AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 29, Number 5, 2013ª Mary Ann Liebert, Inc.
DOI: 10.1089/aid.2012.0094

Short Communication
Antibody Responses to Human Immunodeficiency Virus
Envelope from Infections with Multiple Subtypes
Utilize the 1F7-Idiotypic Repertoire
Matthew S. Parsons,1–4 Robert J. Center,4 Jean-Pierre Routy,1,2,5 Danielle Rouleau,6 Roger LeBlanc,1,7Mark A. Wainberg,8 Cécile L. Tremblay,9 Marcel D. Zannou,10 Stephen J. Kent,4Michael D. Grant,11 and Nicole F. Bernard1–3

Abstract

A common idiotype of anti-HIV antibodies (Abs), designated as 1F7, was recently observed on anti-HIV broadly
neutralizing Abs (BnAbs). The presence of the 1F7-idiotype on BnAbs suggests that continuous selection of 1F7idiotypic Abs may allow these clones to achieve the somatic hypermutation necessary for broad neutralization. As
the selection of type-specific BnAbs occurs in the setting of infections with a wide array of HIV subtypes, weinvestigated Abs from subjects infected with diverse subtypes for the selection of 1F7-idiotypic Abs. We observed
the 1F7-idiotype on antiviral Abs in infections with various HIV subtypes. Furthermore, gp140-specific 1F7idiotypic Abs recognized the gp140 antigens from several HIV subtypes. These results demonstrate that the 1F7idiotype is a common characteristic of Abs from infections with diverse HIV subtypes, and suggests that earlycross-reactivity of 1F7-idiotypic clones may act in conjunction with somatic hypermutation to produce BnAbs.

P

rimary infection with the human immunodeficiency
virus (HIV) induces antibodies (Abs) against viral envelope (Env) glycoproteins. These Abs arise against early viralvariants and in vitro screening demonstrates they can neutralize contemporaneous virus.1 Due to the frequent introduction of mutations and shifts in glycosylation patterns,
emergent viral variants less subject to Ab-mediated effector
functions such as neutralization and Ab-dependent cellularcytotoxicity (ADCC) gain a replicative advantage and rapidly
outcompete Ab-sensitive variants.2,3 Anti-HIV Env-specificAbs maintain neutralizing activity against early viral variants,
but constantly trail newly evolved and replicating autologous
contemporaneous viruses (ACV).1,2 Although the exact
mechanisms underlying this failure of humoral immune re-sponses to keep pace with constantly evolving HIV are unknown, one possible explanation is that anti-HIV Ab
responses suffer a form of original antigenic sin known as
deceptive imprinting or repertoire freeze.4
According to the repertoire freeze hypothesis, after escaping the effector functions of Abs directed against early viralvariants, ACV retain sufficient binding capacity for these Abs
to suppress induction of new Ab responses that could potentially control viral replication.4 Instead, original antigenic
sin allows memory B cells and Abs produced by Ab-secreting
cells to outcompete naive B cells for antigen.5 This leads torecall responses, which induce additional rounds of somatic
hypermutation and affinity maturation in previously selected
cells.6,7 This hypothesis is supported by several lines of1

Research Institute of the McGill University Health Centre, Montréal, Québec, Canada.
Division of Clinical Immunology and Allergy, McGill University Health Centre, Montréal, Québec, Canada.
Division of Experimental Medicine, McGill University, Montréal, Québec, Canada.
4
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.
5
Immunodeficiency Service and Division of Hematology, McGill University Health Centre, Montréal, Québec, Canada.
6
Départements de Microbiologie et Infectiologie, Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada.
7
Clinique LORI, Montréal, Québec, Canada.
8
McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montréal, Quebec, Canada.
9Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), Hôpital Saint-Luc, Montréal, Québec, Canada.
10
Faculté des Sciences de la Santé, Université d’Abomey-Calavi, Cotonou, Bénin.
11
Immunology and Infectious Diseases Program, Division of BioMedical Sciences, Faculty of Medicine, Memorial University of
Newfoundland, St. John’s, Newfoundland and Labrador, Canada.
2
3

