Spatiotemporal hierarchy in antibody recognition against transmitted HIV-1 envelope glycoprotein during natural infection
© Jin et al. 2016
Received: 17 September 2015
Accepted: 4 February 2016
Published: 17 February 2016
Majority of HIV-1 infection is established by one transmitted/founder virus and understanding how the neutralizing antibodies develop against this virus is critical for our rational design an HIV-1 vaccine.
We report here antibody profiling of sequential plasma samples against transmitted/founder HIV-1 envelope glycoprotein in an epidemiologically linked transmission pair using our previously reported antigen library approach. We have decomposed the antibody recognition into three major subdomains on the envelope and showed their development in vivo followed a spatiotemporal hierarchy: starting with the ectodomain of gp41 at membrane proximal region, then the V3C3V4 and the V1V2 of gp120 at the membrane distal region. While antibodies to these subdomains appeared to undergo avidity maturation, the early anti-gp41 antibodies failed to translate into detectable autologous neutralization. Instead, it was the much delayed anti-V3C3V4 and anti-V1V2 antibodies constituted the major neutralizing activities.
Our results indicate that the initial antibody response was severely misguided by the transmitted/founder virus and future vaccine design would need to avoid the ectodomain of gp41 and focus on the neutralizing targets in the V3C3V4 and V1V2 subdomains of gp120.
Neutralizing antibodies are the major component of protective immunity against viral infection in humans. Polyclonal by nature, they exert their function by targeting the crucial antigenic domains on the viral envelop glycoprotein. Identifying the neutralizing antibodies and their recognized antigenic domains have therefore become the first crucial step for better understanding of the protective antibody response and the rational design of immunogens capable of eliciting the neutralizing antibodies [1–5]. In human immunodeficiency virus type I (HIV-1) infection, viral glycoprotein gp160 that mediates infection of CD4+ T lymphocytes is the sole target for neutralizing antibodies. The gp160 is composed of exterior, receptor-binding gp120 and the fusion-mediating, transmembrane gp41 subunits. The unique feature of gp160 is its extensive glycosylation and genetic diversity manifested by rapid generation and high turnover of viral variants during infection . Sequence and structural analysis has revealed the glycosylation and mutations are largely distributed in the hypervarible regions V1–V5 on the exterior surface of gp160 and function to protect the virus from antibody recognition and neutralization [1–5, 7, 8].
Majority of HIV-1 infection is established by one transmitted/founder virus with distinct genetic and phenotypic properties compared to those in the later stages of infection [9–12]. The development of neutralizing antibodies against this virus, however, follows an unusual pathway of inefficiency [2, 4, 13–18]. Most of the antibodies generated during the first few weeks lack neutralizing activities but reactive to gp41 as well as some non-HIV-1 antigens [19–21]. Only after a few months into the infection, autologous neutralizing antibodies become detectable, largely directed to gp120 and invariably strain-specific [4, 13, 14, 22]. Cross-reactive and broadly neutralizing antibodies (bnAbs) capable of neutralizing heterologous viruses across many genetic subtypes can only be generated after years into the infection and most notably in individuals who remain healthy despite prolonged period of infection [1–5, 15, 23]. Isolation and characterization of bnAbs from these individuals have identified five major targets on the gp160. These include the CD4-binding site (CD4bs), the glycan-associated V1V2 and V3/C3 subdomains of gp120, the membrane proximal external regions (MPER) of gp41, and the interface between gp120 and gp41 [1–5, 15]. But how exactly the autologous and bnAbs are generated during the course of HIV-1 infection remain largely unknown. Several elegant studies highlighted the critical role of interplay between viral evolution and antibody development. At the monoclonal levels, germline ancestors for neutralizing antibodies require stimulation by evolving or incoming viral variants during infection [24–29]. Different B cell lineages within the same individuals also appeared to work in concert to drive the development of neutralizing antibodies . At the polyclonal levels, however, dissecting the mechanism underlying the development of neutralizing antibodies is much more complex as polyclonal antibodies function through a dynamic and complex mixture of monoclonal antibodies with diverse targets on the gp160. Studies based on short peptides, chimeric and epitope-specific mutant viruses have identified a few subdomains of gp120 are the major targets for neutralizing activities in polyclonal sera [30–33]. However, the detailed understanding on the scope, specificities and dynamic features of polyclonal antibody recognition against the transmitted/founder virus remain elusive.
