BORAr..~G FRGS - P1 (R) I Kod Projek

A PHASE

PROGRESS REPORT FUNDAMENTAL RESEARCH GRANT SCHEME (FRGS)

Laporan Prestasi Skim Geran Penyelidikan Fundamental (FRGS) Pindaan 212013

: 112011

YEAR : 2014

RESEARCH TITLE : Nef Antibody Phage Display as a Screening Tool for Novel Therapeutics Against HIV

START DATE : June 2011 END DATE : July 2013 - Extended for 6 months (January 2014)

PROJECT LEADER Syed Atif Ali, PhD

PROJECT MEMBERS: 1. Professor Narazah M Yusoff (including GRA) 2. Professor Lokman M Noh

Project progress according to milestones achieved up to this period

Number of articlesl manuscriptsl books (Please attach the First Page of Publication)

Conference Proceeding (Please attach the First Page of Publication)

Intellectual Property (Please specify)

0-25% 26 - 50%

Indexed Journal

International

1. Teow SY, Mualif SA, Omar TC, Wei CY, Yusoff NM, and Ali SA. 2013. Production and Purification of Polymerization-Competent HIV -1 Capsid Protein p24 (CA) in NiCo21 (DE3) Escherichia coli. BMC Biotechnology. 10.1186/1472-6750-13-107 (Impact/actor 2.2).

Two more papers under preparation

Patent under preparation

51 -75% 76 -100%

100%

Non-Indexed Journal

National

C

Human Capital

Citizen

PhD Student

Master Student

Undergraduate Student

Total

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Number Others 1---------.---------1 (please specify)

On-going Graduated

Malaysian

1

2

Non Malaysian Malaysian

Non Malaysian

Budget Approved (Peruntukan diluluskan) Amount Spent (Jumlah Perbelanjaan)

RM 188,500 RM 188,4632

Balance (8aki) Percentage of Amount Spent (Peratusan Belanja)

RM 36.8 99.98%

,

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," .. -' -~~-~~.~~~' ~~ .. >..~:...:...~ ... ~' ~----~' :~--~' -~~---,,,,,,:,,,~---.~ .. --.. ~~~~~-~~, .. -----,~ j D Nef is an accessory protein of HIV-1 virus, which plays a critical role in the pathogenesis of AIDS. Nef augmer

the HIV infection by dysregulating the immune system of the infected human(s). Nef is present on the surface of the infected cells and it is also secreted in exosomes (Nef-exosomes) into intracellular spaces. Nef exerts it p~th,Qg~ni9 .~ffE!ct§ ,. wh_eJ:L HJV:-l[Jf~ft§c!. 9§.1!? .QJ. N~f:~?'<~~9JI1.~~ .. ~orne into contac;twith un-infected (by-stander) cells. Through its interaction with host-cell's CXCR4 receptor, Nef induces ap'6ptosls, and aSci result, 'populatlon of immune cells drops with time. Immune dysregulatory effects of Nef can be inhibited by preventing Nef from interacting with host cells via antibody, peptides, or chemical compounds.

The rationale of the proposed project was to identify the Nef motifs involved in the interaction with immune cells. We proposed to develop an antibody phage display library against Nef and use the library to identify Nef epitopes.

The project was divided into two aims (Aim-1A1B and Aim 2) to be completed in three years' time. However, the project was funded for two years. We did manage to complete Aim-1A and Aim~1B within two years as proposed. Below is a summary of what has been achieved in the past two years.

Aim-1: Identify the Nef Epitopes Exposed on the Surface of secreted Nef~vesicles and HIV 1~infected Cells.

Slop

IIU.

pMJ<B.HNef-SHis ~1q,

64kDa 49kDa

37 kDa

26 kDa

19 kDa

Fig. 2.A. Prokaryotic expression vector pMXB-Hnef-6His. B. Expression and purification of Nef-6His from E. coli Bl21. M: BenchMark'M Pre-Stained Protein Ladder - 41llj Lane 1: Nef - purified from cobalt resin onlYj Lane 2: Nef - passed through chitin beads onlYj Lane 3: Nef- purified through chitin beads and cobalt resin.

A. Production of monoclonal antibody phage library against HIV-1 Nef as screening tool to identify putative Nef motifs.

Conventionally, monoclonal antibodies (MAbs) are generated from hybridoma cells (antibody secreting S­cells fused with myeloma cells). Hybridoma generation is a time-consuming process and not suitable to generate libraries. Monoclonal Phage Display librarY (MPDL) is generated by harvesting antibody-encoding mRNA from the B-cells and then cloning these sequences to coat protein III of M13 phage. Resulting phages display 3-5 molecules of minimal antibody (single chain Fv) on their surface. A MPDL is composed

of 1x107-1x1010 unique monoclonal antibodies. It is this huge diversity of MPDL that makes it a powerful tool to identify potential motifs.

