Characterization of human immunodeficiency virus type 1 CRF01_AE env genes derived from recently infected Thai individuals

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

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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

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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 <0.8 was estimated to be from recently (<127 days) seroconverted individual. b Infectivity of Env-recombinant virus was evaluated using U87.CD4.CXCR4 (U87.X4) and U87.CD4.CCR5 (U87.R5) cells. Relative infectivity of the virus was calculated by comparing it with the luciferase activity of pNL-envCT (pNL4-3)-infected U87.X4 cells, which was defined as 100 relative light units (RLU). c Co-receptor usage of the Env-recombinant virus was evaluated by its infectivity in U87.X4 and U87.R5 cells. d Subtype classification of env gene was examined using RIP 3.0 (www.hiv. lanl.gov).

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(Fig. 2). In contrast, the average numbers of amino acid residues in V3, V4 and V5 regions among 35 CRF01_AE Env gp120 derived from chronically infected individuals were 35, 26 and 9 amino acids, respectively [28]; therefore, the numbers of amino acid residues in V3, V4 and V5 regions were comparable between the CRF01_AE Env gp120 derived from recently and chronically infected Thai individuals. In addition, the average number of PNLG sites in gp160 among 14 early CRF01_AE Env was 28, and was comparable to that in CRF01_AE Env gp160 described in previous reports [13,29]. We next studied the diversity of deduced amino acid sequences in gp160, gp120, gp41 as well as in the C2eV5 (spanning C2, V3, C3, V4, C4 and V5 regions) and V3 regions of gp120 among 14 early CRF01_AE Env proteins. The average differences (%) of the amino acid sequences in gp160, gp120, gp41, gp120 C2eV5 and gp120 V3 regions among 14 early CRF01_AE Env were 11.0%, 13.4%, 7.4%, 12.9% and 11.4%, respectively (Table 2). Highest amino acid diversity was observed in the C2eV5 region of gp120 (Table 2). However, the overall diversity of amino acid sequences among 14 early CRF01_AE Env proteins was markedly smaller than that among 35 CRF01_AE Env proteins derived from chronically infected Thai individuals described in a previous report [13]. Namely, the average differences (%) of the amino acid sequences in gp160, gp120, gp41, gp120 C2-V5 and gp120 V3 regions among 35 CRF01_AE Env derived from chronically infected individuals were 18.5%, 24.1%, 12.1%, 24.5% and 35.3%, respectively [13]. 3.3. Neutralization susceptibility of 14 early CRF01_AE Env-recombinant viruses to NHMAbs, pooled patient serum and HIV-1 entry inhibitors To evaluate the immunological characteristics of 14 early CRF01_AE Env proteins, we studied the susceptibility of recombinant viruses containing early CRF01_AE env genes to neutralization by NHMAbs and pooled patient serum. In addition, neutralization susceptibility of the recombinant viruses to HIV-1 entry inhibitors was tested. The results showed that all recombinant viruses were susceptible to neutralization by a NHMAb to gp41, 4E10, while 9 of 14 (64%) recombinant viruses were susceptible to neutralization by a NHMAb to gp41, 2F5 (Table 3). In contrast, all recombinant viruses were resistant to neutralization by a NHMAb recognizing the mannose cluster of Env gp120, 2G12. A similar tendency was seen in the neutralization susceptibilities of 14 early CRF01_AE Env (Table 3) and 35 CRF01_AE Env derived from chronically infected individuals [28] to 4E10, 2F5 and 2G12 (data not shown). We next studied the neutralization susceptibility of early CRF01_AE Env-recombinant viruses to b12, a NHMAb recognizing the epitope overlapped with the CD4 binding site (CD4BS) on Env gp120. The results showed that 5 of 14 (36%) recombinant viruses were susceptible to b12-mediated neutralization (Table 3). In contrast, 34 of 35 (97%) CRF01_AE Env-recombinant viruses derived from chronically infected individuals were resistant to neutralization by b12 in our previous report [28], suggesting a difference in b12 susceptibility between CRF01_AE Env derived

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Fig. 1. Deduced gp160 amino acid sequence of 14 early CRF01_AE env genes. Nucleotide sequence of 14 CRF01_AE env genes was translated, aligned and compared with the consensus sequence, as described in Materials and methods. Positions of Env signal peptide, gp120 and gp41, as well as the variable (V1, V2, V3, V4 and V5) and conserved (C1, C2, C3, C4 and C5) regions of gp120 are denoted above the aligned sequences. In addition, the membrane-proximal external region (MPER) of gp41 is indicated. The numbering of amino acid residues begins with the first residue of the Env signal peptide. Dots denote amino acid identity, whereas dashes represent gaps introduced to optimize alignment. PNLG sites are shown by underlining.

