this prediction remains to be tested in vivo, by rigorous
comparative testing in appropriate animal models and ulti-
mately human vaccine trials.
Three centralized env genes have thus far been generated and
investigated as DNA and/or protein immunogens. Two
comprise group M consensus env genes (CON6 and CONS)
(Gao et al., 2005; Weaver et al., in press; Liao et al., in press),
whereas the third represents a reconstructed ancestral subtype B
env gene (An1-EnvB) (Doria-Rose et al., 2005). All three
centralized env genes were shown to express functional
envelope glycoproteins that were incorporated into virus
particles and mediated cell fusion, although infectivity was
markedly reduced for the two group M consensus env genes
(Gao et al., 2005; Doria-Rose et al., 2005; Liao et al., in press).
Mice vaccinated with CON6 env DNA developed potent and
broadly cross-reactive (cross-clade) Tcell responses (Gao et al.,
2005; Weaver et al., in press), whereas guinea pigs immunized
with CON6 gp120 or a cleavage site and fusion peptide deleted
gp140ΔCF protein developed only low titer neutralizing
antibody responses (Gao et al., 2005). Rabbits immunized
with full-length (non-modified) gp160 An1-EnvB DNA and
boosted with the cognate gp120 protein also developed
neutralizing antibody responses, but again breadth and potency
were limited (Doria-Rose et al., 2005). In contrast, guinea pigs
immunized with gp140 oligomers of a cleavage site, fusion
peptide and gp41 immunodominant region deleted ConS
glycoprotein (ConS gp140ΔCFI) elicited cross-reactive anti-
bodies that neutralized primary Env containing viruses from
three different clades (Liao et al., in press). Together, these
results provided proof of concept for the utility of centralized
gene products as components of an AIDS vaccine. However,
studies of additional centralized immunogens, including
consensus and ancestral genes from the same subtype, are
necessary to examine the influence of the reconstruction process
on their immunogenic properties.
Among the many subtypes and circulating recombinant
forms of HIV-1 group M, subtype C is the most prevalent. This
subtype is responsible for more than half of all infections
worldwide, dominates the epidemic in Southern Africa (the
region with the highest HIV-1 prevalence globally) and fuels the
rapidly expanding epidemics in India and China (Osmanov et
al., 2002; Nkolola and Essex, 2006; Novitsky et al., 2002).
Subtype C viruses also appear to share unique biological
properties. Most notably, the V3 region and adjacent env
domains of subtype C strains are under markedly different
evolutionary pressures than the corresponding env regions in
other subtypes, suggesting differences in epitope presentation
and/or accessibility (Gaschen et al., 2002). In addition, recently
transmitted subtype C viruses have shorter hypervariable
regions and individuals infected with these viruses develop
higher titer autologous neutralizing antibodies than individuals
acutely infected with subtype B (Derdeyn et al., 2004; Chohan
et al., 2005; Li et al., 2006). Finally, subtype C strains use the
CCR5 co-receptor almost exclusively (Morris et al., 2001;
Peeters et al., 1999). Together, these findings suggest subtype-
specific differences in envelope antigenicity and possibly also
immunogenicity, which represents a rationale for exploring
subtype C specific vaccines (Nkolola and Essex, 2006). Here,
we describe the first centralized subtype C Env immunogens,
including both ancestral (AncC) and consensus (ConC) env
sequences, and compare their antigenic and immunogenic
properties to those of a wild-type subtype C env control.
Design of subtype C consensus and ancestral env sequences
Both ancestral (AncC) and consensus (ConC) env sequences
were reconstructed from alignments of full-length subtype C
env sequences available in the 2001 Los Alamos HIV Sequence
Database. The ancestral env sequence was inferred from
maximum likelihood phylogenetic analyses and represents the
most likely sequence at the interior node of the subtype C
cluster (Gaschen et al., 2002). The consensus env sequence was
generated by selecting the most common amino acid at each
position in the protein alignment. Hypervariable regions of both
centralized env genes were aligned by anchoring on common
glycosylation sites, and only minimal common elements
spanning the region were retained. Fig. 1 depicts the deduced
amino acid sequences of the AncC and ConC env genes.
Overall, the two protein sequences are 95.5% identical and
share all major known functional domains. The AncC env gene
encodes one additional N-linked glycosylation site near the base
of the V1/V2 loop (boxed); all other 25 N-linked glycosylation
sites are conserved between the two proteins.
Expression and virion incorporation of AncC and ConC Env
Codon usage optimization is known to increase HIV/SIV
protein expression in vitro as well as in vivo (Andre et al.,
1998; Haas et al., 1996; Schneider et al., 1997; Schwartz et al.,
1992). We therefore synthesized the full-length AncC and
ConC env genes as codon-usage-optimized versions of their
amino acid sequences (Andre et al., 1998). We also
constructed truncated gp145 and gp120 env genes by
introducing premature stop codons immediately following
the membrane-spanning domain and the gp120/gp41 cleavage
site, respectively. As shown previously for a codon-usage-
optimized env gene of a wild-type subtype C strain (Gao et al.,
2003), full-length and truncated AncC and ConC genes
expressed high levels of glycoprotein upon transfection into
439D.L. Kothe et al. / Virology 352 (2006) 438–449
293T cells (Fig. 2). Optimized gp160 and gp145 Env proteins
were processed, albeit incompletely; this was most likely due
to the saturation of cellular proteases involved in the cleavage
of the gp160 Env precursor (Binley et al., 2002). Cleavage
products were seen for both gp160 and gp145 constructs upon
longer exposure (not shown).