In order to develop anti-Nef MPDL, we first cloned the nef gene from HIV-1 (NL4.3) virus into a mammalian vector called pQBI. This vector meant to be used for DNA immunization of the Balb/C mice. We also included a 6-His tag on C-terminal of the nef gene. We named this vector pQBI-Nef-6His (see Fig. 1). This resulted in the dual use of this vector, to immunize the mice, and to express and purify Nef in mammalian cells such as HEK293.

For antibody screening purposes, we need a Nef-specific ELISA. Nef ELISA is not available commercially, so we developed our own. To develop Nef ELISA, the first step was to obtain highly purified Nef protein. Nef protein is available commercially but it is only 60-65% pure. Additionally, Nef protein is prone to self-aggregation· and has a short shelf-life. To develop a bacterial expression vector, we cloned the nef gene into the backbone of pMXB10 vector (NEB). In this vector, the nef-6His gene is expressed under the control of a T7 promoter. We named this vector pSA-HNef-6His (see Fig. 2A). Expression of HIV Nef in

3

bacterial host is challenging due to the presence of rare codons, E 2-

for which, the E. coli does not have tRNA. We solved this ~ problem by expressing nef gene in the presence of pACYC-RIL g 1-

plasmid, designed to provide tRNA for rare codons. With

Fig 1. Eukaryotic vector pQBI-Nef-6His.

•

•

• extensive optimization, we managed to produce up to 20mg/L of 98% purified Nef (see Fig. 28) in E. coli (we are submitting a full length paper on this work). We have used the purified Nef to develop Nef-ELISA with a sensitivity of 50pg (see Fig. 3).

O~--~I~--~I--~I--__ I~--~I--~I

a 10 100 200 300 400 sao Nef (pg/mL)

Fig. 3. Nef ELISA can detect as low as SOpg/mL Nef

Next step was to immunize the animals. Our initial proposal was

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107

1 2 3 4 5 6 Weeks

Fig. 4. Antibody titer of mice Immunized with Nef

to prime the animals with nef-encoding mammalian vector (pQBI-Nef) and boost with Nef.;exosomes. It proved difficult and very low· titers were obtained even after 6 boosts. We then decided to prime the animals with pQBI-Nef vector and boost with 1 aOug of purified Nef protein. This strategy worked, and we managed to obtain higher titers following 4 booster doses (see Fig. 4). Animals were sacrificed, and mRNA encoding MAb genes were harvested from the Trizol-treated spleens. The mRNA was converted into the cDNA by reverse transcription and variable heavy (VH) and light chain (VL) genes were amplified and assembled following SOE-PCR. The assembled genes were then cloned at SflllNotl in phage display vector called pHEN2. Ligated vector/insert was used to transform electrocompetent TG-1

cells. Successful clones were selected on LB agar plates sup'plemented with Ampicillin. This concludes the

cfu/mL. This gives ~s a moderate size library. E c:: o Zl ~ i-

S 0 .,. "'P" ... "T"' .,. • I Pre­pan

2 4 ChI. 5

Rounds of selected biopanning of phage-scFV

Next we screened the library for specific Nef binder phages. We coated the immuno-tubes with the purified Nef protein and added with phage library. Binder with no or low affinity were washed away by successive washes and phages with highest affinity for Nef were extracted (see Fig. 5). After each round of selection, the infected E. coli TG-2 cells were plated uniformly across the entire surface of selection agar plates. The plating of the bio- Fig. 5. Phage pools from the unpanned mouse anti-Nef

scFv library stock and the phage pool obtained after panned library allowed every clone the opportunity to be subsequent rounds of panning were tested for binding to screened. This was important as some clones may grow slowly Nef by ELISA. than others due to the antibody toxicity within the bacteria. This L-...-..: ______________ --'

affinity selection process allowed for enrichment of recombinant phage-scFv particles against the immobilized Nef. The phage pool contained a collection of recombinant ScFv with varying affinities for Nef and, therefore, individual clones were screened by ELISA to determine the binding patterns of the clones (see Fig.S).

Clones

Fig. 6. Fifty individual colonies were randomly selected and analyzed by Nef-ELISA. Eight (8) of the expressed clones showed significant binding to a Nef-coated ELISA plate suggesting a significant positive selection of phage­scFv from the fourth round of 'biopanning'.