778

IDIOTYPIC RESTRICTION OF HIV ANTIBODIES
evidence including the observation that anti-HIV Abs fromchronic infection exhibit extensive mutations.8 Furthermore,humoral immune responses against HIV in humans andagainst other viruses, such as simian immunodeficiency virus(SIV) and the chimeric simian human immunodeficiency virus (SHIV) in macaques, are characterized by Abs expressing
a common idiotype, designated as 1F7.9,10 This idiotype appears on anti-HIV Abs during primary infection and persists
throughout chronic infection.11 Maintenance of these Abs
appears maladaptive for ongoing Ab-mediated ACV neutralization, as depletion of 1F7-idiotypic Abs in SHIV-infected
Rhesus macaques allows novel anti-SHIV Abs to arise that
better neutralize ACV.12,13
Although previous data suggest that idiotypic-driven
repertoire freeze has a detrimental effect on the ability ofhumoral immune responses to contribute to the control of
ACV, a greater understanding of this phenomenon may help
elucidate the mechanisms required to induce protective antiHIV Ab responses. Approximately 25% of HIV-infectedindividuals produce Abs capable of neutralizing a broad
spectrum of viral isolates.14 Although these broadly neutralizing antibodies (BnAbs) are incapable of slowing progression
to AIDS,15 when purified and passively transferred to Rhesus
macaques prior to SHIV challenge, they protect against viral
infection.16–19 At least six of these BnAbs express the 1F7idiotype.11 Many BnAbs demonstrate extensive somatic hy-

779
permutation, a phenomenon associated with their broad
neutralization of HIV.20,21 Carriage of the 1F7-idiotype on
BnAbs suggests that repertoire freeze-induced maintenanceand continued selection, somatic hypermutation, and affinity
maturation may play a key role in the development of theirbroadly neutralizing capability. Although the evolution of
1F7-idiotypic Abs into BnAbs most certainly involves theseprocesses, the preferential selection of 1F7-idiotypic Abs toultimately develop into the BnAbs that neutralize diverse HIV
strains could also reflect the ability of less extensively mutated
Abs within this repertoire to recognize regions that are conserved across several HIV variants. If so, 1F7-idiotypic Abs
should be generated in the setting of infection with any or
most of multiple different HIV clades, and antigen-specificAbs within the 1F7-idiotypic repertoire should exhibit somedegree of cross-reactivity between different HIV subtypes.
To evaluate the hypothesis that 1F7-idiotypic Abs are acommon feature of infections with several HIV subtypes, we
assessed plasma-derived anti-HIV Env Abs for the presence ofthe 1F7-idiotype using a previously described ELISA.11
Briefly, plates were coated overnight at 4C with 200 ng/wellof HIV-1Bal gp120 (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) or HIV-1
gp41 (Prospec-Tany Technogene Ltd.) in coating buffer(15 mM Na2CO3; 35 mM NaHCO3). The following day, plates
were washed three times with phosphate-buffered saline

FIG. 1. Investigation of 1F7-idiotype expression on antibodies (Abs) induced by infection with multiple HIV subtypes.
Plasma samples from 17 individuals infected with HIV subtype B (A) and 19 subjects infected with non-subtype B HIV (B)were tested for binding to HIV gp120 (left) or gp41 (right) by ELISA. Each data point represents results for a single subject
after subtracting the background level of TEPC183 isotype control binding. The line through the scatter plots represents themean for the group. (C) Bar graphs depict relative levels of binding of the anti-1F7-idiotypic and the TEPC183 isotype control
Abs to plasma anti-gp120 Abs (left) and anti-gp41 Abs (right) from 19 HIV non-subtype B-infected subjects. The first threerows of graphs depict these results from individuals infected with subtypes C, G, and AG, respectively. The last row ofgraphs depicts the results for individuals infected with nine other clades or circulating recombinant forms (CRF), for which
there was only one sample available per clade or CRF.
(Continued /)780

PARSONS ET AL.

(PBS); 0.1% Tween-20, and blocked for 1 h at 37C with 200 ll/
well of PBS; 0.1% Tween-20; 5% bovine serum albumin (BSA).
After three washes, 100 ll of plasma at a 1:50 dilution in PBS,
0.1% BSA, 0.2% Tween-20, and 0.5% NP-40 were added towells for 90 min at 37C. The following sequential additions
were made with six washes between each step: (1) 200 ng/
well 1F7 (a kind gift from Dr. Heinz Kohler, University of
Kentucky) or isotype control TEPC183 (Sigma-Aldrich) for
90 min at 37C, (2) 100 ll/well horseradish peroxidase (HRP)conjugated goat antihuman IgG or HRP-conjugated goat
antimouse IgM ( Jackson Labs) for 1 h at 37C, and (3) 3¢3¢5¢5¢tetramethylbenzidine substrate (Sigma-Aldrich) for 30 min atroom temperature in the dark. Color development was stopped with 50 ll/well of 1 M H2SO4 and plates were read on a

FIG. 1.