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Fig. 1. (continued).

from recently and chronically infected Thai individuals. In addition, we studied the neutralization susceptibility of early CRF01_AE Env-recombinant viruses to pooled serum derived from 10 recently infected Thai individuals from which viral env genes were amplified; however, the replication of no recombinant viruses was inhibited by the pooled serum (Table 3). The pooled serum also showed no neutralizing activity to pNL-envCT and pNL-BaLenv, while the replication of 4 randomly selected recombinant viruses containing early CRF01_AE env genes was efficiently inhibited by pooled serum derived from chronically HIV-1-infected Thai individuals (Table 3), suggesting that the serum derived from recently infected individuals had no or quite low anti-HIV-1 neutralizing activity. There was a possibility that the serum samples had been collected from acutely HIV-1infected individuals in whom potent anti-HIV-1 neutralizing antibody responses had not been induced. Finally, we studied the neutralization susceptibility of early CRF01_AE Envrecombinant viruses to HIV-1 entry inhibitors. The replication of all recombinant viruses containing X4 or X4R5 early CRF01_AE env genes was inhibited by a CXCR4 antagonist, AMD3100, while that of all recombinant viruses containing R5 and X4R5 env genes was inhibited by a CCR5 antagonist, TAK779 at various levels (Table 3). In addition, the replication of most recombinant viruses was inhibited at various levels by a fusion inhibitor, T-20, whereas a recombinant virus containing an early CRF01_AE env gene, RTA24, was T-20 resistant (Table 3).

3.4. Correlation between the neutralization susceptibility and amino acid sequences of early CRF01_AE Env proteins The length of variable regions of Env gp120 and N-linked glycosylation of particular amino acid residues affect the protein structure and neutralization susceptibility of HIV-1 Env proteins [30,31]; therefore, we next studied the possible correlations between neutralization susceptibility and the characteristics of amino acid sequences of early CRF01_AE Env proteins. Possible correlations between the neutralization susceptibility of 14 early CRF01_AE Env-recombinant viruses and the characteristics of Env amino acid sequences were studied, including the number of amino acid residues in gp120 variable regions, number of PNLS sites in particular Env regions and amino acid mutations in epitope regions. As a result, no correlations were found between the susceptibilities of the recombinant viruses to neutralization by NHMAbs, b12 and 2F5, or by AMD3100 and the above characteristics of Env amino acid sequences (data not shown). However, a correlation was observed between the lengths of gp120 V1/V2 regions and the IC50 values of TAK-779 in inhibiting viral replication (Fig. 3A), indicating that the length of V1/V2 regions was negatively correlated with TAK-779 susceptibility of the recombinant viruses. In addition, the IC50 values of 4E10 in inhibiting viral replication were correlated with the lengths of the gp120 V4 region, indicating a negative

Fig. 2. Comparison of the lengths of V1 and V2 regions between CRF01_AE Env gp120 derived from chronically and recently infected Thai individuals. The numbers of amino acid residues in the V1 (A) and V2 (B) regions of gp120 on 35 CRF01_AE Env derived from chronically infected individuals (Genbank accession numbers EU743757eEU743759) [13] (Chronic), as well as on 14 early CRF01_AE Env derived from recently infected Thai individuals (Recent), were manually counted and plotted. Horizontal solid lines show median values, and the number of samples studied (n) is shown below the panels. Differences among groups were analyzed with the unpaired t test, and p values ( p) are shown in the panels.

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Table 2 Diversity of deduced amino acid sequences among 14 early CRF01_AE env genes. % Differencea

n b

gp160 gp120 gp41 C2-V5 V3

14 14 14 14 14

Min

Mean

Max

0.1 0.2 0 0.4 0

11.0 13.4 7.42 12.9 11.4

19.7 24.1 14.4 25.7 22.9

a

Interclone distances were calculated by pairwise comparisons of deduced amino acid sequences. b Including Env signal peptide.

correlation between the 4E10 susceptibility of recombinant viruses and the length of the gp120 V4 region (Fig. 3B). Moreover, the IC50 values of 4E10 in inhibiting viral replication were inversely correlated with the lengths of gp120 V1/ V2 regions or the total numbers of PNLG sites in V1, V2, V3, V4 and V5 (V1/V2/V3/V4/V5) regions (Fig. 3C and D), indicating that the length of V1/V2 regions and the total number of PNLG sites in gp120 variable regions were correlated with the 4E10 susceptibility of the recombinant viruses. No mutations were found in the N-terminal heptad repeat of gp41 in which the T-20 binding site is located [32] in the Env clone, RTA24 (Fig. 1); therefore, T-20 resistance of the recombinant virus containing RTA24 could not be explained by the introduction of mutations in this region. In contrast, a

