Microbes and Infection 16 (2014) 142e152 www.elsevier.com/locate/micinf
Original article
Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals Nithinart Chaitaveep a,b, Piraporn Utachee c, Shota Nakamura d, Thippawan Chuenchitra b, Pattama Ekpo e, Naokazu Takeda c,d, Kovit Pattanapanyasat e, Masanori Kameoka c,d,f,* a
Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand b Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand c Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Nonthaburi, Thailand d Research Institute for Microbial Diseases, Osaka University, Osaka, Japan e Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand f Department of International Health, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe, Hyogo 654-0142, Japan Received 20 August 2013; accepted 17 October 2013 Available online 26 October 2013
Abstract Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals. Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Recent HIV-1 infection; Transmitted/founder virus; CRF01_AE; Envelope glycoproteins; Neutralization; Captured BED-ELISA
1. Introduction Human immunodeficiency virus type 1 (HIV-1) is a major causative agent of acquired immune deficiency syndrome (AIDS). HIV-1 is a blood-borne virus that spreads through contaminated blood and other body fluids. After the sexual transmission of HIV-1, dendritic cells are considered to interact with the virus at mucosa and transfer it to CD4þ T * Corresponding author. Department of International Health, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe, Hyogo 654-0142, Japan. Tel./fax: þ81 78 796 4594. E-mail address: [email protected] (M. Kameoka).
cells [1]. CD4þ T cells are then productively infected with the virus in systemic lymphoid tissues, leading to the peak viral load [2]. In parallel with viral productive infection, anti-HIV-1 host immune responses are elicited [3], and viral load subsequently rapidly declines [4]. In the early phase of HIV-1 infection, the virus is reported to be genetically homogeneous within an individual [5]. It is reported that the viral population is homogenized in the early phase of infection [6] by the selection pressure of neutralizing antibody responses against autologous viruses [7]. Transmitted/founder virus in the early phase of HIV-1 infection induces primary antiviral immune responses and is the target of such immune reactions; therefore, is considered to be the target of anti-HIV-1 vaccines.
1286-4579/$ - see front matter Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2013.10.015
N. Chaitaveep et al. / Microbes and Infection 16 (2014) 142e152
HIV-1 is characterized by extensive genetic heterogeneity [8] and is divided into four groups: M (major), O (outlying), N (new or non-M, non-O) and P (pending). The viruses in group M are further classified into many subtypes and circulating recombinant forms (CRFs). Among them, subtypes A, B, C, D and G, as well as CRF01_AE and CRF02_AG, are the major subtypes and CRFs responsible for the worldwide HIV-1 pandemic [9]. While subtype B of HIV-1 is the predominant subtype in the Americas, Europe and Australia, there is a growing epidemic of non-B subtypes and CRFs in Africa and Asia. CRF01_AE is prevalent throughout Southeast Asia [9] and is responsible for more than 80% of infection cases in Thailand [10]. The envelope glycoproteins (Env), gp120 and gp41, of HIV-1 play a central role in viral transmission and mediate attachment and incorporation of the virus into target cells through specific interactions with the CD4 receptor and chemokine co-receptors. In addition, Env is a major target of humoral immune responses against HIV-1 and is therefore a candidate for a vaccine antigen [11]. Env gp120 and gp41 are the most variable HIV-1 proteins with typical intersubtype and intrasubtype differences, reaching 35% and 20%, respectively [8]; therefore, the humoral immune responses against Env potentially somewhat vary among different subtypes and CRFs. The captured BED-enzyme-linked immunosorbent assay (ELISA) has been used as a simple method to estimate HIV incidence [12]. It was developed as an assay to detect recent HIV-1 seroconversion. The assay measures