To examine whether the AncC and ConC env expressed
glycoproteins could be packaged into HIV-1 particles, gp160
and gp145 constructs were co-transfected with an env-deficient
optimized and rev-dependent env constructs of a wild-type
subtype C strain (96ZM651.8), along with the env-minus
backbone vector, were analyzed in parallel as controls.
Pseudovirion preparations were purified by centrifugation
and examined by Western blot analysis using plasma from an
derived gp160 and gp145 glycoproteins were incorporated into
virus particles (Fig. 3). Indeed, AncC and ConC gp160
containing pseudovirions (lanes 1 and 3) contained at least
ten-fold more envelope glycoprotein than pseudovirions gener-
ated with the rev-dependent 96ZM651.8 subtype C env control
(Fig. 3, lane 6). Again, a large fraction of particle associated
and truncated gp41 proteins were visible in the AncC and ConC
Env containing pseudovirion preparations (Fig. 3). Taken
together, both full-length and truncated centralized Envs were
Fig. 1. Alignment of ancestral (AncC) and consensus (ConC) subtype C Env protein sequences. Amino acid sequence differences are highlighted in red. A potential N-
linked glycosylation site present in AncC but absent in ConC is boxed. Variable regions (V1–V5) were reconstructed using a minimal loop approach, reasoning that
shorter loops may be of advantage for exposure of neutralizing epitopes (Srivastava et al., 2003). The gp120/gp41 cleavage site and the transmembrane domain (TM)
Fig. 2. Expression of AncC and ConC envelope glycoproteins. Full-length and
truncated env genes were transfected into 293T cells and lysates examined by
Western blot analysis using plasma from a patient infected with HIV-1 subtype
C. The sizes of the gp160, gp145 and gp120 expression products are indicated.
440 D.L. Kothe et al. / Virology 352 (2006) 438–449
packaged efficiently into HIV-1 virions and are thus suitable for
inclusion into virus-like particle based vaccines.
Infectivity and co-receptor usage of AncC and ConC Env
To determine whether AncC and ConC envelope glycopro-
teins were capable of mediating fusion and entry into
appropriate target cells, purified pseudovirion preparations
were analyzed for infectivity on JC53-BL cells (Derdeyn et
al., 2000; Platt et al., 1998; Wei et al., 2002). JC53-BL cells
express high levels of CD4, CCR5 and CXCR4 receptor
molecules and are stably transfected with beta-galactosidase
and luciferase reporter cassettes under the control of the HIV-1
long terminal repeat (LTR). These cells can thus be used to
determine the infectious titer of pseudovirion preparations by
determining luciferase activity or by staining cultures for beta-
galactosidase expression and counting the number of blue cells.
As shown in Fig. 4, both AncC and ConC Env glycoproteins
conferred infectivity to HIV-1/SG3Δenv when complemented
in trans; however, in five independent experiments, the relative
infectivity of the AncC Env containing particles was approx-
imately 4-fold lower than that of ConC Env containing
pseudovirions and approximately 6-fold lower than that of
wild-type 96ZM651.8 Env containing pseudovirions. These
differences were statistically significant.
Using antagonists of the CXCR4 (AMD3100) and CCR5
(TAK779) co-receptors, the JC53-BL cell assay was also used
to determine the co-receptor usage of the AncC and ConC env
gene products. As shown in Fig. 5, both centralized glycopro-
teins required CCR5 as the co-receptor for entry (pseudovirions
containing the CXCR4 tropic NL4.3 Env and the CCR5-tropic
YU2 Env are shown for control). Thus, the centralized subtype
C envelope glycoproteins resemble primary subtype C Envs in
their co-receptor preference.
Sensitivity of AncC and ConC Env containing pseudovirions to
neutralization by plasma from individuals chronically infected
with HIV-1 subtype C
To determine whether AncC and ConC envelope glycopro-
teins retained neutralizing epitopes also found in contemporary
subtype C viruses, we tested pseudovirion preparations for
(96ZM651.8-opt) envgenes(gp160)withthe HIV-1/SG3Δenvbackbonevector.
(infectious units, IU) per nanogram of p24 pseudovirion stock (IU/ng p24). Bars
indicate standard errors (values are averaged from five independent experi-
ments). P values are indicated. The infectivity of pseudovirions generated by co-
transfection of the HIV-1/SG3Δenv backbone with a non-codon-optimized
standard envelope (YU2 gp160) is included for control.
Fig. 5. Co-receptor usage of AncC and ConC Env containing pseudovirions.
JC53-BL cells were pretreated with AMD3100 (inhibitor of CXCR4), TAK779
(inhibitor of CCR5), both or neither (media) before being infected by AncC,
ConC, 96ZM651.8-opt, NL4.3 and YU2 Env containing pseudovirions. Virus
infectivity, asa percentageof the untreatedcontrol,isplottedon the vertical axis.