Here, we report antibody profiling of sequential plasma samples against transmitted/founder HIV-1 envelope glycoprotein in an epidemiologically linked transmission pair. Using our previously reported approach based on combinatorial antigen library displayed on the surface of the yeast Saccharomyces cerevisiae, we were able to delineate polyclonal antibody recognition in both qualitative and quantitative terms . Through sequential analysis of plasma-reactive antigenic sequences over the first 2 years of infection, we decomposed the polyclonal antibody recognition into three major subdomains and showed their development in vivo followed spatiotemporal hierarchy: starting at the ectodomain of gp41, then at the V3C3V4 and V1V2 of gp120. While antibodies to all three subdomains appeared to undergo avidity maturation, the early anti-gp41 antibodies demonstrated no detectable autologous neutralization and only those delayed anti-V3C3V4 and anti-V1V2 antibodies constituted the major neutralizing activities. Our results indicate that the initial antibody response was severely misguided by the transmitted/founder virus and future vaccine design would need to avoid the ectodomain of gp41 and focus on the neutralizing targets in the V3C3V4 and V1V2 subdomains of gp120.
Construction and validation of combinatorial antigen library from the transmitted HIV-1 envelopes
Neutralization sensitivity of transmitted/founder P08 and P11 pseudoviruses to various bnAbs
Spatiotemporal hierarchy in antibody development against distinct subdomains of HIV-1 envelope during natural infection
Avidity maturation of antibody recognition against distinct subdomains of HIV-1 envelope during natural infection
V3C3V4 and V1V2 but not gp41 contain the major targets for autologous neutralization
We report here the systematic characterization of antibody recognition against transmitted/founder HIV-1 envelope glycoprotein during natural infection in an epidemiologically linked transmission pair infected by highly homologous CRF01_AE strains. Based on several complementary approaches to determine the specificities of binding as well as neutralizing antibodies, we were able to decompose the complex plasma antibody recognition into three discrete subdomains on the HIV-1 envelope: ectodomain of gp41, V3C3V4 and V1V2 of gp120. The development of these subdomain-specific antibodies appeared to follow a spatiotemporal hierarchy with distinct dynamic, biochemical and neutralizing properties. While antibodies to all three subdomains appeared to undergo avidity maturation, the early and strong anti-gp41 antibodies failed to translate into detectable autologous neutralization. Instead, it was the much delayed anti-V3C3V4 and anti-V1V2 antibodies constituted the major neutralizing activities. In particular, it reinforced the early discoveries in that the majority of the initial antibody response was severely misguided by the transmitted/founder virus towards its gp41 subdomain and therefore missed the most critical window of opportunity to contain or clear the virus replication through recognizing the neutralizing epitopes in the V3C3V4 and V1V2 subdomains [19, 20]. By the time when the neutralizing antibody response was indeed mounted in a substantial manner, it was much too late and virus had already established its permanent residence in the target cells. Such defects in mistargeting and mistiming have provided some explanations for the failure of human immune system to contain viral replication during early infection, and strongly recommend that future vaccine design would need to avoid the ectodomain of gp41 and focus more on those neutralizing targets in the V3C3V4 and V1V2 subdomains of gp120.
