Advance Publication by J-STAGE Japanese Journal of Infectious Diseases
Impact of maraviroc-resistant mutation M434I in the C4 region of HIV-1 gp120 on sensitivity to antibody-mediated neutralization
Samatchaya Boonchawalit, Shigeyoshi Harada, Noriko Shirai, Hiroyuki Gatanaga, Shinichi Oka, Shuzo Matsushita, and Kazuhisa Yoshimura
Received: June 25, 2015. Accepted: July 2, 2015 Published online: July 10, 2015 DOI: 10.7883/yoken.JJID.2015.310
Advance Publication articles have been accepted by JJID but have not been copyedited or formatted for publication.
Impact of maraviroc-resistant mutation M434I in the C4 region of HIV-1 gp120 on sensitivity to antibody-mediated neutralization
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Shinichi Oka3, Shuzo Matsushita1, Kazuhisa Yoshimura1, 2, 3*
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Samatchaya Boonchawalit1, 2, Shigeyoshi Harada2, Noriko Shirai1, Hiroyuki Gatanaga3,
Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto
860-0811, Japan, 2AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan, 3AIDS Clinical Center, National
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Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
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Running head: Impact of MVC-resistant Env to NAb-sensitivity
* Correspondence to:
Kazuhisa Yoshimura MD, PhD,
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AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama,
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Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-4582-2814 Fax: +81-3-5285-1165 E-mail:
[email protected]
Abstract: 136 words, Main text: 3,508 words, 5 figures and 3 tables
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This work was presented in part at the 20th International AIDS Conference, Melbourne,
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Australia, 20-25 July 2014, Abstract MOPE010.
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Keywords:
HIV-1, Maraviroc resistant mutations, Neutralizing antibody, Enhancing neutralization,
（著者名）
サマッチャヤ1, 2、原田 恵嘉2、白井 慎一3、松下 修三1、吉村 和久1, 2, 3*
ブンチャワリト 岡
1
〒862-0081
2
〒162-8640
熊本県熊本市中央区本荘 2-2-1 東京都新宿区戸山 1-23-1
ce
ター
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（所属）
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M434I mutation
〒162-8655
東京都新宿区戸山 1-23-1
教子1、潟永
博之3、
熊本大学エイズ学研究センター
国立感染症研究所
エイズ研究セン
国立国際医療研究センター
エイズ
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治療・研究開発センター
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SUMMARY We previously reported that a maraviroc (MVC) resistant HIV-1 generated using in
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vitro selection exhibited high sensitivity to several neutralizing monoclonal antibodies
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(NMAbs) and autologous plasma IgGs. The MVC-resistant variant acquired four sequential mutations, T297I, M434I, V200I and K305R in gp120. In this study, we examined the mutation most responsible for conferring enhanced neutralization
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sensitivity of the MVC resistant variant to several NMAbs and autologous plasma IgGs. The virus with the first resistant mutation, T297I, became sensitive to all NMAbs compared to the passage control virus. The neutralization sensitivity greatly increased
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following the second mutation M434I in the C4 region. The M434I mutation conferred the greatest neutralizing sensitivity among four MVC-resistant mutations, and the single M434I mutation was sufficient for the enhanced neutralization by NMAbs, autologous
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plasma IgGs and heterologous sera relative to the parental virus.
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INTRODUCTION CCR5 inhibitors work by allosterically altering the conformation of CCR5 at the
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target cell surface, thereby disrupting its interaction with HIV gp120 (1, 2). Although
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MVC, which is the first and currently only approved CCR5 antagonist for the treatment of CCR5-tropic (R5) HIV-1 infected patients and another CCR5 inhibitor, vicriviroc (VCV) can efficiently suppress HIV-1 replication, resistant variants can arise both in
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vitro and in vivo using drug-bound CCR5 for cellular entry (3-18). The presence of MVC resistant HIV-1 variants may be due to the outgrowth of pre-existing CXCR4-tropic (X4) viruses (19, 20) or the selection of MVC resistant R5 mutants (12,
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17). MVC resistant mutations are commonly detected in the gp120 V3 loop and are strain-specific (3, 12, 16, 21, 22).