the proportion of HIV-1-specific IgG in blood samples with respect to total IgG. The target viral antigen is a branched peptide containing immunodominant sequences from the Env gp41 of subtypes B, E (CRF01_AE) and D. Recent seroconverters have a lower proportion of HIV-specific IgG in their sera than those with long-term infection [12]; therefore, study participants are classified as recent seroconverters if their blood samples have a normalized optical density (ODn) below a threshold cutoff based on a calibrator specimen on the assay. In this report, serum samples from Royal Thai Army (RTA) conscripts diagnosed as having been recently infected with HIV-1 by captured BED-ELISA were subjected to a study of the viral env gene (early env gene) derived from viruses in the early phase of HIV-1 infection. Fourteen infectious recombinant viruses containing full-length early CRF01_AE env genes were established, and their neutralization susceptibilities to neutralizing antibodies and viral entry inhibitors were studied. 2. Materials and methods 2.1. Serum samples Serum samples were collected from RTA conscripts (male, 21 years old) who entered the military between 2009 and 2011 and were tested for anti-HIV-1 antibodies by ELISA, followed by immunoblot analysis to confirm the diagnosis of HIV-1 infection. Samples derived from HIV-1-positive individuals were then subjected to captured BED-ELISA [12] to identify
143
recent HIV-1 infection. The captured BED-ELISA was performed, as described previously [12]. An 0.8 cutoff value for the ODn was used to distinguish recent from chronic infection status. Based on the previous data, we determined the mean period from initial seroconversion to an ODn of 0.8 (the recency period) as 127 days. This study was conducted with approval from the institutional review broad of RTA Medical Department. 2.2. Amplification of HIV-1 env gene (early env gene) from serum samples derived from recently HIV-1infected Thai individuals An HIV-1 genomic fragment containing a full-length env gene was amplified essentially as described previously [13,14]. Prior to extracting RNA from serum samples, viral particles were concentrated from 1 to 2 ml serum by ultracentrifugation for 2 h at 65,000 rpm at 4 C using a TLA-100.3 rotor with optima TLX ultracentrifuge (Beckman Coulter, Fullerton, CA, USA). RNA was then extracted from the concentrated viral particles using the QIAamp viral RNA mini-kit (Qiagen, Hilden, Germany). Viral RNA was reverse transcribed to cDNA using the SuperScript III First-Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA) with a reverse primer, Kenv-R1, 50 -CCAATCAGGGAAGAAGCCTTG-30 [corresponding to nucleotide (nt) 8736 to 8716 of CRF01_AE reference strain, CM240 (GenBank accession no. U54771)]. The HIV-1 genomic fragment, encoding full-length Env precursor gp160, Rev and Vpu as well as partial fragments of Tat and Nef, was then amplified by nested polymerase chain reaction (PCR) using BIO-X-ACT DNA polymerase (Bioline, Luckenwalde, Germany) or PrimeSTAR GXL DNA Polymerase (Takara Bio, Shiga, Japan) with primer sets and conditions described in a previous study [14]. 2.3. Establishment of Env-recombinant proviral constructs The establishment of an Env-recombinant, luciferase reporter proviral construct containing an early env gene was carried out essentially as described [13,15]. Briefly, the SalINotI fragment, encoding full-length HIV-1 Env, Tat, Rev and Vpu, of a pNL4-3 [16]-derived luciferase reporter proviral construct, pNL-envCT [15] was ligated into pCI-neo (Promega, Madison, WI, USA) to generate an HIV-1 env shuttle/ expression vector, pCI-envCT. Meanwhile, based on the analysis of the partial N-terminal and C-terminal nucleotide sequences of the amplified HIV-1 genomic fragment described below, primers were designed to amplify a full-length HIV-1 env gene. The forward primer contained the recognition site for BspEI immediately upstream of the Env signal peptide, while the reverse primer contained the recognition site for NotI immediately downstream of the stop codon of the env gene. The nucleotide sequences of the primers are available upon request. The full-length env gene was then PCRamplified from the HIV-1 genomic fragment using PfuUltra hotstart DNA polymerase (Agilent Technologies, Santa Clara,