Victor X5 Plate Reader (Perkin Elmer). Samples were considered positive for the 1F7-idiotype when the average OD
obtained for wells incubated with the anti-1F7-idiotypic Ab
was more than two times the average OD obtained for wells
treated with the TEPC183 isotype control. Plasma samples
obtained from 26 individuals in the Montreal Primary Infection (PI) Cohort (n = 17 were infected with subtype B andn = 9 with non-subtype B HIV-1), as well as 10 plasma samples from non-subtype B-infected patients enrolled at theCentre National Hospitalier Universitaire de Cotonou, Benin
were tested for HIV-specific Abs bearing the 1F7-idiotype.
All participants provided informed consent and the institutional review board of all participating sites approved
this study.

(Continued)

IDIOTYPIC RESTRICTION OF HIV ANTIBODIES
Assessment of plasma-derived anti-HIV Env Abs for the
1F7-idiotype revealed that the idiotype is common to infections with diverse HIV subtypes. We detected the 1F7-idiotype on anti-gp120 and anti-gp41 Abs from 16/17 and 17/17
HIV-1 subtype B-infected individuals, respectively (Fig. 1A).
Among the individuals infected with HIV non-subtype B
viruses were those infected with AB, AD, AE, AG, A/AG, AE/
K, AG/K, CRF06, CRF011/F, C, F, and G subtypes. The 1F7idiotype was detected on anti-gp120 and anti-gp41 Abs from
15/19 and 18/19 individuals, respectively (Fig. 1B). Figure 1C
illustrates the breakdown of the detection of the 1F7-idiotype
on Abs from individuals infected with each clade.
We next determined whether HIV Env from subtype B induced and selected Abs capable of recognizing epitopes that are
relatively conserved between HIV subtype B variant and nonsubtype B Env glycoproteins. We screened plasma samples
from five HIV subtype B-infected individuals, who were attending the Melbourne Sexual Health Clinic (Melbourne,
Australia), for the presence of Abs recognizing gp140 fromvarious HIV subtypes, including subtypes A, AE, and B. As
well, we screened these cross-clade reactive Abs for the presenceof the 1F7-idiotype. Procurement of the gp140 from subtypes A,
AE, and two B viruses has been previously described.22 The
only difference between the ELISA protocol utilized for theseexperiments and the one used for the experiments depicted inFig. 1 is the antigens coated on the plate. All five individuals had
Abs that reacted with the four HIV gp140s tested (Data notshown). Subtype cross-reactive Abs bound to each of these781
gp140 antigens were 1F7-idiotype positive (Fig. 2). These results
demonstrate that infection with a single HIV subtype can induce
Env-specific 1F7-idiotype-positive Abs that can recognize Env
epitopes from diverse HIV subtypes. As these Env-specific 1F7idiotypic Abs developed in the absence of exposure to theseother HIV subtypes, the 1F7-idiotypic Abs can recognize Env
epitopes conserved across multiple HIV subtypes.
This study is the first to demonstrate the presence of the 1F7idiotype on Abs induced during infection with diverse HIV
subtypes. This observation is important for understanding theability of HIV to evade humoral immune responses. A common
feature of infections with diverse HIV subtypes is the inability of
the humoral immune response to efficiently respond to actively
replicating ACV.1,2 1F7-idiotypic Abs develop in primary infection and are maintained and expanded during chronic
infection.11 It has been proposed that the persistence of 1F7idiotype-positive Abs against early viral variants negatively
impacts the induction of Abs able to contribute to the control of
ACV. This contention is supported by data demonstrating thatsuppression of 1F7-idiotypic Abs allows development of newAbs better able to neutralize ACV.12,13 As such, the observationthat 1F7-idiotypic Abs are present in infections with diverse HIV
subtypes suggests that the repertoire freeze associated with thepresence of the 1F7-idiotype on antiviral Abs may be an important factor limiting the efficacy of humoral immune responses in a wide array of HIV infections.
This report also supports the contention that Env-specific1F7-idiotypic Abs induced during infection with one HIV