Fig. 3. Protein profiles of AncC and ConC Env containing pseudovirions. Full-
length (gp160) and truncated (gp145) AncC and ConC as well as codon-usage-
optimized (opt) and wild-type (wt) 96ZM651.8 env genes were co-transfected
with the HIV-1/SG3Δenv backbone vector and subjected to Western blot
analysis using plasma from a patient infected with HIV-1 subtype C. The env-
minus SG3 backbone vector (SG3Δenv) was included for control. Molecular
weights of major viral proteins are indicated. The truncated gp41 cleavage
product (Δgp41) of the membrane anchored gp145 protein is also shown.
Pseudovirions derived from optimized and wild-type env genes were loaded at
25 and 250 ng of p24 per lane, respectively.
441 D.L. Kothe et al. / Virology 352 (2006) 438–449
sensitivity to neutralization by plasma from nine individuals
chronically infected with subtype C strains. The results are
summarized in Fig. 6. Interestingly, AncC and ConC Envs were
equally sensitive to neutralization by patient plasma, although
IC50titers varied widely from 1:170 to 1:5450. Moreover, these
titers were very similar to those obtained for 96ZM651.8 Env
pseudovirions, indicating that both centralized and contempo-
rary subtype C envelope glycoproteins contain neutralizing
epitopes that are accessible to patient antibodies on the surface
of virus particles. Thus, the overall structure and function of
centralized Envs appear to resemble those of contemporary
subtype C strains.
Humoral immune responses in AncC and ConC env DNA
immunized guinea pigs
To examine whether centralized subtype C immunogens
were capable of eliciting humoral immune responses in a
small animal model, we immunized guinea pigs (three animals
per group) three times at three-week intervals with 400 μg of
AncC and ConC env DNA. Plasmids containing the codon-
optimized 96ZM651.8 env gene as well as an empty vector
were tested in parallel as positive and negative controls. Two
weeks following the third immunization, sera were collected
from each animal and assayed for the presence of binding
antibodies to purified 96ZM651.8 (Fig. 7) and ConC (data not
shown) gp120 glycoprotein using an ELISA assay. All DNA
vaccines elicited env-specific binding antibodies, which were
measurable after the second injection, but reached endpoint
titers of up to 1:500,000 after the third immunization.
Moreover, centralized env vaccines elicited binding antibody
responses that were slightly higher than those induced by the
contemporary control, but these differences were not statisti-
We next asked whether antibodies in sera from guinea pigs
immunized with the AncC and ConC DNA vaccines were
capable of binding to a greater number of contemporary
subtype C envelopes than antibodies in sera from animals
immunized with the wild-type control. Fourteen expression
plasmids containing primary (non-codon-optimized) env genes
of divergent subtype C viruses were transfected into 293T
cells, cell lysates harvested and tested for reactivity with
individual guinea pig sera from each immunization group by
Western blot analysis (Fig. 8A). Codon-optimized AncC and
ConC env genes as well as the wild-type 96ZM651.8-opt env
were included for control. This analysis revealed that sera
from guinea pigs immunized with the centralized env vaccines
were more cross-reactive than sera from animals immunized
with the 96ZM651.8 wild-type control; however, the observed
differences were only minor. As shown in Fig. 8A, AncC and
ConC immunized animals recognized up to nine of the
fourteen subtype C envelopes (the most cross-reactive serum
of each group is shown). In contrast, one of three 96ZM651.8
immunized guinea pigs recognized seven (Fig. 8A), whereas
the other two reacted with less than five primary subtype C
envelopes. Fig. 8B shows that the selected primary envelopes
are representative of the full spectrum of subtype C diversity.
Thus, both centralized env genes induced binding antibodies
that recognized a slightly greater number of linear Env
epitopes than those induced by a wild-type subtype C env
Finally, sera from immunized guinea pigs were tested
individually for the presence of neutralizing antibodies. This
was done in a single round neutralization assay using
pseudovirions containing the cognate AncC, ConC and
96ZM651.8-opt Env proteins, as well as two neutralization
sensitive envelopes representing subtypes B (SF162) and C
(TV-1), respectively (Li et al., 2005). Each serum was
analyzed at a 1:10 dilution and its neutralization activity
determined relative to the baseline activity of the pre-immune
serum from the same animal (Grundner et al., 2005). Sera that
Fig. 6. Sensitivity of AncC and ConC Env containing pseudovirions to
neutralization by patient plasma. Plasma from nine HIV-1 subtype C infected
individuals were tested for their ability to neutralize AncC, ConC and
96ZM651.8-opt Env containing pseudovirions. Neutralization was scored as
the plasma dilution required to reduce virus infectivity by 50% (IC50). Vertical
boxes represent the 25th to 75th percentiles of the IC50values, the line in the box
the median and the lines emerging from the box the highest and lowest serum
dilutions observed for the group, respectively.