Next we incubated select high-affinity-phages with increasing concentration of specific Nef peptides in individual reactions, and performed competitive binding experiments. Phages with affinity for a particular Nef peptide failed to bind to the immobilized Nef in the immuno-tubes and thus collected as unbound fraction. Unbound fraction was concentrated and expanded for future use. By this method, we identified ScFv-phages against ten 30-11" . Nef peptides overlapping 10 amino acids and spanning the em. 206 amino acid Nef protein. Each binder was later confirmed for its specificity towards specific Nef epitope by competitive ELISA (see Fig.7). Selected binders were then propagated and sequenced. At this step of the project, we had obtained a

repertoire of ScFv-phages able to recognize at least one of 10 Nef peptides across the entire 206 amino acids. All 10 ScFv clones were individually transformed into E.coli expression host (BL21-DE3) and ScFv expressed and purified on Cobalt columns using I MAC (immobilized metal affinity chromatography). Purified ScFv were then conjugated with Biotin

1.0 0.9 0.8 0.7

~ 0.6 8 0.5

0.4 0.3 0.2 0.1 0.0

100 1000

Peptide (ng/mL)

Nef peptides _1-30 __ 20-50 _ 40-70 _ 60-90 __ 80-110 _ 100-130 _ 120-150 _ 140-170 _ 160-190 _ 190-206

(biotinylated ScFv) and used to identify Nef motifs in HIV­infected Jurkat-T and THP-1 monocytes and the secreted Nef-containing exosome-Iike vesicles from the infected cells.

Fig. 7. Monoclonal phage inhibition ELISA was performed on a select anti-NefscFv clone in presence of 10 peptides representing various Nef motifs. The phage were incubated for 1 hour with Nef peptides Ilg/mL, 1 Ilg/mL and 200 ng/mL) and added to a Nef-coated plate. Binding phage-scFv were detect .... using an anti-M13 HRP-Iabelled antibody.

B. Identification of Nef epitopes exposed on the surface of HIV~1 infected cells amd secreted Nef vesicles.

The next objective of the study was to use biotinylated ScFv and use them to identify Nef epitopes on the surface of infected cells and secreted exosomes. We infected Jurkat T-cells and THP1 monocytes with HIV-1 (NL4.3) and harvested the infected cells and culture supernatant containing Nef vesicles. Cells and vesicles were washed and treated individually with biotinylated ScFv, each specific to a particular Nef peptide (as determined previously by peptide binding assays). Following incubation, biotinylated ScFv were counter stained with anti­biotin-FITC antibodies and subjected to flow cytometry analysis. Data

MGGKWSKSSVIGWPAVRERMRRAEPAADGV

RRAEPAADGVGAVSRDLEKHGAITSSNTAA

A

GAITSSNTAANNAACAWLEAQEEEEVGfPV ~:::::~. ~ QEEEEVGFPVTPQVPLRPMTYK.~~; '.'D.:'SHf 1 :g, ·nF.!J.,vr;LSHfLKEKGGLEGLIHSQRRQDIL -1 __ _ III

~ IHSQRRQDILDLWIYHTQGYFPDWQNYTPG III Z FPDWQNYTPGPGVRYPLTFG;'i'::'ir.id1··h~F

ti"!·:;.c ... l.,'-: P\;:~ LHGMDDPEREVLEWRFDSRLA -1 __ _

LEWRFDSRLAFHHVARELHPEYFKNC*

MGGKWSKSSVIGWPAVRERMRRAEPAADGV

RRAEPAADGVGAVSRDLEKHGAITSSNTAA

Mean fluorescence

c

GAITSSNTAANNAACAWLEAQEEEEVGFPV ~:::-~ QEEEEVGFPv~PQVPLRPMTYKAAVDLSHFi ~ YKAAVDLSHFLKEKGGLEGLIHSQRRQDIL III

~ IHSQRRQDILDLWIYHTQGYFPDWQNYTPG III Z fPDWQNYTPGPGVRYPLTFGWCYKLVPVEP

WCYKLVPVEPDKVEEANKGENTSLLRPVSL ___ _

NTSLLliPVSLHGMDDPEREVLEWRFDSRLA •••

LEWRFDSRLAFHHVARELHPEYFKNC*

Mean fluorescence

B

Mean fluorescence

o

Mean fluorescence

Fig. 8. Flowcytometric analysis of motif-specific ScFv binding to A. Jurkat-T cells; B. THP-1 monocytes; C. Nef-exosomes from Jurkat T cells; D. Nef-exosomes from THP-l monocytes.

