HIV Type 1 Subtype A Envelope Genetic Evolution in a Slow Progressing Individual with Consistent Broadly Neutralizing Antibodies

SEQUENCE NOTES

HIV Type 1 Subtype A Envelope GeneticEvolution in a Slow Progressing Individual

with Consistent Broadly Neutralizing Antibodies

Tessa Dieltjens,1 Nathalie Loots,1 Katleen Vereecken,1 Katrijn Grupping,1 Leo Heyndrickx,1

Emmanuel Bottieau,2 Guido Vanham,1,3 David Davis,4 and Wouter Janssens1

Abstract

Studies of viruses taken from individuals with broad cross-neutralizing antibodies against primary isolates mayreveal novel antibody specificities and their associated epitopes that could be useful for immunogen design. Wereport on the Env antigenic variability of a slow progressing HIV-1 subtype A-infected donor with consistentbroad cross-neutralizing antibodies during the second decade of disease progression after vertical transmission.The Env evolution is characterized by a genetic shift to variants with altered V1–V5 loop sequences, marked byconsecutive changes in V1, V4–V5, and C3 and largely conserved V2 and V3 loop sequences. Major V1 Envsequence expansion, variation by a duplication event, and cumulative addition of cysteine residues and potentialN-glycosylation sites over time may contribute to escape from antibody pressure directed to Env receptordomains by changing the exposure of neutralization-sensitive epitopes. Conservation of functional epitopes maycorrelate with the continued presence of broad cross-neutralizing antibodies.

Amajor goal in the generation of an effective HIV-1vaccine is to induce antibodies that are capable of po-

tently neutralizing the broad spectrum of HIV-1 variants.Using in vitro neutralization assays some rare individualshave been identified whose plasma antibodies are capable ofbroadly neutralizing representatives of the major HIV-1 sub-types and circulating recombinant forms (CRFs).1 Studies ofviruses of these individuals may reveal novel antibody spe-cificities and their associated viral epitopes that could beuseful for vaccine immunogen design.

In a recent study of Zhang et al. broadly neutralizing anti-bodies (bNAbs) were elicited in rabbits that were immunizedwith gp140 (designated R2), selected for its unusual CD4-independent phenotype and the bNAb response of thedonor’s plasma.2 In the present study we report on the evo-lution of Env amino acid variation in follow-up samples(1997–2007) of a Rwandese adolescent (hereafter referred asITM1), infected by mother-to-child transmission in 1986. BothITM1 and his mother (MOT) first consulted a physician atthe Institute of Tropical Medicine (ITM; Antwerp, Belgium)on arrival in Belgium in 1994. During follow-up the viral loadof ITM1 fluctuated between 15,000 and 25,000 HIV RNAcopies=ml while his CD4 T cell count gradually decreased

from 520 to less then 300 cells=mm3 (Table 1). Patient ITM1has never received antiretroviral therapy.

MOT started antiretroviral therapy shortly after arrival atour Institute because her CD4 T cell count was 130 cells=mm3

at that time. Patient ITM1 was selected in a previous study forthe highest degree of cross-neutralization of his plasma in aperipheral blood mononuclear cell (PBMC) neutralizationassay with heterologous viruses as described by Davis et al.,3

neutralizing 75–100% of four representatives of each clade (A,B, C, D, as well as CRF01_AE) analyzed. The bNAb responseobserved for ITM1 was conserved for all follow-up plasmasamples.

The cross-neutralizing activity was confirmed by Mono-gram Biosciences in a pseudovirus=U87 neutralization assay.4

The continued presence of broad cross-neutralizing anti-bodies may indicate the inability of the virus to escape fromthe immunologic pressure due to the compulsory conserva-tion of functional epitopes. A study of the viral evolution andthe autologous humoral responses could be informative aboutthe specific virus–host interactions. To determine the evolu-tion of the envelope in this subtype A-infected adolescent,during the second decade after infection, we performed ananalysis of the genetic changes in the virus isolates extracted

1Department of Microbiology, Unit of Virology, Institute of Tropical Medicine, Antwerp, Belgium.2Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.3Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.4Department of Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands.

AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 25, Number 11, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=aid.2008.0161

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from several follow-up plasma samples. Further, we describethe amino acid changes in the gp120 region and discuss theautologous neutralization data over a period of 10 years.

The complete env sequence was amplified by reverse tran-scriptase polymerase chain reaction (RT-PCR) starting fromplasma RNA. The env fragment obtained by nested PCR wascloned in an expression vector. Cotransfection of 293T cellswith an HIV-1 Env expression vector and pNL4-3.Luc.R-E�5

(obtained from NIH AIDS Research and Reference ReagentProgram and contributed by Nathaniel Landau) was evalu-ated for the generation of luciferase-encoding HIV-1 virionspseudotyped with the desired HIV-1 Env proteins. Cloned envgenes were sequenced and analyzed using DNASTAR soft-ware (Lasergene, WI). Autologous neutralization was mea-sured by a reduction in luciferase gene expression after asingle round of infection of TZM-bl cells with pseudotypedviruses. ID50 was calculated as the reciprocal plasma dilutioncausing a 50% reduction of relative light units compared tothe virus control.6

The newly obtained env sequences from ITM1 and MOTvirus isolates were aligned (Clustal W) with representativesof all subtypes as suggested on the HIV Databases website.Phylogenetic analysis positioned ITM1 and MOT sequences ina monophyletic cluster in subtype A (Fig. 1). Env nucleotidedistances were as follows: Intra-ITM1: 0.1–7.2%; ITM1-MOT:9.3–10.5%. Two distinct clusters of ITM1 sequences were ap-parent, each harboring sequences that originated from the2001 plasma sample (¼ ITM1-01). Isolates from 1997 (¼ ITM1-97) (Env nucleotide distances: 0.3–1.1%) clustered with ITM1-01 clones 1, 54, and 58 (cluster I; distances 0.1–0.3%); the rangeof distances for these ITM1-97 and ITM1-01 cluster I cloneswas 1.8–2.3%. ITM1-01 clones 5 and 59 (cluster II; distances:0.7–1.8%) clustered with later time samples with a distancerange of 3.0–6.3%. Env nucleotide distances between ITM1-01cluster I and II clones were between 5.2% and 5.6%.

Variability over time in the HIV-1 env gene lengths wasexamined in the infectious pseudotyped viruses. MOT-94clone 20 (2607 bp) was within the range of subtype A envreferences (2511–2610 bp) available from the HIV Databases(http:==www.hiv.lanl.gov). The length of the ITM1 clones

gradually increased from 2574 bp (ITM1-97; 1997) to 2604 bp(ITM1-01; 2001), 2622 bp (ITM1-03; 2003), and 2655 bp (ITM1-05; 2005), and eventually remained more or less constant inthe last time point with 2652 bp (ITM1-07; 2007). The expan-sion of the Env was mainly due to a major evolution ofthe V1 loop length (Fig. 2). V1 loop diversity in ITM1 in-cludes cumulative addition of cysteine residues, potentialN-glycosylation sites (PNGS), and a duplication event.

As compared to ITM1-97 clones, either one or two cysteineswere added in ITM1-03 clones; later samples had two moreadditional cysteine residues. An amino acid sequence CA-NYTGTAYNC present in ITM1-03 was duplicated in ITM1-05and retained as length variation in future samples (Fig. 2). Thenumber of PNGS gradually increased from ITM1-97 andITM1-01 (n¼ 3) to ITM1-05 (n¼ 7), and decreased in ITM1-07(n¼ 3). A continuous increase of the V1 loop length was ob-served, starting from 24 amino acids (aa) in ITM1-97 and ex-panding to 50 aa in ITM1-05 and ITM1-07 (Table 1). Within theV2 and V3 loop only a small amount of variation is seen overtime. The V2 loop shows a relocation of glycosylation sites incluster II isolates, but no expansion of the number of PNGS(n¼ 2 or 3) or V2 sequence length (aa¼ 42 or 43). The V3region of cluster II variants is highly conserved and an iden-tical length (aa¼ 35) and number of PNGS (n¼ 1) is found.