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CA, USA), digested with BspEI and NotI, and then replaced with the env gene of pNL4-3 in pCI-envCT. The SalI-NotI fragment of pCI-envCT containing the newly cloned env gene was then cloned back into pNL-envCT to generate the Env-recombinant proviral construct. 2.4. DNA sequencing and data analysis Sequencing analysis of the amplified HIV-1 genomic fragment and the early env gene cloned into the proviral construct was carried out using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM 3130XL genetic analyzer (Applied Biosystems, Foster City, CA, USA), and data were assembled using SeqScape v2.5 software (Applied Biosystems). In addition, for early env genes cloned in Envrecombinant proviral constructs, the following genotypic analyses were carried out. The deduced amino acid sequences of gp160 were aligned by the MAFFT program [17]. The Showalign program in the EMBOSS suite v.5.0.0 was then used to construct consensus sequences and to display the alignments [18]. Pairwise distance matrices were calculated using the PROTDIST program from the Phylogeny Inference Package (PHYLIP) version 3.6, incorporating the Dayhoff PAM matrix (distributed by Dr. Joe Felsenstein, Department of Genome Sciences, University of Washington, Seattle, WA, USA). Frequency analysis of these pairwise distances was then performed. In addition, the potential N-linked glycosylation (PNLG) site and the subtype classification of HIV-1 env genes were examined using N-Glycosite and the Recombinant Identification Program (RIP) 3.0 (www.hiv.lanl.gov), respectively. 2.5. Preparation of Env-recombinant virus Viral supernatants were prepared by transfecting 293T cells with the Env-recombinant proviral construct using FuGENE HD transfection reagent (Roche, Basel, Switzerland), essentially as described [19]. pNL-envCT and pNL-Luc-BaLenv [20], containing the env gene of pBa-L (GenBank accession no. AB253432), were used to prepare CXCR4-tropic (X4) and CCR5-tropic (R5) subtype B HIV-1, respectively. The viral titer was determined by measuring the concentration of HIV-1 Gag p24 antigen in viral supernatants by ELISA (HIV-1 p24 Antigen Capture Assay; Advanced Bioscience Laboratory, Rockville, MD, USA). 2.6. Evaluation of viral infectivity and co-receptor usage U87.CD4.CXCR4 and U87.CD4.CCR5 cells [21] were provided by Dr. HongKui Deng and Dr. Dan R. Littman through the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, NIAID, NIH. U87 cell lines were infected with Env-recombinant virus (10 ng of p24 antigen). Forty-eight hours after infection, luciferase activity in infected cells was measured using the Steady Glo Luciferase assay kit (Promega) with LB960 microplate luminomater
(Berthold, Bad Wildbad, Germany), according to the manufacturer’s protocol. 2.7. Neutralization assay Neutralization susceptibilities of the early Env-recombinant viruses were examined for neutralizing human monoclonal antibodies (NHMAbs) against gp120, 2G12 [22] and IgG1 b12 [23]; NHMAbs against gp41, 2F5 [22] and 4E10 [24]; heatinactivated (56 C for 1 h) pooled patient serum, a CXCR4 antagonist, AMD3100 [25]; a CCR5 antagonist, TAK-779 [26]; and a fusion inhibitor, T-20 [27]. Briefly, viral supernatant (2 ng of p24 antigen) was treated with 2-fold serially diluted NHMAbs or patient serum at 37 C for 1 h, followed by mixing with cells. Alternatively, U87.CD4.CXCR4 or U87.CD4.CCR5 cells were treated with 2-fold serially diluted AMD3100, TAK-779 or T-20 at 37 C for 1 h, followed by mixing with viral supernatant (4 ng of p24 antigen). The mixture of cells and viral supernatant was then incubated for 48 h, and luciferase activity in infected cells was measured as described above. The neutralization level was evaluated as a reduction in luciferase activity in infected cells. The 50% inhibitory concentration (IC50) of NHMAbs, AMD3100, TAK-779 and T-20 for suppressing viral replication, and the reciprocal serum dilution, at which viral replication was suppressed by 50% (50% inhibitory dilution, ID50), were calculated by the doseeresponse curve using a standard function of GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). NHMAbs, b12, 2G12, 2F5 and 4E10, were purchased from Polymun Scientific (Vienna, Austria), while AMD3100 was purchased from SigmaeAldrich (St. Louis, MO, USA). In addition, TAK-779 and T-20 (provided by Roche) were obtained through ARRRP. 