The majority of HIV-infected individuals mount a neutralizing antibody (NAb)
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response against the infecting virus envelope (Env). However, the virus can evade this antibody
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autologous
response
by
several
means,
including
generation
of
epitope-specific mutations, decoration with a dense glycan shield, hypervariable loops that mask conserved Env features and high intrinsic conformational dynamics that render it a poorly defined antigen (23). Interestingly, the increased accessibility to epitopes of some neutralizing monoclonal antibodies (NMAbs) has been reported for
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entry inhibitor resistant variants. Other CCR5 inhibitors, AD101 or VCV escape viruses were more sensitive to anti-Env NMAbs and sera from some HIV-1 infected patients
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compared to the parental virus (24). Moreover, the MVC resistant virus obtained from a
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patient who experienced virologic failure while on MVC combination therapy showed neutralization sensitivity to anti-CD4 binding site (CD4bs) b12 and gp41 2F5 NMAbs and HIV-IgG (13). These previous studies imply that mutation(s) in Env that confer
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drug resistance may increase the accessibility to epitopes for neutralizing antibodies. In our recent study, we induced in vitro selected-MVC resistant virus using the PM1/CCR5 cell line. Four accumulated MVC-resistant mutations, T297I, M434I,
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V200I and K305R, were detected sequentially. The MVC resistant virus became more sensitive to Env NMAbs: anti-V3, CD4 induced epitope (CD4i) and CD4bs and autologous plasma IgGs compared to the passage control (PC) virus (22). However, it is
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still not clear which single or combination of mutation(s) affect the accessibility of
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NMAbs and autologous plasma IgGs to the epitopes in Env. In this study, we assessed which single or combination of mutation(s) confers the
greatest enhanced neutralization sensitivity of the MVC resistant variant. We constructed several infectious clones with single or a combination of the four MVC resistant mutations, V200I, T297I, K305R and M434I, and determined the effect on the
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neutralization sensitivity to three different kinds of NMAb, autologous plasma IgGs and
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heterologous sera from HIV-1 infected subjects.
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MATERIALS and METHODS
Viruses: The in vitro selected-MVC resistant and PC infectious clones were previously described (22). Briefly, MVC resistant virus was generated by culturing a
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HIV-1 subtype B R5 tropic primary isolate (KP-5) in PM1/CCR5 cells with increasing concentrations of MVC for 48 passages. The same HIV-1 primary isolate was also
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cultured in PM1/CCR5 cells without MVC for 48 passages to obtain the PC virus.
Construction and expansion of Env chimeric infectious clones: The gp160 region of the in vitro selected MVC resistant, and PC were amplified using primers EnvFv
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(5-AGCAGAAGACAGTGGCAATGAGAGCGAAG-3)
and
EnvR
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(5-AGCAGAAGACAGTGGCAATGAGAGCGAAG-3). Each point mutation was introduced into the MVC resistant gp160 region by site-directed mutagenesis, using PrimeSTAR mutagenesis Basal Kit (TAKARA Bio Inc, Shiga, Japan) and the specific mutagenesis primer sets.