At the current stage, we are uncertain about the underlying mechanisms leading to the spatiotemporal hierarchy for antibody recognition against the three major envelope subdomains. The overwhelming response against gp41 during early infection could be due to the pre-existing gp41 cross-reactive memory B cells that acquired reactivity with autologous gp41 [19, 44, 45]. A recent study showing majority of gut-derived anti-gp41 antibodies cross-reacted with commensal bacteria supports this hypothesis . It could also be due to the shedding of gp120 leading to the exposure of preferred structures during early infection although the exact step and timing of such preference during viral replication are currently unknown. Generally speaking, gp41 exhibits at least three distinct conformational states during the viral fusion process: the prefusion, the prehairpin intermediate, and the postfusion conformation. It is believed that the conformational differences among the three states are so great that each of them likely presents distinct antigenic surface to the immune system [46–48]. So far, only the prehairpin intermediate was found to be the target of bnAbs such as 2F5, 4E10 and 10E8 while the other two states were largely recognized by non-neutralizing antibodies. In particular, the non-neutralizing antibodies against gp41 appeared to group in two clusters based on the location of their respective epitopes. Cluster I antibodies recognize the immunodominant C–C loop of gp41 (aa590–600), and the cluster II antibodies react with the downstream immunodominant segment (aa644–663) [46–49]. But whether the two clusters of antibodies specifically react with prefusion and postfusion conformation remain to be determined. As the antibody recognition found in our study subjects overlapped with cluster I antibodies, the conformational state against which they were initially generated was unlikely to be the prehairpin intermediate. Whatever the conformational state was recognized, it must be the one to be avoided in our vaccine design to prevent non-neutralizing epitopes as well as severe misguidance and mistiming found during natural infection.
Neutralizing activity of P08 and P11 plasma samples against a panel of pseudoviruses with distinct genotypic and phenotypic features from China and abroad
Our study has unraveled the complex and dynamic feature of antibody development against transmitted/founder HIV-1 envelope glycoprotein during natural infection. The major binding and neutralizing antigenic subdomains identified here will provide critical reference for our better understanding of the spatiotemporal feature of protective antibody response during natural infection and assist our rational design of vaccines that will empower the strengths while minimize the weaknesses of human immune recognition.
Study subjects and plasma samples
Two acutely infected individuals, P08 and P11, were chosen for the study. P08 was 37 and P11 38 years old when identified through China’s largest acute infection cohort for man who have sex with man (MSM) that followed several thousands of high risk individuals over the last decade. P08 and P11 were epidemiologically linked transmission pair and P08 infected P11 during acute infection based on epidemiologic and clinic documentation. When enrolled on day 30 after infection for P08 and day 18 for P11, both individuals were negative for HIV-1 antibody measured by enzyme-linked immunosorbent assay (ELISA) and indeterminate Western blot test, and therefore fell into Fiebig II-IV substage of acute infection . P08 and P11 had baseline CD4 lymphocyte count of 339 and 369 per cubic millimeter (FACS Calibur, BD) and plasma viral load of 30,600 and 889,000 RNA copies per milliliter (Cobas AmpliPrep/Cobas TaqMan HIV-1 version 5.1 Assay, Roche), respectively. Both individuals progressed to diseases relatively fast and by 2 years into the infection, the CD4 lymphocyte count dropped to 147 for P08 and 181 for P11 per cubic millimeter and plasma viral load remained as high as 12,500 for P08 and 40,429 for P11 RNA copies per milliliter. Sequential plasma and peripheral blood mononuclear cells (PBMCs) were collected over the first 2 years of infection and stored at −80 °C until use. Neither P08 nor P11 received any antiretroviral therapy during the study period. This study was reviewed and approved by the institutional research ethics committee at the No. 1 Hospital of China Medical University in Shenyang, Liaoning Province, China.
Full-length envelopes, phylogenetic analysis, pseudoviruses and neutralization assay
The full-length envelope genes from P08 (P08-gp160) and P11 (P11-gp160) were obtained through PCR amplification of single HIV-1 RNA molecules directly from the plasma samples. The reference envelopes from subtype A (KER2018.11 and RW020.2), subtype B (JRFL, Bal.01, YU2.DG) and subtype C (ZA012.29, ZM106.9, ZM55.28a) were kindly provided by John Mascola of Vaccine Research Center at National Institute of Health (NIH). The representative envelopes from HIV-1 infected individuals in China came from our previously studies including those from CRF01_AE, subtype B′, subtype B′C, and CRF07_BC and CRF_08BC. These full-length envelope sequences were aligned using the Clustal W program together with selected subtypes/CRFs of geographical importance from the Genbank database. Phylogenetic analysis was conducted using the neighbor-joining method and the reliability of the branching orders was tested by bootstrap analysis of 1000 replicates .