2.8. Statistical analysis Statistical analysis was carried out using the standard function of GraphPad Prism 5 software (GraphPad Software) with an unpaired t-test or Spearman’s rank correlation test. 2.9. Nucleotide sequence accession numbers The nucleotide sequences of 14 early CRF01_AE env genes have been deposited in the GenBank database under accession numbers KF268035eKF268048. In addition, the deduced amino acid sequences of 35 previously reported CRF01_AE env genes with Genbank accession numbers, EU743757eEU743759 and EU743763eEU743794 [13], were examined. 3. Results 3.1. Establishment of early CRF01_AE Env-recombinant viruses Full-length early HIV-1 env genes were amplified from serum samples derived from 10 recently HIV-1-infected RTA
N. Chaitaveep et al. / Microbes and Infection 16 (2014) 142e152
conscripts, and amplified env genes were cloned into the luciferase reporter proviral construct to generate early Envrecombinant proviral DNA. The infectivity of the generated recombinant virus was then evaluated by measuring luciferase activity in infected cells (data not shown), and 14 early Envrecombinant viruses showing high infectivity were selected for further characterization (Table 1). All early env genes cloned in the recombinant viruses were classified into CRF01_AE env genes and consisted of 13 R5 and dual-tropic (X4R5) env clones (Table 1). In addition, the early Envrecombinant viruses showed various levels (0.35e2.06-fold) of infectivity relative to pNL-envCT (pNL4-3) (Table 1). 3.2. Characterization of the deduced amino acid sequences of 14 early CRF01_AE env genes The deduced amino acid sequences of 14 early CRF01_AE env genes and their consensus sequence are shown in Fig. 1. The average numbers of amino acid residues in V1, V2, V3, V4 and V5 regions among 14 early CRF01_AE Env gp120 were 27, 42, 35, 27 and 8 amino acids, respectively. The numbers of amino acid residues in V1 and V2 (V1/V2) regions of 14 early CRF01_AE Env gp120 were significantly lower than those of 35 previously established CRF01_AE Env gp120 derived from 19 chronically infected Thai individuals [13] Table 1 Phenotypic properties of 14 early env genes derived from recently infected Thai individuals. ID of env gene
ODn Relative infectivity (RLU)b values U87.X4 U87.R5 of BEDa ELISA
CoSubtyped receptor usagec
RTA2 RTA3 RTA4 RTA5 RTA6 RTA8 RTA9 RTA11 RTA13 RTA16 RTA21 RTA23 RTA24 RTA27
0.077 0.441 0.080 0.080 0.080 0.675 0.681 0.131 0.290 0.272 0.681 0.185 0.681 0.099
R5 X4R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5
a
0 105 0 0 0 0 0 0 0 0 0 0 0 0
87 39 75 128 149 167 36 134 206 117 35 100 185 143
CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE
The normalized optical density (ODn) was calculated by the OD of sample divided by the median OD of calibrator on the captured BED-ELISA. The sample with an ODn of 10 >10 8.3 >10 >10 4.4 7.1 5.0 3.6 3.4 7.6 2.6 >10 3.8
8.4 3.8 2.4 2.8 2.2 2.3 2.5 0.9 3.6 2.3 4.5 2.3 2.5 2.9 1.8 3.5 5.5
1.9 0.9 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
0.4 0.3 9.1 >10 >10 >10 >10 >10 >10 5.5 >10 >10 0.9 >10 5.1 2.8 >10
IC50 of entry inhibitors (mg/ml)b
ID50 of recently infected patient serumb,c
ID50 of chronically infected patient serumb,c
AMD3100
TAK-779
T-20
Original article
Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals Nithinart Chaitaveep a,b, Piraporn Utachee c, Shota Nakamura d, Thippawan Chuenchitra b, Pattama Ekpo e, Naokazu Takeda c,d, Kovit Pattanapanyasat e, Masanori Kameoka c,d,f,* a
Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand b Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand c Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Nonthaburi, Thailand d Research Institute for Microbial Diseases, Osaka University, Osaka, Japan e Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand f Department of International Health, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe, Hyogo 654-0142, Japan Received 20 August 2013; accepted 17 October 2013 Available online 26 October 2013