A remarkable aa change in the tip of the V3 loop is seen inITM1-07 (GPGQ?GPGR). This change is not associated witha switch in coreceptor usage, as all the infectious pseudotypedviruses of ITM1 are using CCR5 as the coreceptor. The clusterI isolate ITM1-97 clone 13 differs from the HIV-1 group M V3loop consensus at a highly conserved position 299 (HXB2 Envnumbering, HIV Databases) where a leu (L) is present insteadof pro (P) (Fig. 2). The diversity in the V4 and V5 loop is aresult of substitutions, insertions, and deletions that have onlya small influence on the length and number of PNGS in theseregions (length¼V4: 30–32 aa; V5: 7–11 aa; PNGS¼V4: 3–5,V5: 1–2). The C1–C5 region is highly conserved; only the C3region shows some variation over time.

To determine whether the evolution of Env correlated withthe development of HIV-1 antibodies in ITM1, we analyzedthe neutralization sensitivity of the infectious pseudotyped

Table 1. Clinical Data and Neutralizing Antibody Responses from Follow-up ITM1 Plasma Samples

Subject plasma ITM1-97 ITM1-03 ITM1-05 ITM1-07

Viral load (copies=ml) 14,629 23,800 16,200 20,500CD4 count (cells=mm3) 523 243 237 297

Autologous neutralization (ID50)a V1 loopb

Pseudoviruses (PV)Length (aa) PNGS

PV MOT-94_20 <25 39 44 69 27 4PV ITM1-97_13|I <25 32 31 28 24 3PV ITM1-01_59 25 43 40 39 30 3PV ITM1-03_03 II <25 <25 38 44 35 4PV ITM1-05_25 <25 <25 <25 31 50 7PV ITM1-07_12 <25 <25 <25 <25 50 3

Heterologous neutralization (ID50)a

Control pseudoviruses

PV SF 162 (subtype B) 1533 543 698 713PV 92BR025 (subtype C) 962 248 309 430

a50% neutralization titers measured in a pseudovirus assay using TZMbl cells.6bLength (¼number of amino acids) and number of PNGS (¼potential N-linked glycosylation sites) within the V1 loop.

1166 DIELTJENS ET AL.

0.1

91

79 ITM1-03_7 ITM1-03_4

100

100

96100

91

100

97

86

ITM1

ITM1ITM1-07_12*

ITM1-05_25*ITM1-07_5

ITM1-04_7

ITM1-03_5ITM1-04_5

ITM1-03_3*

ITM1-04_2

100

100

100

100

100

100

100

Cluster II

Cluster I

-07_10

ITM1-97_16ITM1-97_12

97 13*

ITM1-01_54 ITM1-01_1ITM1-01_58 ITM1-01_5ITM1-01_59*

A1_93RW037A A1_92RW025A

A1_UG031 A1_RW_SF1703

J SE7887

97

100

98

100

98

100

MOT

ITM1- _ITM1-97_15

MOT-94_27 MOT-94_20*

G_DRCBL G_HH8793_12_1

_J_SE7022

G_SE6165 G_92NG083 D_A280

D_94UG114 D_ELI

100

97100

100

99

98

90

C ETH2220

B_671_00T36 B_1058_11

C_SK164B1

D_01CM_4412HAL

B_HBX2 B_BK132

C_BR025-d C_95IN21068

100

100

10096

91

C_ETH2220

F2 MP257

H_VI991 H_VI997

H_056 K_EQTB11C

K_MP535 F2_CM53657 F2_02CM_0016BBY

100

79100

100100

100

100

F1_MP411 F2_MP255

F1_93BR020_1

F2_MP257

F1_VI850 F1_FIN9363

100

96

100

FIG. 1. Phylogenetic tree based on HIV-1 env sequences. Tree topologies were inferred by neighbor joining. The number oftrees out of 1000 replications supporting a particular phylogenetic group in more than 70% is placed alongside the nodeconsidered. Expression vectors with functional Env are indicated by an asterisk.