single amino acid mutation, A to T, was detected at position 678 in the 2F5 core epitope, ELDKWA [33], in the membraneproximal external region (MPER) of gp41 in the Env clones, RTA 2, 3, 6, and 24 (Fig. 1), whereas recombinant viruses containing the corresponding env genes were resistant to 2F5mediated neutralization, suggesting an important role of this mutation in conferring 2F5 resistance. However, although a single amino acid mutation, E to A, was detected at position 673 in the 2F5 core epitope in RTA8, 11 and 27 (Fig. 1), the Env clone was susceptible to neutralization by 2F5 (Table 3). In addition, no mutation was found in the core epitope in RTA5 (Fig. 1), although the Env clone was resistant to neutralization by 2F5 (Table 3), suggesting the existence of other determinants affecting the 2F5 susceptibility of certain Env clones. Finally, although a single mutation, D to S or N to S, was detected in the 4E10 core epitope, NWFDIT [34], in RTA3, 4, 5 and 13 (Fig. 1), the recombinant viruses containing the corresponding env genes were susceptible to neutralization by 4E10 (Table 3), indicating that such a mutation played no major role in determining the 4E10 susceptibility of Env clones. 4. Discussion In this report, 14 full-length CRF01_AE env genes were amplified from serum samples derived from recently HIV-1infected RTA conscripts and were cloned into a reporter proviral construct. The immunological characteristics of early env

Table 3 Neutralization susceptibility of 14 early CRF01_AE Env-recombinant viruses to NHMAbs, pooled patient serum and entry inhibitors.a env gene

IC50 of NHMAbs (mg/ml)b

Coreceptor usage

Target cells

2F5

4E10

2G12

IgG1 b12

pNL4-3d pBa-Ld RTA2 RTA3

X4 R5 R5 X4R5

RTA4 RTA5 RTA6 RTA8 RTA9 RTA11 RTA13 RTA16 RTA21 RTA23 RTA24 RTA27

R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5 R5

U87.X4 U87.R5 U87.R5 U87.R5 U87.X4 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5 U87.R5

4.1 3.4 >10 >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

<20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20 <20

368e 242e ND ND ND 58.3 ND ND ND ND ND 272.3 ND ND 925.7 ND 82.7

8.9 ND ND ND 2.1 ND ND ND ND ND ND ND ND ND ND ND ND

NDf 4.5 5.9 12.5 ND 8.9 4.1 5.4 3.3 8.9 6.1 5.5 3.2 3.6 13.1 3.7 21.6

92 12.2 17.6 9.4 25.6 4.7 16.8 16.4 33.6 21.9 18.6 2.9 5.9 137 49.8 >175 315

a Neutralization susceptibility of X4 or R5 virus was examined using U87.CD4.CXCR4 (U87.X4) or U87.CD4.CCR5 (U87.R5) cells, respectively, whereas that of X4R5 virus was examined using both cell lines. b IC50 of NHMAbs and entry inhibitors for suppressing viral replication and the reciprocal dilution of pooled patient serum at which viral replication was suppressed by 50% (50% inhibitory dilution, ID50) were calculated using GraphPad Prism 5 software. c Pooled serum samples derived from 10 recently or 19 chronically HIV-1 infected Thai patients were subjected to the study. d Recombinant virus containing subtype B X4 (pNL4-3) or R5 (pBa-L) Env. e Values were retrieved from our previous report [28]. f Not done.

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Fig. 3. Correlations between the neutralization susceptibility of early CRF01_AE Env-recombinant virus and the length of gp120 variable regions or the number of PNLG sites. Neutralization susceptibility of CRF01_AE Env-recombinant virus to a CCR5 antagonist, TAK-779 (A) and to a NHMAb, 4E10 (BeD) was determined as described in Materials and methods. The correlations were examined between the neutralization susceptibility of the early CRF01_AE Envrecombinant virus to TAK-779 or 4E10 and the length of the indicated variable regions of gp120 (AeC) or the number of PNLG sites in the gp120 V1/V2/ V3/V4/V5 regions (D). Correlations were evaluated using Spearman’s rank correlation test, and the correlation coefficient (r) and p values ( p) are shown above the panels.