Abstract Transmitted/founder virus is responsible for the establishment of human immunodeficiency virus type 1 (HIV-1) infection and induces primary anti-HIV-1 immune responses; therefore, it is important to study the viral population to understand the early events of HIV-1 infection. We amplified HIV-1 env genes from sera derived from recently infected Thai individuals, and established envelope glycoproteins (Env)-recombinant viruses. Generated Env-recombinant viruses were tested for their neutralization susceptibility to neutralizing human monoclonal antibodies (NHMAbs) and entry inhibitors, as well as being subjected to genotypic analysis. Most recombinant viruses were susceptible to neutralization by NHMAbs to Env gp41, whereas approximately one-third of the recombinant viruses were susceptible to a NHMAb against the CD4 binding site of gp120. In addition, all env genes were classified into CRF01_AE genes and showed low genetic divergence. Taken together with our previous studies on CRF01_AE env genes derived from chronically infected Thai individuals, these results suggested that the immunological and genetic characteristics of CRF01_AE Env derived from recently infected Thai individuals were different from those derived from chronically infected individuals. Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Keywords: Recent HIV-1 infection; Transmitted/founder virus; CRF01_AE; Envelope glycoproteins; Neutralization; Captured BED-ELISA
1. Introduction Human immunodeficiency virus type 1 (HIV-1) is a major causative agent of acquired immune deficiency syndrome (AIDS). HIV-1 is a blood-borne virus that spreads through contaminated blood and other body fluids. After the sexual transmission of HIV-1, dendritic cells are considered to interact with the virus at mucosa and transfer it to CD4þ T * Corresponding author. Department of International Health, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe, Hyogo 654-0142, Japan. Tel./fax: þ81 78 796 4594. E-mail address: [email protected] (M. Kameoka).
cells [1]. CD4þ T cells are then productively infected with the virus in systemic lymphoid tissues, leading to the peak viral load [2]. In parallel with viral productive infection, anti-HIV-1 host immune responses are elicited [3], and viral load subsequently rapidly declines [4]. In the early phase of HIV-1 infection, the virus is reported to be genetically homogeneous within an individual [5]. It is reported that the viral population is homogenized in the early phase of infection [6] by the selection pressure of neutralizing antibody responses against autologous viruses [7]. Transmitted/founder virus in the early phase of HIV-1 infection induces primary antiviral immune responses and is the target of such immune reactions; therefore, is considered to be the target of anti-HIV-1 vaccines.
1286-4579/$ - see front matter Ó 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.micinf.2013.10.015
N. Chaitaveep et al. / Microbes and Infection 16 (2014) 142e152
HIV-1 is characterized by extensive genetic heterogeneity [8] and is divided into four groups: M (major), O (outlying), N (new or non-M, non-O) and P (pending). The viruses in group M are further classified into many subtypes and circulating recombinant forms (CRFs). Among them, subtypes A, B, C, D and G, as well as CRF01_AE and CRF02_AG, are the major subtypes and CRFs responsible for the worldwide HIV-1 pandemic [9]. While subtype B of HIV-1 is the predominant subtype in the Americas, Europe and Australia, there is a growing epidemic of non-B subtypes and CRFs in Africa and Asia. CRF01_AE is prevalent throughout Southeast Asia [9] and is responsible for more than 80% of infection cases in Thailand [10]. The envelope glycoproteins (Env), gp120 and gp41, of HIV-1 play a central role in viral transmission and mediate attachment and incorporation of the virus into target cells through specific interactions with the CD4 receptor and chemokine co-receptors. In addition, Env is a major target