viruses using ITM1 plasma of four different time points(ITM1-97, ITM1-03, ITM1-05, and ITM1-07) (Table 1). Overall,we observe a low potency of the antibodies to neutralize theautologous virus (ID50< 100). Within most of the time pointsanalyzed, the titers against earlier virus were greater thanagainst contemporaneous or later virus, indicating a changein immune pressure and viral escape. Compared with autol-ogous viruses, higher ID50 titers against the heterologouscontrol viruses (SF162 and 92Br025) are seen for all follow-upITM1 plasma samples.

Our data show the longitudinal analysis of the HIV-1subtype A envelope evolution after mother-to-child trans-mission. Changes in the V1, V4, and V5 loop imply that thesevariable loops are a likely target of immunological or repli-cative selective pressure during virus evolution or that theyare at least involved in protecting a more conserved functionalregion. The length and composition of the V1 loop, includingintroduction of cysteine residues that may participate in ad-ditional or alternative loop formation, as well as the posi-tioning of the N-glycosylation sites, may have an impact onthe orientation of the V1 loop. Several studies suggest thatV1and V2 loops shield conserved neutralization-sensitive re-gions in the Env glycoprotein, including the V3 loop and theCD4 binding site.7–11

Changes in V1 are known to influence the neutralizationsensitivity of HIV-1 subtype A.9 The C3 region was previouslyshown to be a target of autologous neutralizing antibodies in

subtype C-infected patients12; in addition, here we observeimmunologic pressure on this region, manifested by severalamino acid substitutions in the different sequences. Theautologous neutralization data of our patient ITM1 suggest acontinuous escape of the virus from antibody pressure over10 years. The de novo humoral responses against early pseu-doviruses persisted in being relatively low. A genetic shiftoccurred in 2001, 15 years postinfection, with selection ofcluster II variants of ITM1-01. Sequence data of later timepoints suggest suppression of cluster I viruses.

Overall, our data indicate changes in V1, V4–V5, and C3,which may contribute to viral escape. The evolving humoralimmune response remains rather low in potency againstthe autologous virus; therefore the contribution of the bNAbresponses to the slow disease progression observed overthe course of infection of ITM1 is unclear. Other factorssuch as viral fitness, cellular immunity, and host factors maybe involved in the slow disease progression after HIV-1infection.13–15

Accession Numbers

The HIV-1 env nucleotide sequence data were deposited inthe EMBL, GenBank, and DDBJ nucleotide sequence data-bases under the following accession numbers: FM165626–FM165647. For this article the GenBank nomenclaturewas converted into VI1383¼MOT-94; PIC771¼ ITM1-97;

FIG. 2. Amino acid alignment of gp120 from ITM1 follow-up and MOT-94 pseudoviruses. PNGS are highlighted in gray,dots are included for alignment purposes, additional cysteines are bold, and duplication is underlined. Sequence numberingfollows the HxB2 convention. I and II indicate cluster I and cluster II viruses.

1168 DIELTJENS ET AL.

PIC13072¼ ITM1-01; VI2809¼ ITM1-03; VI2992¼ ITM1-04;VI3050¼ ITM1-05; and VI3196¼ ITM1-07.

Acknowledgments

This project was supported by the Research Foundation,Flanders (FWO project G 0302.01). Tessa Dieltjens is sup-ported by a Ph.D. scholarship from the Institute for the Pro-motion of Innovation by Science and Technology in Flanders(IWT). We thank the ITM AIDS Reference Centre (ARC) andLaboratory (ARL) for patient follow-up and clinical data.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Tessa Dieltjens

Department of MicrobiologyInstitute of Tropical Medicine

Nationalestraat 1552000 Antwerp, Belgium

E-mail: [email protected]

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