genes that possibly resemble those derived from transmitted/ founder CRF01_AE viruses were then studied. Viral genes were amplified from serum samples in which titers of anti-HIV antibodies were quite low, as assessed by the captured BED-ELISA system. In addition, anti-HIV-1 neutralizing activities were undetectable in the pooled serum samples (Table 3); therefore, the samples had possibly been derived from acutely HIV-1infected individuals, although direct evidence was difficult to obtain. Previous reports show the predominance of R5 viruses among founder/transmitted viruses [35] or among viruses in the early phase of HIV-1 infection [36,37]. Consistent with these results, most early env genes derived from recently infected Thai individuals showed an R5 phenotype. Viral population in the early phase of HIV infection is genetically homogeneous within an individual [5,37], and the genetic diversity of HIV-1 env genes is increased in the course of infection [38]. In this report, we failed to study the intrasubject diversity of viral env genes in each serum sample; however, 14 early CRF01_AE env genes derived from 10 recently infected Thai individuals showed less divergence than 35 CRF01_AE env genes derived from 19 chronically infected individuals [13] (data not shown), suggesting that the genotype of CRF01_AE virus in the early phase of viral infection was considerably

homogeneous compared to the virus in the chronic phase of infection. In addition, the length of gp120 V1/V2 regions of early CRF01_AE Env derived from recently infected Thai individuals was significantly shorter than that of CRF01_AE Env derived from chronically infected individuals (Fig. 2), while no difference was observed in the average number of PNLG sites in Env gp120 derived from recently and chronically infected individuals [13]. Previous reports show that the length of variable regions and the number of PNLG sites are shorter or fewer in Env gp120 derived from recently infected individuals than those derived from chronically infected individuals [39,40]; therefore, our results were partly inconsistent with previous reports. A difference was observed in the susceptibility to b12, a HNMAbs against CD4BS on gp120, of CRF01_AE Env derived from recently and chronically infected Thai individuals. Consistently, a previous report showed that subtype B Env derived from transmitted viruses was more susceptible to neutralization by b12 than Env derived from viruses in the chronically infected phase [36,41]. However, another report showed that subtype A Env derived from recently infected individuals was reported to show low susceptibility to b12 [42], which is inconsistent with our results. These results

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suggested an intersubtype difference in the susceptibility of the virus derived from recently infected individuals to antibodies against CD4BS. Correlations were observed between 4E10 susceptibility and the length of or number of PNLG sites in gp120 variable regions (Fig. 3). 4E10 recognizes the MPER of Env gp41 and so these results were unexpected; however, previous reports showed that the mutations in gp120 amino acid residues that alter the efficiency of proteolytic cleavage of Env precursor gp160 also affect 4E10 susceptibility of HIV-1 Env [43]. 4E10 binds to the epitope in the MPER of gp41 during the process of viral and cellular membrane fusion that is triggered by the conformational change of Env gp120 upon its interaction with CD4 [44]. We consider that amino acid mutations in gp120 variable regions affect protein stability and the quaternary structure of Env gp120 and gp41 in the HIV-1 entry process, and eventually affect the susceptibility of Env proteins to neutralization by 4E10. In addition, an inverse correlation was observed between the length of V1/V2 regions and the susceptibility of recombinant viruses to a CCR5 antagonist, TAK779, (Fig. 3). Previous reports show that mutations in V1/V2 regions affect viral neutralization susceptibility to antibodies against the co-receptor binding site of gp120 [11] as well as the efficiency of viral infection through the CCR5 co-receptor [30]. Such effects of V1/V2 regions on the co-receptor binding site of gp120 may be due to the interaction of V1/V2 and V3 regions in the trimeric structure of HIV-1 Env [11]. We consider that the length of V1/V2 regions affects the interaction of V1/V2 and V3 regions as well as the protein structure of Env gp120 in the HIV-1 entry process, and eventually alters viral susceptibility to TAK-779. Finally, panels of subtypes A, B and C env genes were previously established from acutely or recently infected individuals, and phenotypic and genotypic characterizations of these early env genes were carried out [42,45,46]; however, such a panel of early CRF01_AE env genes has not been established. We believe that this is the first report to clone early CRF01_AE env genes from recently infected Thai individuals. Recent vaccine developments involve clinical trials using CRF01_AE Env as a vaccine antigen, including a clinical trial conducted in Thailand, RV144 [47]; therefore, we consider that the established early CRF01_AE Envrecombinant viruses are useful to evaluate the efficacy of immune responses elicited by vaccine candidates [48]. In addition, these recombinant viruses as well as cloned early CRF01_AE env genes are useful to study the phenotypic properties of transmitted/founder CRF01_AE virus, including its antigenicity and mucosal tissue tropism. These studies were carried out in part for subtype B virus using early env genes derived from founder/transmitted viruses [49,50]. We hope to conduct such work using the established early CRF01_AE Env-recombinant viruses in a future study. Acknowledgments We are grateful to Major General Boonyarak Poonchai, a Former Director of AFRIMS, Thailand, for his valuable help

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