of humoral immune responses against HIV-1 and is therefore a candidate for a vaccine antigen [11]. Env gp120 and gp41 are the most variable HIV-1 proteins with typical intersubtype and intrasubtype differences, reaching 35% and 20%, respectively [8]; therefore, the humoral immune responses against Env potentially somewhat vary among different subtypes and CRFs. The captured BED-enzyme-linked immunosorbent assay (ELISA) has been used as a simple method to estimate HIV incidence [12]. It was developed as an assay to detect recent HIV-1 seroconversion. The assay measures the proportion of HIV-1-specific IgG in blood samples with respect to total IgG. The target viral antigen is a branched peptide containing immunodominant sequences from the Env gp41 of subtypes B, E (CRF01_AE) and D. Recent seroconverters have a lower proportion of HIV-specific IgG in their sera than those with long-term infection [12]; therefore, study participants are classified as recent seroconverters if their blood samples have a normalized optical density (ODn) below a threshold cutoff based on a calibrator specimen on the assay. In this report, serum samples from Royal Thai Army (RTA) conscripts diagnosed as having been recently infected with HIV-1 by captured BED-ELISA were subjected to a study of the viral env gene (early env gene) derived from viruses in the early phase of HIV-1 infection. Fourteen infectious recombinant viruses containing full-length early CRF01_AE env genes were established, and their neutralization susceptibilities to neutralizing antibodies and viral entry inhibitors were studied. 2. Materials and methods 2.1. Serum samples Serum samples were collected from RTA conscripts (male, 21 years old) who entered the military between 2009 and 2011 and were tested for anti-HIV-1 antibodies by ELISA, followed by immunoblot analysis to confirm the diagnosis of HIV-1 infection. Samples derived from HIV-1-positive individuals were then subjected to captured BED-ELISA [12] to identify
143
recent HIV-1 infection. The captured BED-ELISA was performed, as described previously [12]. An 0.8 cutoff value for the ODn was used to distinguish recent from chronic infection status. Based on the previous data, we determined the mean period from initial seroconversion to an ODn of 0.8 (the recency period) as 127 days. This study was conducted with approval from the institutional review broad of RTA Medical Department. 2.2. Amplification of HIV-1 env gene (early env gene) from serum samples derived from recently HIV-1infected Thai individuals An HIV-1 genomic fragment containing a full-length env gene was amplified essentially as described previously [13,14]. Prior to extracting RNA from serum samples, viral particles were concentrated from 1 to 2 ml serum by ultracentrifugation for 2 h at 65,000 rpm at 4 C using a TLA-100.3 rotor with optima TLX ultracentrifuge (Beckman Coulter, Fullerton, CA, USA). RNA was then extracted from the concentrated viral particles using the QIAamp viral RNA mini-kit (Qiagen, Hilden, Germany). Viral RNA was reverse transcribed to cDNA using the SuperScript III First-Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA) with a reverse primer, Kenv-R1, 50 -CCAATCAGGGAAGAAGCCTTG-30 [corresponding to nucleotide (nt) 8736 to 8716 of CRF01_AE reference strain, CM240 (GenBank accession no. U54771)]. The HIV-1 genomic fragment, encoding full-length Env precursor gp160, Rev and Vpu as well as partial fragments of Tat and Nef, was then amplified by nested polymerase chain reaction (PCR) using BIO-X-ACT DNA polymerase (Bioline, Luckenwalde, Germany) or PrimeSTAR GXL DNA Polymerase (Takara Bio, Shiga, Japan) with primer sets and conditions described in a previous study [14]. 