Fig. 7. Humoral immune responses in AncC and ConC env DNA vaccinated
guinea pigs. Female Hartley guinea pigs (three per group) were vaccinated three
times at three-week intervals with 400 μg of AncC, ConC, 96ZM651.8-opt and
empty vector DNA. Animals were bled two weeks following the third
vaccination and assayed for the presence of binding antibodies to 96ZM651.8
gp120 protein. Sera were serially diluted and the last dilution giving absorbance
values greater than twice the OD value of the negative control was identified as
theendpointtiter. Verticalboxesindicate themeanendpointtiter ±SDfor each of
the groups indicated.
442D.L. Kothe et al. / Virology 352 (2006) 438–449
reduced viral infectivity by less than 50% were considered
neutralizing antibody negative. Despite high titer cross-
reactive binding antibodies, only one ConC immunized guinea
pig serum neutralized the homologous ConC Env, and then
only by 51% (not shown). All other eight sera failed to
neutralize both their autologous and the other two heterolo-
gous envelope glycoproteins. Sera from one AncC and two
ConC immunized animals exhibited weak neutralizing activity
against SF162, but in each case the observed reduction in viral
infectivity was less than 70%. No neutralizing activity was
observed against the second isolate, TV-1. Thus, despite the
fact that AncC, ConC and 96ZM651.8 envelopes were quite
sensitive to neutralization by patient plasma (Fig. 6), they
failed to elicit appreciable homologous and heterologous
neutralizing antibodies in guinea pigs when delivered in the
form of a DNA vaccine.
Fig. 8. Cross-reactivity of AncC and ConC induced Env binding antibodies.(A) Sera from guinea pigs vaccinated with AncC, ConC or 96ZM651.8-opt env DNAwere
tested for cross-reactivity with Env glycoproteins from divergent subtype C strains (as shown in B). Western blot strips containing equal amounts of env transfected
293T cell lysates were tested. The most cross-reactive serum per immunization group is shown. Expressed glycoproteins (gp160 and gp120) are indicated. (B)
Phylogenetic relationship of divergent subtype C env sequences. Sequences used for Env protein expression (as shown in A) are highlighted in red; reference strains
from the database (and their country of origin) are shown in black. The tree was inferred by the neighbor joining method and rooted using the subtype B strain YU2;
443 D.L. Kothe et al. / Virology 352 (2006) 438–449
Cellular immune responses in AncC and ConC env DNA
BALB/c mice (four animals per group) were immunized
intramuscularly four times at three-week intervals with 50 μg of
AncC, ConC, 96ZM651.8-opt env (and vector control) plasmid
DNA. Two weeks after the last DNA immunization, all mice
were sacrificed, their splenocytes isolated and tested in a
gamma-interferon (INF-γ) ELISpot assay for the number of
spot forming cells (SFCs). Env peptides (15-mers overlapping
by 11) of the cognate ConC Env as well as a wild-type subtype
C (Chn19) Env control (20-mers overlapping by 10) (Chen et
al., 2000) were used to stimulate T cell responses. All env
expressing DNA vaccines elicited T cell responses, whereas
splenocytes of mice immunized with the empty vector control
developed no appreciable Env-specific SFC responses (Fig. 9).
When tested with ConC overlapping peptides, 841 ± 102 and
785 ± 215 SFCs were detected in splenocytes of AncC and
ConC env immunized mice, respectively. 96ZM651.8 immu-
nized mice had slightly lower responses (636 ± 99 SFCs), but
this difference was not statistically significant. As expected,
responses to Chn19 wild-type peptides were lower in all groups.
In this study, we compared the function, antigenicity and
immunogenicity of reconstructed subtype C ancestral and
consensus env gene products. A central question regarding the
use of such immunogens is whether the expressed glycoproteins
are functional and whether the algorithms used for their
reconstruction affect their antigenicity and immunogenicity.
An ancestral sequence is an attempt to re-create the sequence of
a parental strain for a particular lineage. However, this
reconstruction is dependent on the particular phylogenetic
model used, tends to generate sequences artificially enriched for
adenine (A), and does not include recently fixed escape
mutations (Gaschen et al., 2002). Consensus sequences tend
to represent fixed escape mutations but have the potential of
linking polymorphisms not found in natural infections (Doria-
Rose et al., 2005). Finally, both ancestral and consensus
sequences are vulnerable to sampling bias. Thus, both
consensus and ancestral sequences represent only imperfect
approximations of the evolutionary history of the virus. This is
particularly true in hypervariable regions, which evolve by
insertion and deletion. As a consequence, consensus or ancestral
genes constructed for any one HIV-1 group or subtype have to
be tested individually to examine their function and utility as
We characterized ancestral and consensus env genes of HIV-
1 subtype C and show that both centralized constructs express
functional envelope glycoproteins. AncC and ConC Envs
incorporated efficiently into virus particles (Fig. 3), mediated
virus entry via the CCR5 co-receptor (Figs. 4 and 5), and were
sensitive to neutralization by plasma from individuals with
chronic HIV-1 subtype C infection (Fig. 6). Moreover, AncC
and ConC env expressing DNA vaccines elicited humoral and
cellular immune responses in guinea pigs and mice, respective-
ly, indicating that they were immunogenic in vivo (Figs. 7 and
9). Thus, both ancestral and consensus reconstruction
approaches yielded biologically active subtype C Env proteins.