FIG. 2. Investigation of 1F7-idiotype expression on anti-HIV Env glycoprotein antibodies (Abs) exhibiting cross-clade
reactivity. ELISA plate wells were coated with 200 ng/well of gp140 proteins from HIV clades A (strain: 92UG037), A/E(strain: 93TH966), and B (strains: AD8 and NL4.3). Plasma samples from five HIV subtype B-infected individuals were added
to the wells followed by the anti-1F7-idiotypic murine IgM or the TEPC183 isotype control. The binding of murine IgM wasdetected with an HRP goat antimouse IgM Ab. The bar graphs depict the relative levels of binding of anti-1F7-idiotypic and
TEPC183 Abs to gp140-bound plasma-derived Abs from five HIV subtype B-infected individuals.

782
subtype are capable of recognizing epitopes on Env fromother subtypes. The first evidence of this phenomenon wasprovided by the demonstration that a series of six BnAbs bear
the 1F7-idiotype.11 Here, we show that 1F7 recognizes Abs
within the general polyclonal antiviral Ab response to multiple
HIV subtypes. This observation offers insight into pathways
and selection mechanisms, such as early cross-clade crossreactivity, repeated selection within an idiotypic framework,and high levels of somatic hypermutation, that may be involved in generating potentially protective Ab responses.
Although the phenomenon of repertoire freeze is undesirable during chronic viral infection, the observation that 1F7idiotypic Abs are reactive with Env from different HIV clades
and that BnAbs develop from the 1F7-idiotypic node suggeststhat a greater understanding of repertoire freeze may aid inunderstanding how BnAbs develop during the natural history of HIV infection and how to induce protective Abs via
vaccination. We hypothesize that early 1F7-idiotypic Abs
represent precursors to BnAbs, and that repeated rounds of
selection and somatic hypermutation drive their development
into BnAbs. Future experiments simultaneously mapping theevolution of viruses and 1F7-idiotypic Abs in individuals whodevelop BnAbs would test this hypothesis.
This study demonstrates that Abs raised during diverse
HIV infections are selected from a common idiotypic node.
These results suggest the phenomenon of idiotypic-driven
repertoire freeze is common among infections with diverse
HIV-1 subtypes. Idiotype-associated repertoire freeze may
serve as a pragmatic framework for understanding both theability of HIV to evade humoral immune responses and theselection of potentially protective Abs. As BnAbs have been
demonstrated to develop from the 1F7-idiotypic repertoire,
vaccine constructs should be designed to select and develop
antiviral Abs from this repertoire. Understanding the role of
1F7-idiotypic repertoire freeze in selecting anti-HIV Abs may
contribute toward the design of an effective HIV vaccine, as
broadly reactive Ab responses appear inextricably linked tothis repertoire.
Acknowledgments
This work was supported by Canadian Institutes for Healthresearch grants MOP-79515, MOP-93770, and HPR-85528 andthe Fonds de la Recherche en Santé du Québec (FRSQ) AIDS
and Infectious Disease Network. J-P.R. and N.F.B. are members of the Research Institute of the McGill University HealthCentre, an institution funded in part by the FRSQ. M.S.P is
supported by a CIHR Vanier Scholarship, and was a visiting
scholar at the University of Melbourne with the support of a
CIHR Michael Smith Foreign Study Supplement.
Author Disclosure Statement
M.S.P. and M.D.G. are both members of the scientific advisory board for Network Immunology Incorporated.
References
1. Richman DD, Wrin T, Little SJ, and Petropoulos CJ: Rapid
evolution of the neutralizing antibody response to HIV type1 infection. Proc Natl Acad Sci USA 2003;100(7):4144–4149.
2. Wei X, Decker JM, Wang S, et al.: Antibody neutralizationand escape by HIV-1. Nature 2003;422(6929):307–312.