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All MVC resistant and PC Env infectious clones were constructed from the pNL4-3 proviral plasmid by overlapping PCR as described previously (22, 25). Briefly, fractions
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of the NL4-3 provirus containing EcoRI restriction site (nucleotides 5284 to 6232) and
sets;
NL(5284)F
NL(6232)Rv
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XhoI restriction site (nucleotides 8779 to 9045) were amplified with specific primers (5’-GGTCAGGGAGTCTCCATAGAATGGAGG-3’)
(5’-CTTCGCTCTCATTGCCACTGTCTTCTGCT-3’)
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and EcoRI
NL(9045)R
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fragment and NL(8779)F (5’- GCTATAAGATGGGTGGCAAGTGGTCAAAA-3’) and (5’-GATCTACAGCTGCCTTGTAAGTCATTGGTC-3’)
for
XhoI
fragment. Overlapping PCR was used to join the mutated and wild type gp160 coding
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sequence to the EcoRI and XhoI fragments that had been amplified from pNL4-3. The mutated and PC gp160 containing EcoRI and XhoI enzyme restriction sites were then cloned into EcoRI/XhoI digested-NL4-3 proviral plasmid to generate the recombinant
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Env/NL4-3 proviral DNA. The Env region from each constructed infectious clone was
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sequenced using a 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA). 293T cells were transfected with KP-5-PC and mutated KP-5 Env/NL4-3 proviral DNAs using LipofectamineTM 2000 transfection reagent (Invitrogen, Carlsbad, CA). Supernatants were collected 48 hours after transfection and stored at -80oC until use.
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Viral titer was determined using the Cell Counting Kit-8 assay (WST-8 assay, Dojindo
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Laboratories, Kumamoto, Japan).
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Cells and culture conditions: The CD4-positive T-cell line PM1 expressing high CCR5 co-receptor, PM1/CCR5, was kindly provided by Dr. Yosuke Maeda (22, 26). PM1/CCR5 cells were maintained in RPMI-1640 (Sigma-Aldrich, St. Louis, MO)
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supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, South Logan, UT) and 0.1 mg/ml of G418 (Nakarai Tesque, Kyoto, Japan). 293T cells were
maintained
in
Dulbecco’s
modified
Eagle’s
medium
(Sigma-Aldrich)
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supplemented with 10% heat-inactivated fetal bovine serum.
Antibodies and inhibitors: Anti-V3 NMAbs: KD-247 (27, 28) and 447-52D (29),
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anti-CD4bs: 0.5 (3D6) (30, 31) and b12 (32) and anti- CD4i: 4E9C (30) were used for susceptibility
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neutralization
assays.
KD-247
was
kindly
provided
by
The
Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). 0.5∂ and 4E9C were established at the Center for AIDS Research, Kumamoto University (Kumamoto, Japan). The b12 and 447-52D NMAbs were purchased from Polymun Scientific
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Immunbiologische Forschung GmbH (Klosterneuburg, Austria). The CCR5 inhibitor
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MVC was kindly provided by Pfizer (Groton, CT).
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Patient plasma IgGs and sera: Autologous plasma samples were collected and purified using Protien A-Sepharose (Affi-gel Protein A; Bio-Rad,Hercules, CA) (22, 30, 33). The purified plasma IgGs were designated KP-5-IgG-1, -2, -5 and -7.
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Sera from HIV-1 infected patients were provided by the National Center for Global Health and Medicine（Tokyo, Japan）. Heterologous sera were obtained from ART naïve patients. Phlebotomy was performed following signed informed consent in accordance
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with the study protocol. The study was reviewed and approved by the Ethics committee for clinical research & advanced medical technology at the Kumamoto University Medical School (Kumamoto, Japan) and the National Center for Global Health and
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Medicine.
Neutralization susceptibility assay: The sensitivity of the chimeric infectious viruses
to MVC and anti-HIV-1 Env antibodies was determined using PM1/CCR5 cells (22). Briefly, 2x103 PM1/CCR5 cells were infected with 100 median tissue culture infective dose (TCID50) chimeric Env/NL4-3 infectious clones in U-bottom 96-well microplates
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in the presence of various concentrations of MVC, NMAbs, autologous plasma IgGs or heterologous sera, and incubated at 37oC for 7 days. The IC50 was determined using the
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Cell Counting Kit-8 assay. A reciprocal neutralization titer of >100 was used to define
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sensitivity.
Statistical analysis: Neutralizing antibody titers were summarized using geometric
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mean titers and statistical comparisons were performed using Student’s t test. A p value