Using a similar approach, Lian and colleagues recently
constructed a consensus env gene from published subtype C
sequences from Botswana (Lian et al., 2005). However, this
centralized Env failed to bind CD4 and to mediate fusion,
indicating that not all re-constructed consensus sequences
express functional gene products. In our study, we found no
evidence of functional impairment of the consensus env
glycoprotein. ConC Env containing pseudovirion preparations
were significantly more infectious than pseudovirions contain-
ing the AncC Env (Fig. 4), and ConC env DNAvaccine induced
higher titer binding antibodies (Fig. 7). Moreover, ConC and
AncC immunized guinea pig sera recognized the same number
of divergent Env proteins on immunoblots (Fig. 8). Thus, at
least for the subtype C env consensus gene, previously noted
theoretical concerns (Doria-Rose et al., 2005) and functional
limitations (Lian et al., 2005) were not substantiated.
A major goal of AIDS vaccine development is the elicitation
of broadly cross-reactive neutralizing antibodies against HIV-1
envelope glycoproteins. Fig. 6 shows that both the AncC and
ConC env gene products retained neutralizing epitopes present
in contemporary subtype C viruses, perhaps because they
contain very short hypervariable loops that minimize camou-
flage and glycosylation (Srivastava et al., 2003). However,
neither DNA immunogen (nor the wild-type control) was
capable of eliciting appreciable neutralizing antibodies against
their homologous as well as heterologous Env glycoproteins.
Given that non-modified (full-length) env DNA vaccines rarely
elicit neutralizing antibodies, these results are not unexpected.
Rasmussen et al. (2006) immunized mice with a codon-
optimized env gene from a contemporary subtype C isolate and
did not detect neutralizing antibodies in pooled sera against the
Fig. 9. Tcell immune responses in AncC and ConC env DNAvaccinated mice.
Balb/c mice (four per group) were vaccinated four times at three-week intervals
with 50 μg of AncC, ConC, 96ZM651.8-opt or empty vector DNA. Two weeks
following the last vaccination, splenocytes were isolated from individual mice
and stimulated with consensus and wild-type (Chn19) subtype C overlapping
Env (or no) peptide pools. Total responses are expressed as spot forming cells
(SFC) per million splenocytes. The values for each column are the mean ± SEM
of IFN-γ SFCs.
444 D.L. Kothe et al. / Virology 352 (2006) 438–449
homologous strain. Similarly, no appreciable neutralizing
antibody titers against SF162 were observed in rabbits
immunized five times with the full-length AN1 Env B gene
(Doria-Rose et al., 2005). Gao et al. (2005) were able to detect
neutralizing antibodies to the subtype B strain SF162 in CON6
immunized guinea pigs; however, in this study, the centralized
immunogen was delivered as a secreted Env protein (gp120 or
gp140ΔCF) rather than plasmid DNA. This was also the case
for the group M consensus CONS Env which induced high titer
cross-clade neutralizing antibody responses when delivered as
gp140ΔCFI glycoprotein oligomers (Liao et al., in press). Thus,
additional modes of delivery will need to be tested to examine
the full neutralizing antibody induction potential of AncC and
ConC Env immunogens.
In addition to suboptimal vaccine delivery, there may be an
inherent, as yet unexplained inability of subtype C env
immunogens to induce neutralizing antibodies. Seaman et al.
(2005) found in primates that among contemporary subtype A,
B and C env immunogens in a DNA prime/recombinant
adenovirus (rAd) boost regimen, wild-type subtype C env was
by far the weakest inducer of neutralizing antibodies, and this
was also observed when these same env immunogens were
delivered in the form of gp140 protein oligomers and studied in
guinea pigs (Liao et al., in press). Also, subtype B, but not
subtype C, V3 peptides induced neutralizing antibodies against
Tier 1 subtype B and C primary isolates, again suggesting
subtype-C-specific differences in V3 loop presentation (Haynes
et al., 2006; B. Haynes and B. T. K Korber, unpublished).
Atomic level understanding of susceptible Env epitopes and
their interaction with neutralizing antibodies will likely be
necessary to design novel immunogens that are capable of
inducing cross-reactive neutralizing antibodies (Burton et al.,
2004; Huang et al., 2005; Pancera et al., 2005). In this regard, a
better understanding of the structural basis of subtype-C-
specific neutralizing antibody responses will be important.
Once this is accomplished, the theoretical advantages of
centralized envelope glycoprotein backbones will have to be
The primary rationale of ancestral and consensus vaccines is
the expectation that centralized immunogens induce broader,
more cross-reactive Tcell responses (Gaschen et al., 2002). We
did not observe differences in the T cell response of ancestral,
consensus and wild-type subtype C Env immunized mice when
peptide pools were used for stimulation (Fig. 9); however,
determining the breadth of Tcell responses was not the purpose
of the study. The mouse studies were intended to document
basic in vivo immunogenicity of AncC and ConC env DNA
vaccines. As shown in Fig. 9, this was accomplished because
the observed T cell responses were at least as high as those
elicited by the wild-type env control. Inbred mice are not an
appropriate animal model to compare the breadth and quality of
CD4+ and CD8+ T cell responses to centralized immunogens.