2.3. Establishment of Env-recombinant proviral constructs The establishment of an Env-recombinant, luciferase reporter proviral construct containing an early env gene was carried out essentially as described [13,15]. Briefly, the SalINotI fragment, encoding full-length HIV-1 Env, Tat, Rev and Vpu, of a pNL4-3 [16]-derived luciferase reporter proviral construct, pNL-envCT [15] was ligated into pCI-neo (Promega, Madison, WI, USA) to generate an HIV-1 env shuttle/ expression vector, pCI-envCT. Meanwhile, based on the analysis of the partial N-terminal and C-terminal nucleotide sequences of the amplified HIV-1 genomic fragment described below, primers were designed to amplify a full-length HIV-1 env gene. The forward primer contained the recognition site for BspEI immediately upstream of the Env signal peptide, while the reverse primer contained the recognition site for NotI immediately downstream of the stop codon of the env gene. The nucleotide sequences of the primers are available upon request. The full-length env gene was then PCRamplified from the HIV-1 genomic fragment using PfuUltra hotstart DNA polymerase (Agilent Technologies, Santa Clara,
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CA, USA), digested with BspEI and NotI, and then replaced with the env gene of pNL4-3 in pCI-envCT. The SalI-NotI fragment of pCI-envCT containing the newly cloned env gene was then cloned back into pNL-envCT to generate the Env-recombinant proviral construct. 2.4. DNA sequencing and data analysis Sequencing analysis of the amplified HIV-1 genomic fragment and the early env gene cloned into the proviral construct was carried out using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM 3130XL genetic analyzer (Applied Biosystems, Foster City, CA, USA), and data were assembled using SeqScape v2.5 software (Applied Biosystems). In addition, for early env genes cloned in Envrecombinant proviral constructs, the following genotypic analyses were carried out. The deduced amino acid sequences of gp160 were aligned by the MAFFT program [17]. The Showalign program in the EMBOSS suite v.5.0.0 was then used to construct consensus sequences and to display the alignments [18]. Pairwise distance matrices were calculated using the PROTDIST program from the Phylogeny Inference Package (PHYLIP) version 3.6, incorporating the Dayhoff PAM matrix (distributed by Dr. Joe Felsenstein, Department of Genome Sciences, University of Washington, Seattle, WA, USA). Frequency analysis of these pairwise distances was then performed. In addition, the potential N-linked glycosylation (PNLG) site and the subtype classification of HIV-1 env genes were examined using N-Glycosite and the Recombinant Identification Program (RIP) 3.0 (www.hiv.lanl.gov), respectively. 2.5. Preparation of Env-recombinant virus Viral supernatants were prepared by transfecting 293T cells with the Env-recombinant proviral construct using FuGENE HD transfection reagent (Roche, Basel, Switzerland), essentially as described [19]. pNL-envCT and pNL-Luc-BaLenv [20], containing the env gene of pBa-L (GenBank accession no. AB253432), were used to prepare CXCR4-tropic (X4) and CCR5-tropic (R5) subtype B HIV-1, respectively. The viral titer was determined by measuring the concentration of HIV-1 Gag p24 antigen in viral supernatants by ELISA (HIV-1 p24 Antigen Capture Assay; Advanced Bioscience Laboratory, Rockville, MD, USA). 2.6. Evaluation of viral infectivity and co-receptor usage U87.CD4.CXCR4 and U87.CD4.CCR5 cells [21] were provided by Dr. HongKui Deng and Dr. Dan R. Littman through the AIDS Research and Reference Reagent Program (ARRRP), Division of AIDS, NIAID, NIH. U87 cell lines were infected with Env-recombinant virus (10 ng of p24 antigen). Forty-eight hours after infection, luciferase activity in infected cells was measured using the Steady Glo Luciferase assay kit (Promega) with LB960 microplate luminomater
(Berthold, Bad Wildbad, Germany), according to the manufacturer’s protocol. 2.7. Neutralization assay Neutralization susceptibilities of the early Env-recombinant viruses were examined for neutralizing human monoclonal antibodies (NHMAbs) against gp120, 2G12 [22] and IgG1 b12 [23]; NHMAbs against gp41, 2F5 [22] and 4E10 [24]; heatinactivated (56 C for 1 h) pooled patient serum, a CXCR4 antagonist, AMD3100 [25]; a CCR5 antagonist, TAK-779 [26]; and a fusion inhibitor, T-20 [27]. Briefly, viral supernatant (2 ng of p24 antigen) was treated with 2-fold serially diluted NHMAbs or patient serum at 37 C for 1 h, followed by mixing with cells. Alternatively, U87.CD4.CXCR4 or U87.CD4.CCR5 cells were treated with 2-fold serially diluted