These envelope clones were also used to generate pseudoviruses by co-transfection with backbone construct pNL43R-E-luciferase into the 293 cells. Forty-eight hours later, the culture supernatant containing the pseudoviruses was collected and tested for luciferase activity to standardize viral input in the subsequent neutralization analysis. Neutralizing activities of plasma samples from P08 and P11 and neutralizing sensitivity of P08 and P11 pseudoviruses to various bnAbs were analyzed as previously described . In brief, 100 TCID50 of pseudoviruses was incubated with serially diluted plasma, or various bnAb in a 96-well plate in triplicate for 1 h at 37 °C. Approximately 2 × 104 TZM-bl cells were then added and the cultures were maintained for an additional 48 h at 37 °C. Neutralizing activity was measured by the reduction in luciferase activity compared with controls (Bright-Glo luciferase assay system, E2650, Promega). Half-maximal inhibitory concentrations or dilutions (IC50 or ID50) were reported as the concentration for bnAbs or dilution for plasma required to inhibit infection by 50 % compared with the controls. The values were calculated using the dose–response inhibition model with a variable slope in GraphPad Prism, version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). The bnAb PG9 and PG16 were kindly provided by Wayne Koff at International AIDS Vaccine Initiative (IAVI), VRC01 by John Mascola of Vaccine Research Center at NIH, 3BNC117 by Michel Nussenzweig at Rockefeller University, and Ibalizumab by David Ho at Aaron Diamond AIDS Research Center of Rockefeller University. The rest of the bnAbs were obtained from NIH reference and reagents program.
Generation and verification of envelope chimeras
Chimeric gp160 envelopes were generated using an overlapping PCR strategy with gp120, gp41, V3C3V4, V1V2 subdomains amplified separately and then together with the flanking regions from the backbone of CNE6. The resultant envelope chimeras were cloned into the pcDNA3.1 (Invitrogen), verified by sequencing before used for pseudovirus production. Sensitivity of chimeric pseudoviruses to plasma neutralization was measured as described above.
Construction and expression of transmitted/founder HIV-1 envelope combinatorial libraries on the surface of yeast Saccharomyces cerevisiae
Construction of yeast library displaying the combinatorial antigens was carried out as described previously . In brief, the full-length P08 and P11 envelope gene from the day 30 and day 18 after infection, respectively, was amplified by single molecule PCR, purified (QIAquick DNA purification kit, QIAGEN) and digested by DNase I into fragments about 50 base pair (bp) in length. The digested fragments were reassembled to approximately 100–600 bp fragments through controlled number of PCR cycles, added A-tails (DNA A-tailing kit, TAKARA) and ligated to the modified yeast surface displayed vector pCTCON2-T. The ligation products were transformed into the Escherichia coli competent cells, amplified, extracted and then further transformed into the competent yeast cell line EBY100 using electroporation. Transformed yeast cells were partially spread on SDCAA Amp plates and incubated overnight at 30 °C to estimate the number and insert sequences of colonies for quality control purpose. Conditions for yeast growing and induction of surface antigen expression in solution have been previously described . In short, EBY100 yeasts were first grown in SDCAA media at 30 °C for 48 h. At the exponential growth phase, yeasts were transferred to SGCAA media for induction of antigen expression at 20 °C for 48 h before incubating with either plasma samples or monoclonal antibodies for subsequent analysis .