This will have to be done in primates or humans with diverse
Both ancestral and consensus env genes are a reflection of
the particular HIV-1 sequences that were used for their
reconstruction and consequently subject to change as additional
sequences become available. The AncC and ConC env genes
described here are representative of envelopes derived from
chronically infected individuals. It is possible that centralized
gene products derived from acute HIV-1 infection sequences
would be of greater relevance to AIDS vaccine design. This is
because HIV-1 strains from recently infected individuals may
share biological properties that are distinct from those of viruses
dominating in chronic infections. Studies are underway to
compile an acute HIV-1 infection database and to generate such
second generation centralized immunogens for comparative
In summary, we have shown here that ancestral and
consensus subtype C envelope genes express functional
envelope glycoproteins that are immunogenic in laboratory
animals and induce humoral and cellular immune responses
comparable to those of a contemporary HIV-1 subtype C DNA
vaccine. As new avenues of vaccine design and delivery are
uncovered, their application to centralized gene-derived immu-
nogens will determine the full extent of B and T cell responses
that can be elicited by these synthetic gene products. The results
described here will serve as a baseline for future studies of
modified and improved centralized immunogens. The codon-
optimized AncC and ConC env genes have been submitted to
the National institutes of Health Research and Reference
Program (Rockville, MD) and are available to interested
Materials and methods
HIV-1 subtype C ancestral and consensus env gene
sequences were codon-usage optimized as described (Gao et
al., 2003) and are available at GenBank under accession
numbers DQ401076 and DQ401075, respectively. The genes
were cloned in pcDNA3.1.
Env gene expression
293T cells (5 × 105cells/well) were seeded in 6-well plates
12–14 h prior to transfection. Monolayers were transfected with
each env gene using FuGene 6 Transfection Reagent (Roche
Applied Science, Indianapolis, IN). Forty-eight hours post-
transfection, cells were washed with Tris–saline (pH 7.5,
50 mM Tris–Cl and 150 mM NaCl) and resuspended in 150 μl
of lysing buffer (20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1%
Triton X-100, 1% deoxycholic acid, 2 mM EDTA, 2 μg/ml
aprotinin, 1 mg/ml pepstatin A, 2 mg/ml leupeptin and 1 mM
PMSF). The amount of total protein was quantified using the
BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
For each expressed gene, 5 μgof cell lysate was boiled for 5 min
in the presence of reducing buffer and separated on a 4–20%
SDS–polyacrylamide gel (Bio-Rad Laboratories, Hercules,
CA). Proteins were transferred to a PVDF nitrocellulose
membrane (GE Healthcare Life Sciences, Piscataway, NJ) by
electroblotting and incubated with blocking buffer (5% nonfat
dry milk in PBS) for 1 h. Western blots were probed with
445D.L. Kothe et al. / Virology 352 (2006) 438–449
plasma from an HIV-1 (subtype C)-infected individual and
developed with HRP-labeled species-specific antibodies
(SouthernBiotech, Birmingham, AL) using the enhanced
chemiluminescence (ECL) detection system (GE Healthcare
Life Sciences, Piscataway, NJ).
Generation of pseudovirions
To test whether subtype C ancestral and consensus
glycoproteins were capable of incorporating into virus particles,
we transfected each gene with an env-minus HIV-1 backbone
vector (pSG3Δenv). Plasmids were co-transfected into a 100-
mm-diameter Petri dish containing a 60% confluent layer of
293T cells in complete Dulbecco's modified Eagle media
(DMEM) using FuGene 6 (Roche Applied Science, Indianapo-
lis, IN) at a 3:1 ratio as specified by the manufacturer. Forty-
eight hours post-transfection, virus-containing supernatant was
collected, clarified by low-speed centrifugation, passed through
a 0.2-μm filter and pelleted through a 20% sucrose cushion.
Pelleted virus was normalized by p24 for SDS–PAGE and
Western blot analysis.
Infectivity and co-receptor usage determination
Infectivity assays were performed as described (Derdeyn et
al., 2000). Briefly, JC53-BL cells were seeded in 24-well plates
at 50,000 cells/well in DMEM supplemented with 10% FBS, L-
glutamine and Pen/Strep and incubated overnight. Pseudotyped
virus stocks containing AncC, ConC or 96ZM651.8 Env were
added to each well in the presence of DEAE-Dextran
hydrochloride (80 μg/ml) (Sigma-Aldrich, St. Louis, MO) in
a final volume of 250 μl. Following a 48-h incubation, plates
were washed, stained and the number of blue cells counted to
determine the infectious virus titer.