AMD3100, TAK-779 or T-20 at 37 C for 1 h, followed by mixing with viral supernatant (4 ng of p24 antigen). The mixture of cells and viral supernatant was then incubated for 48 h, and luciferase activity in infected cells was measured as described above. The neutralization level was evaluated as a reduction in luciferase activity in infected cells. The 50% inhibitory concentration (IC50) of NHMAbs, AMD3100, TAK-779 and T-20 for suppressing viral replication, and the reciprocal serum dilution, at which viral replication was suppressed by 50% (50% inhibitory dilution, ID50), were calculated by the doseeresponse curve using a standard function of GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). NHMAbs, b12, 2G12, 2F5 and 4E10, were purchased from Polymun Scientific (Vienna, Austria), while AMD3100 was purchased from SigmaeAldrich (St. Louis, MO, USA). In addition, TAK-779 and T-20 (provided by Roche) were obtained through ARRRP. 2.8. Statistical analysis Statistical analysis was carried out using the standard function of GraphPad Prism 5 software (GraphPad Software) with an unpaired t-test or Spearman’s rank correlation test. 2.9. Nucleotide sequence accession numbers The nucleotide sequences of 14 early CRF01_AE env genes have been deposited in the GenBank database under accession numbers KF268035eKF268048. In addition, the deduced amino acid sequences of 35 previously reported CRF01_AE env genes with Genbank accession numbers, EU743757eEU743759 and EU743763eEU743794 [13], were examined. 3. Results 3.1. Establishment of early CRF01_AE Env-recombinant viruses Full-length early HIV-1 env genes were amplified from serum samples derived from 10 recently HIV-1-infected RTA
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conscripts, and amplified env genes were cloned into the luciferase reporter proviral construct to generate early Envrecombinant proviral DNA. The infectivity of the generated recombinant virus was then evaluated by measuring luciferase activity in infected cells (data not shown), and 14 early Envrecombinant viruses showing high infectivity were selected for further characterization (Table 1). All early env genes cloned in the recombinant viruses were classified into CRF01_AE env genes and consisted of 13 R5 and dual-tropic (X4R5) env clones (Table 1). In addition, the early Envrecombinant viruses showed various levels (0.35e2.06-fold) of infectivity relative to pNL-envCT (pNL4-3) (Table 1). 3.2. Characterization of the deduced amino acid sequences of 14 early CRF01_AE env genes The deduced amino acid sequences of 14 early CRF01_AE env genes and their consensus sequence are shown in Fig. 1. The average numbers of amino acid residues in V1, V2, V3, V4 and V5 regions among 14 early CRF01_AE Env gp120 were 27, 42, 35, 27 and 8 amino acids, respectively. The numbers of amino acid residues in V1 and V2 (V1/V2) regions of 14 early CRF01_AE Env gp120 were significantly lower than those of 35 previously established CRF01_AE Env gp120 derived from 19 chronically infected Thai individuals [13] Table 1 Phenotypic properties of 14 early env genes derived from recently infected Thai individuals. ID of env gene
ODn Relative infectivity (RLU)b values U87.X4 U87.R5 of BEDa ELISA
CoSubtyped receptor usagec
RTA2 RTA3 RTA4 RTA5 RTA6 RTA8 RTA9 RTA11 RTA13 RTA16 RTA21 RTA23 RTA24 RTA27
0.077 0.441 0.080 0.080 0.080 0.675 0.681 0.131 0.290 0.272 0.681 0.185 0.681 0.099
R5 X4R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5
a
0 105 0 0 0 0 0 0 0 0 0 0 0 0
87 39 75 128 149 167 36 134 206 117 35 100 185 143
CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE CRF01_AE
The normalized optical density (ODn) was calculated by the OD of sample divided by the median OD of calibrator on the captured BED-ELISA. The sample with an ODn of 10 >10 8.3 >10 >10 4.4 7.1 5.0 3.6 3.4 7.6 2.6 >10 3.8
8.4 3.8 2.4 2.8 2.2 2.3 2.5 0.9 3.6 2.3 4.5 2.3 2.5 2.9 1.8 3.5 5.5
1.9 0.9 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
0.4 0.3 9.1 >10 >10 >10 >10 >10 >10 5.5 >10 >10 0.9 >10 5.1 2.8 >10
IC50 of entry inhibitors (mg/ml)b
ID50 of recently infected patient serumb,c
ID50 of chronically infected patient serumb,c
AMD3100
TAK-779
T-20