Immunofluorescence staining, sorting, sequencing and sequence analysis of bnAb- or plasma-reactive yeast clones by FACS
The entire procedure was conducted as previously described . Induced yeast cells (106–107) were collected by centrifugation (6000 rmp/s, 1 min), washed twice with cold PBS and incubated with either bnAb or patient plasma (1:100 dilution) on ice for 1 h with occasional agitation. After washed three times with cold PBS, the cells were incubated with PE labeled anti-human IgG secondary antibody (1:200 dilution, rabbit anti-human IgG-PE, Santa Cruz) on ice for another 45 min, washed again with PBS for three times, analyzed and sorted for positive clones using FACS Aria II (BD, USA). The positive yeast clones were grown in SDCAA before plasmids were extracted (Yeast plasmid kit, Omega Bio-Tek) for sequencing and sequence analysis (Sequencher 5.0, Gene Codes Corp.). The most frequently recognized amino acid residues within each subdomain were calculated as over 90 % percentile among the selected fragments for each subdomain.
Measurement of Kd and Ka for each antigenic subdomain
The technique relies on measuring the MFI of the bound polyclonal antibodies, at and variety of concentrations of polyclonal antibodies, on the c-myc positive yeast . Specifically, four representative yeast clones were selected from each envelope subdomain based on their coverage and mixed in equal proportion (106 cells) before incubated with a serial 1:3 dilution of sequential plasma samples from P08 and P11. After washed twice with cold PBS, the mixture was resuspended in PE labeled anti-human secondary antibody (1:200 dilution, rabbit anti-human IgG-PE, Santa Cruz) and incubated for another hour. Antibody recognition of yeast clones expressing the distinct envelope subdomains were detected by FACS. The MFI was recorded, plotted and fitted using a nonlinear least square curve against the reciprocal plasma dilution. Kd value was determined using the following equation: y = MFImax × Plasmadilution−1/(Kd + Plasmadilution−1). Ka is the reciprocal of Kd.
Measurement of plasma binding to trimeric and monomeric gp120 and gp140 by ELISA
Trimeric ectodomain of NL4-3 (subtype B) constructed based on BG505 SOSIP.664  was kindly provided by Dr. Yi Shi at Institute of Microbiology, Chinese Academy of Sciences. Monomeric gp120 and monomeric gp140 (CRF01_AE) derived from a CRF01_AE circulating strain CM235 (Genebank #: AAG28611) isolated in Thailand in year 2000 were purchased and produced from 293T cells (Immune Technology Corp, China). Recombinant envelope glycoprotein was coated overnight at 4 °C on the 96-well plate (100 ng/well), blocked for 2 h at 37 °C with 1 % bovine serum albumin (BSA) in PBS before addition of ten serial threefold dilutions of plasma samples. After incubating for 1 h at 37 °C and three-time thorough washes with PBST (PBS with 0.05 % Tween), the secondary antibody conjugated with horseradish peroxidase (1:4000 dilution, anti-human IgG-HRP, Promega) was added before applying substrate for detectioin. Maximum absorbance at 450 nm and corresponding plasma dilution were recorded (Microplate Reader, Bio-Rad). Plasma samples from HIV-1 negative individuals were included as negative controls.
SJ, YJ, QW, HW, XS, and XH conducted experiments. TZ analyzed the antigenic subdomains in the context of gp160 sequence. HS and LZ designed the study and were involved in writing of the manuscript. All authors read and approved the final manuscript.
We thank Drs. K. Dane Wittrup and Annie Gai at Massachusetts Institute of Technology for providing yeast surface display vector pCTCON2 and Drs. John Mascola of Vaccine Research Center at NIH, Wayne Koff at IAVI, Michel Nussenzweig and David Ho at Rockefeller University for providing bnAbs. We also thank patients for their participation.
The authors declare that they have no competing interests.
This work was supported by the funds from National Natural Science Foundation Award 81530065, the National Science and Technology Major Projects (2012ZX10001-006, -004 and -009), Ministry of Science and Technology of China (2014CB542500-03), also in part by the Tsinghua University Initiative Scientific Research Program (20124812029) and Janssen Investigator Award to Linqi Zhang.
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