A modification of the infectivity assay was used to determine
co-receptor usage using antagonists to CXCR4 (AMD3100)
and CCR5 (TAK-779) co-receptors (Zhang et al., 2000;
Spenlehauer et al., 2001). Briefly, JC53-BL cells that had
been seeded overnight were treated for 1 h with AMD3100
(1.2 μM/well), TAK-779 (10 μM/well) or a combination of
these chemokine receptor blocking agents. Two thousand
infectious units of pseudotyped virions were added to each
well in the presence of 80 μg/ml DEAE-Dextran hydrochloride
and incubated at 37 °C. Following a two-day incubation,
supernatant was removed from the wells, and cells were lysed
using a luciferase assay system kit (Promega, Madison, WI).
The light intensity of each cell lysate was measured on a Tropix
luminometer using Tropix WinGlow version 1.24 software. A
reduction in infectivity in the presence of a specific inhibitor
reflects a requirement of the targeted co-receptor for entry.
Pseudovirions containing the AncC, ConC or 96ZM651.8
envelope glycoproteins were probed for the presence of
neutralizing epitopes using a sensitive, single round infectivity
assay (Li et al., 2005). Briefly, JC53-BL cells were seeded at
8000 cells/well in a 96-well plate in 10% DMEM media
overnight at 37 °C with 5% CO2. Plasma from HIV-1 subtype
C-infected individuals were serially diluted and incubated with
2000 units of infectious virus per well for 1 h at 37 °C. Pre-
incubated virus/plasma dilutions were added to the cells in the
presence of DEAE-Dextran and incubated for 2 days at 37 °C.
Control wells containing pseudovirions that had not been pre-
incubated with antibody were included for each virus tested.
Additionally, cell-only wells were included on each plate as a
measure of background. Cells were lysed and analyzed for
luciferase production using the Luciferase Assay System
(Promega, Madison, WI). Neutralization was measured as the
percent reduction of viral infectivity in comparison to control
wells infected with virus alone. The 50% inhibitory concentra-
tion (IC50) was obtained by non-linear regression using Prism 4
(GraphPad Software, San Diego, CA).
Single round neutralization assays were also conducted to
examine sera from immunized guinea pigs for the presence of
neutralizing antibodies. For these assays, guinea pig sera were
incubated at a 1:10 dilution with pseudovirions containing the
AncC, ConC and 96ZM651.8-opt Env proteins, as well as two
neutralization sensitive envelopes representing subtypes B
(SF162) and C (TV-1), respectively (Li et al., 2005).
Neutralization was measured as the percent reduction of viral
infectivity compared to control wells where virus was incubated
with the same animal's pre-immune serum (Grundner et al.,
2005). Sera that reduced viral infectivity by less than 50% were
considered neutralizing antibody negative.
Guinea pig immunization and serum collection
Guinea pigs were housed in the University of Alabama at
Birmingham (UAB) Animal Facility according to Accreditation
of Laboratory Animal Care (AALAC) guidelines. Animals use
protocols were approved by the UAB Institutional Animal Care
and Use Committee. Female Hartley guinea pigs (Harlan
Sprague, Indianapolis, IN) (n =
intramuscularly three times at 3-week intervals with 400 μg
plasmid DNA. Two weeks following the last immunization,
5 ml of blood was collected from each animal via the cranial
vena cava. Sera were obtained by tabletop centrifugation using
Becton Dickinson SST Tubes (BD, Franklin Lakes, NJ) as
directed by the manufacturer. Samples were stored at −20 °C
3/group) were immunized
Endpoint binding titer ELISA
Guinea pig sera were tested for binding antibodies to subtype
C consensus and 96ZM651.8 recombinant gp120 glycoprotein
by enzyme linked immunosorbent assay (ELISA). Recombinant
96ZM651.8 gp120 was obtained from the NIH AIDS Research
and Reference Reagent Program (Bethesda, MD). Subtype C
consensus recombinant gp120 was produced by transfecting
293T with the corresponding plasmid, harvesting culture
supernatant 72 h post-transfection, clarifying the supernatant
by tabletop centrifugation and passing through a 0.2-μM filter.
Recombinant gp120 was purified using Galanthus Nivalis
446D.L. Kothe et al. / Virology 352 (2006) 438–449
Lectin (GNL) (Vector Laboratories, Burlingame, CA), eluted
with 500 mM alpha-methyl mannoside (Vector Laboratories,
Burlingame, CA), dialyzed overnight in PBS and quantified by
BCA Protein Assay (Pierce Biotechnology, Rockford, IL).
Microtiter plates were coated with recombinant gp120 (0.5 μg/
ml in PBS), washed and blocked with 200 μl/well 5% nonfat
milk in PBS-T. Serial five-fold dilutions were made of each
guinea pig serum, added to individual wells and set to incubate
for 1 h at 37 °C. Following a wash, 100 μl of HRP-conjugated
goat anti-guinea pig antibody (ICN Pharmaceuticals, Costa
Mesa, CA) diluted to 1:50,000 in blocking buffer was added to
each well. After an additional 1-hour incubation at 37 °C,100 μl
of liquid TMB (3,3′,5,5′-tetramethylbenzidine) was added to
each well. Reactions were stopped by the addition of 100 μl of
4N sulfuric acid. Absorbances were read at 405 nm on an MRX
Microplate reader (DYNEX Technologies, West Sussex, UK).