PARSONS ET AL.
3. Chung AW, Isitman G, Navis M, et al.: Immune escapefrom HIV-specific antibody-dependent cellular cytotoxicity(ADCC) pressure. Proc Natl Acad Sci USA 2011;108(18):7505–
7510.
4. Kohler H, Muller S, and Nara PL: Deceptive imprinting in
the immune response against HIV-1. Immunol Today 1994;
15(10):475–478.
5. Lambert PH, Liu M, and Siegrist CA: Can successful vaccines teach us how to induce efficient protective immune
responses? Nat Med 2005;11(4 Suppl):S54–S62.
6. Bende RJ, van MF, Triesscheijn M, et al.: Germinal centers inhuman lymph nodes contain reactivated memory B cells.
J Exp Med 2007;204(11):2655–2665.
7. Rada C, Gupta SK, Gherardi E, and Milstein C: Mutation and selection during the secondary response to 2phenyloxazolone. Proc Natl Acad Sci USA 1991;88(13):5508–5512.
8. Breden F, Lepik C, Longo NS, et al.: Comparison of antibody
repertoires produced by HIV-1 infection, other chronic andacute infections, and systemic autoimmune disease. PLoS
One 2011;6(3):e16857.
9. Wang H, Muller S, Zolla-Pazner S, and Kohler H: Human
monoclonal and polyclonal anti-human immunodeficiency
virus-1 antibodies share a common clonotypic specificity.
Eur J Immunol 1992;22(7):1749–1755.
10. Muller S, Margolin DH, and Min G: An HIV-1 infectionrelated idiotype/clonotype (1F7) is expressed on antibodies
directed to envelope glycoprotein in simian immunodeficiency virus- and chimeric simian/human immunodeficiency virus-infected rhesus monkeys. Hybridoma 1997;
16(1):17–21.
11. Parsons MS, Rouleau D, Routy JP, et al.: Selection of humananti-HIV broadly neutralizing antibodies occurs within thecontext of frozen 1F7-idiotypic repertoire. AIDS 2011;25(10):1249–1264.
12. Muller S, Margolin DH, Min G, et al.: Stimulation of antiviral
antibody response in SHIV-IIIB-infected macaques. Scand J
Immunol 2001;54(4):383–395.
13. Muller S, Margolin DH, Nara PL, Alvord WG, and Kohler H:
Stimulation of HIV-1-neutralizing antibodies in simian HIVIIIB-infected macaques. Proc Natl Acad Sci USA 1998;
95(1):276–281.
14. Stamatatos L, Morris L, Burton DR, and Mascola JR: Neutralizing antibodies generated during natural HIV-1 infection: Good news for an HIV-1 vaccine? Nat Med 2009;15(8):866–870.
15. Euler Z, van Gils MJ, Bunnik EM, et al.: Cross-reactive
neutralizing humoral immunity does not protect fromHIV type 1 disease progression. J Infect Dis 2010;201(7):1045–1053.
16. Burton DR, Hessell AJ, Keele BF, et al.: Limited or no protection by weakly or nonneutralizing antibodies against
vaginal SHIV challenge of macaques compared with astrongly neutralizing antibody. Proc Natl Acad Sci USA
2011;108(27):11181–11186.
17. Hessell AJ, Rakasz EG, Poignard P, et al.: Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum
neutralizing titers. PLoS Pathog 2009;5(5):e1000433.
18. Parren PW, Marx PA, Hessell AJ, et al.: Antibody protects
macaques against vaginal challenge with a pathogenic R5
simian/human immunodeficiency virus at serum levels
giving complete neutralization in vitro. J Virol 2001;75(17):8340–8347.

IDIOTYPIC RESTRICTION OF HIV ANTIBODIES
19. Veazey RS, Shattock RJ, Pope M, et al.: Prevention of virustransmission to macaque monkeys by a vaginally appliedmonoclonal antibody to HIV-1 gp120. Nat Med 2003;9(3):343–346.
20. Haynes BF, Moody MA, Liao HX, Verkoczy L, and Tomaras
GD: B cell responses to HIV-1 infection and vaccination:
Pathways to preventing infection. Trends Mol Med 2011;17(2):108–116.
21. Zhou T, Georgiev I, Wu X, et al.: Structural basis for broad
and potent neutralization of HIV-1 by antibody VRC01.
Science 2010;329(5993):811–817.
22. Center RJ, Wheatley AK, Campbell SM, et al.: Induction of
HIV-1 subtype B and AE-specific neutralizing antibodies in783
mice and macaques with DNA prime and recombinant gp140
protein boost regimens. Vaccine 2009;27(47):6605–6612.

Address correspondence to:
Nicole F. Bernard
Research Institute of the McGill University Health Centre
Montréal General Hospital
1650 Cedar Avenue Rm. C10-160
Montréal, Quebec H3G 1A4
CanadaE-mail: nicole.bernard@mcgill.ca