showed that ScFv bound to four (4) putative motifs of Nef present on the surface of HIV-1 infected Jurkat T cells and THP-1 monocytes. A motif comprised of amino acid sequence 'QEEEEVGFPV' appeared to be the most promising, since the mean fluorescence was highest for the ScFv targeted this region (see Fig. 8A & B). The pattern was consistent for both, HIV-1 infected Jurkat T cells and THP-1 monocytes. The ScFv bound to motifs IYKAAVDLSHF', 'WCYKLVPVEP', and 'NTSLLHPVSL' also resulted in higher mean fluorescence but not as high as for the first motif. The ScFv specific to the same peptides also gave high fluorescence when incubated with Nef-exosomes, even though the mean fluorescence was lower compared to antibodies bound to the infected cells (see Fig. 8e & D). We further confirmed these finding by first incubating the ScFv with peptides containing specific motifs and then incubating with the infected cells and/or Nef-exosomes. Pre-incubation with peptides resulted in disappearance of fluorescence suggested that ScFv were indeed binding specifically to the identified motifs. At this stage of the project, we successfully identified 4 Nef motifs that are exposed on the surface of HIV-1 infected cells and Nef-exosomes secreted by these cells. Now that the exposed motifs are identified we aim to et these motifs for ntial anti ediated thera 'c intervention.

E None

nate lrikh

F

July 31st, 2013 Project Leader's Signature: Tandatangan Ketua Projek

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Date:

PROF. MADYALEE KEATTEONG Pengarah . . \ Pejabat Pengurusan 8. KreatMti ~enyeltdlkan

UniYersiti Sains MalaYSia 11800 USM. Pulall Pineng. "l u... t1'-\

Signature: Tandatangan:

Name: Nama:

Tarikh:

88 88 8 ~ ci '.d d 0 c:i

Teow et 01. BMC Biotechnology 2013,13:107 http://www.biomedcentral.com/1472-67S0/13/107 ~

Biotechnology

METHODOLOGY ARTICLE Open Access

Production and purification of polymerization­competent HIV-l capsid protein p24 (CA) in NiCo2l (DE3) Escherichia coli Sin Yeang Teowlt, Siti Aisyah Mualiflt, Tasyriq Che Omar\ Chew Yik Weill Narazah Mohd Yusoff2 and Syed A Alt

Abstract

Background: HIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-l CA

Result: In this work, 6-His-tagged wild type CA from HIV-l (NL4.3) was expressed in rare tRNA-supplemented NiC02l (DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-l CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L- l (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L-l H1V-1 CA A two-step purification strategy (chitin beads + IM~q .Y'!as empl?xed, w.hich efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted Inhtgnlypure HW-l CAThe purified proteirYWas ccfpable . of polymerization when tested in an in vitro polymerization assay.

Conclusions: By using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-l CA can be produced at the lab-scale and thus used for further biochemical studies.

Background Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS), a progressive im­mune disorder that allows life-threatening opportunistic infections, cardiovascular diseases, and cancers to thrive. The capsid protein p24 (CA) plays seminal roles in both late and early stages of the HIV replication cycle (1]. HIV-l CA is considered an important target for develop­ing novel drugs to treat AIDS. For example, a small mole­cule, CAP-I, and two versions of a peptide inhibitor, CAl and NYAD-l, have been reported that target HIV-l CA

-.----.-~~~----~--.--

• Correspondence: aIi2@amdi.usm.edu.my tEqual contributors 10nco\ogical and Radiological Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia Fun list of author information is available at the end of the article

in vitro and interfere with its fUnction in infected cells [2-4]. In another study, two small compounds PF-3450074 and PF-3759857 have shown to be active against HIV-l in low 11M concentration and latter (PF-3759857) against HIV-2 too [5]. More recently, compounds derived from benzodiazepines (BD) and the benzimidazoles (BM) series of chemicals have shown to prevent virion release and inhibited the formation of the mature capsid [6). These studies require milligram quantities of the CA in soluble and active form. However, high commercial cost may limit its use in studies carried out at academic level.

The HIV -1 CA has been produced in bacterium Escherichia coli [7-11], yeast Pichea pastoris [12], plants [13-15], and baculovirus-insect cells [16]. However, eXisting methodologies rely on sequence modifications and several purification rounds involving precipitation

() BioMed Central © 2013 Teow et at; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http"llcreativecommons.org!1icenseslby12.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.