Endpoint titers were determined as the serum titer at which the
absorbance value was 2× the mean OD of the negative serum
Immunization of mice and splenocyte isolation
Female BALB/c mice (6–8 weeks old) were purchased from
Charles River Laboratories (Raleigh, NC) and housed in the
Duke University Animal Facility under AALAC guidelines
with animal use protocols approved by the Duke University
Animal Use and Care Committee. Four mice per group were
immunized intramuscularly in the quadriceps with AncC, ConC
or 96ZM651.8-opt gp160 or empty vector plasmid DNA
(50 μg) four times at 3-week intervals. Two weeks after the
4th DNA immunization, mice were euthanized and spleens
were collected. Spleens from individual mice were minced and
forced through a 70-μm Nylon cell strainer (BD Labware,
Franklin Lakes, NJ). Splenocytes were then washed, treated
with ACK lysis buffer and resuspended in HEPES-buffered
complete RPMI medium with 10% fetal bovine serum,
gentamicin (50 μg/ml), 10 mM non-essential amino acids and
0.053 mM β-mercaptoethanol.
Enzyme linked immune spot (ELISpot) assay
Overlapping Env peptides of ConC (subtype C consensus;
211 peptides, 15-mers overlapping by 10 amino acids) and
Chn19 (subtype C; 87 peptides, 20-mers overlapping by 10
amino acids) were obtained from the NIH AIDS Research and
Reference Reagent Program (Bethesda, MD) and used to
stimulate mouse splenocytes. Single-cell suspensions of
splenocytes were plated in 96-well polyvinylidene difluoride-
backed plates (MultiScreen-IP, Millipore, Billerica, MA) coated
with 50 μl of anti-mouse IFN-γ Mab AN18 (5 μg/ml; Mabtech,
Stockholm, Sweden) overnight at 4 °C. The plates were blocked
with HEPES-buffered complete RPMI medium at 37 °C for 2-h.
Equal volumes (50 μl) of each peptide pool (one pool of 87
peptides for Chn19; 211 peptides for ConC divided into two
pools) and splenocytes (107cells/ml) were added to the wells in
duplicate. Wells containing cells and complete RPMI medium
served as negative controls, whereas wells containing cells and
concanavalin A (5 μg/ml) (Sigma, St. Louis, MO) served as
positive controls. Plates were incubated overnight (14–16 h) at
37 °C with 5% CO2. After the plates were washed 6 times with
phosphate buffered saline (PBS), 50 μl of 1:1000-diluted
biotinylated anti-mouse IFN-γ mAb (Mabtech, Stockholm,
Sweden) was added to each well. Plates were then incubated at
room temperature for 2-h, washed 3 times with PBS, and 50 μl
of streptavidin–alkaline phosphatase conjugate (1:1000 dilu-
tion; Mabtech, Stockholm, Sweden) was added to each well.
After incubation for 1 h at room temperature, plates were
washed 5 times with PBS-T, and 100 μl of BCIP/NBT (Plus)
alkaline phosphatase substrate (Moss, Pasadena, MD) was
added to each well. Following an incubation for 10 min at room
temperature and a final wash with water, plates were air-dried.
Spots were counted using an automated ELISpot plate reader
(Immunospot counting system, CTL Analyzers, Cleveland,
OH) and expressed as spot-forming cells (SFC) per 106
splenocytes. Responses were considered positive if the number
of spots was four times greater than the negative control and at
least 50 SFC/106cells/well.
Deduced Env protein sequences of HIV-1 subtype C strains
used to evaluate binding antibody cross-reactivity were aligned
with geographically diverse subtype C Env sequences from the
database using CLUSTALW (Thompson et al., 1994). The tree
was inferred from a gap stripped alignment using the neighbor
joining method as implemented in CLUSTAL W using
Kimura's correction. Robustness of the branching order was
evaluated using the bootstrap method (1000 replicates) as
implemented in CLUSTAL W.
Analysis of variance (ANOVA) was used to compare
infectivity, neutralization sensitivity and ELISPOT means
across envelope comparison groups. Statistical tests were
performed using Prism 4 (GraphPad Software, San Diego,
CA). The F ratio was used to assess significance at the 5% level.
If the F ratio was significant, pairwise comparisons using a
modification of the t test were computed (Tukey–Kramer test).
A result was considered significant if the P value <0.05 (5%
level of significance). Two-tailed P values are cited.
We thank Susan Allen for sera from individuals infected with
HIV-1 subtype C; the NIAID-sponsored Reagent Resource
Support Program for AIDS Vaccine Development for providing
purified 96ZM651.8 gp120 protein and subtype C specific
peptide pools; and Wendy J. Abbott for artwork and manuscript
preparation. This work was supported in part by grants from the
National Institutes of Health (NO1 AI85338, U19 AI 028147,
P01 AI 061734, P30 AI27767, P30 CA13148, R21 AI055386),
an internal directed research (DR) grant for vaccine design at
Los Alamos National Laboratory, the NIAID Center for HIV/
447D.L. Kothe et al. / Virology 352 (2006) 438–449
AIDS Vaccine Immunology grant AI067854, the Bristol Myers
Freedom to Discover Program and the Howard Hughes Medical
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