Neutralizing Antibody Responses and Evolution of the Viral Envelope in the Course of HIV-1 Korean Clade B Infection

2.1. Study subjects, cells, and plasma samples

Blood samples taken from patients with a suspected HIV infection were referred to the Korea Center for Disease Control (KCDC) from public health centers, hospitals and local blood banks through local Institutes of Public Health and Environment (IPHE) for the final HIV confirmation test. Among these patients, three were diagnosed with preseroconversion status and could be monitored longitudinally. None of these subjects received antiretroviral therapy over the course of this study.

293T/17 and TZM-bl cells were obtained from the National Institute for Biological Standards and Control (catalog No. ARP5011) and the American Type Culture Collection (catalog No. 11268), respectively. An env-deficient HIV-1 backbone vector (pSG3 Env) was obtained through the NIH AIDS Research and Reference Reagent Program (catalog No. 11051).

2.2. Cloning of full-length HIV-1 env genes and pseudovirus production

Genomic DNA was extracted from the peripheral blood mononuclear cells (PBMCs) using QIAamp DNA blood mini kits (QIAgen, Valencia, CA, USA). We amplified a 3.2-kb region of the HIV-1 env gene using nested polymerase chain reaction (PCR), as previously described [9].

The purified PCR products were cloned into the pcDNA3.1/V5-His-Topo vector (Invitrogen Corp., Carlsbad, CA, USA). Pseudoviruses were produced by infecting 293 T cells with the env expression plasmid and pSGΔenv vector using the FuGENE 6 transfection kit (Invitrogen). Pseudovirus-containing culture supernatants were harvested 72 hours after transfection, filtered (0.45 ㎛), and stored at -80 ℃ until use in the neutralization assays.

2.3. Neutralization assay

The activities of the neutralizing antibodies were measured as the reduction in β-galactosidase reporter gene expression after a single round of viral infection in TZM-bl cells, as previously described [1]. In brief, 100 TCID₅₀ pseudoviruses and heat-inactivated plasma mixtures were incubated at 37℃ for 1 hour and then added to a preparation of TZM-bl cells (1 ×10⁴ cells/mL) on 96-well plates. The cells were then harvested after incubation for 48 hours and counted using a β-galactosidase-staining V2600 staining kit (Takara Bio Inc., Tokyo, Japan). The 50% inhibitory concentration (IC₅₀) of the neutralizing antibody was defined as the concentration of plasma dilution required to decrease the number of infected cells by 50% at 48 hours after infection with 100 TCID₅₀, and the IC₅₀ was calculated from the mean value of the repeated results. Two independent experiments were performed in duplicate.

2.4. Sequencing and phylogenetic analyses

The sequencing reaction for the env region was performed using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, CA, USA) in an automated ABI prism 3730 DNA sequencer (Perkin Elmer, CT, USA). The nucleotide and amino acid sequences of the env gene were aligned using the Lasergene software package (DNASTAR Inc., WI, USA). Phylogenetic analyses were performed using PAUP (Phylogenetic Analysis Using Parsimony, version 4.0; http://paup.csit.fsu.edu/). The number and position of potential N-linked glycosylation sites (PNGS) were analyzed using the N-GlycoSite program (www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html). Aligned amino acid sequences were analyzed using Jalview (www.jalview.org).

2.5. Statistical analysis

Statistical analyses were performed using SAS version 9.1 (SAS Institute, Inc., Cary, NC, USA). Spearman’s rank correlation method was used to determine the significance of any association between the neutralizing antibody response and the HIV env sequence.

3.1. Characteristics of study subjects

Three subjects (K1004, K1314, and K2160) with primary HIV infection (PHI) were selected for monitoring over the course of their infections, all of whom were male subjects. The average age of the selected group was 36.3 years. The average course of infection after PHI was 4.8 years, and eight time points were sampled over 85 months (7.1 years) for K1004, six time points over 63 months (5.3 years) for K1314, and five time points were sampled over 25 months (2.1 years) for K2160. The route of transmission for each subject was sexual contact with men who have sex with men (MSM). The mean values for the longitudinal samples were 449 cells/mm³ (range: 147-896 cells/mm³) for CD4⁺ T cells and 1,083 cells/mm³ (range: 599-1,954 cells/mm³) for CD8⁺ T cells (Table 1). Phylogenetic tree analysis of all HIV-1 env sequences showed that all isolates were HIV-1 Korean clade B (Figure 1).

Table 1.

Epidemiological and immunological characteristics of Korean subjects infected with HIV-1 type B

Figure 1.

Phylogenetic relationships between HIV-1 env (gp120) sequences of Korean subjects infected by HIV-1 Korean clade B and env sequences of 17 different type B reference strains from the HIV database in Los Alamos National Laboratory (http://www.hiv.lanl.gov). This phylogenetic tree was constructed using the neighbor-joining method used by the PAUP* 4.10b program. The numbers at the nodes represent the bootstrap values calculated from 1000 bootstrap resamplings. The three infected Korean patients are labeled from according to the sampling time.

3.2. Viral escape pattern of neutralization against autologous viruses over the course of evolution

Pseudoviruses were produced from each individual in order to analyze neutralization reactivity. We determined the neutralization reactivity against autologous HIV-1 env pseudovirus at each sampled time point in order to analyze cross-reactivity at different stages of evolution.

In the evaluations of the neutralizing activities to HIV-1 plasma at different stages, the pseudoviruses that were produced in the early phase showed low neutralizing reactivities to contemporaneous plasma compared with the later phase, which was significant except for K2160 (p < 0.005). In K1004 and K1314, gradual increases were observed after early phase, but these

increased neutralization activities were reduced by 4 years post-HIV infection. K2160, who was monitored for relatively shorter time than the other subjects, showed a gradually increase in neutralization activities that was time dependent (Figure 2). Specifically, K1004, who produced by eight samples at each time point, showed the most complicated neutralization pattern which also showed reversion. There was an increase in the neutralization pattern in the early phase through the fourth time point, but this increase began to decrease through measurement at the seventh time point. In case of K1314, the results show an increased pattern from the first through the fifth time point and reduced neutralization by the last time point, similar to K1004. However, for K2160, who provided five samples at each time point, we were unable to determine the escape pattern in the later phase; only a steadily rising neutralization pattern was identified in the early phase.

Figure 2.

Autologous cross-reactivity of HIV-1 pseudoviruses against neutralizing antibodies in Korean HIV-1 longitudinal subjects over the course of HIV-1 infection. PV, pseudovirus.

3.3. Genetic variation of env gp160 over the course of evolution

Genetic variations mainly existed in gp120 as result of insertions and deletions in this region. In K1004, most PNGS variations were observed in all variable loops over the course of infection, but some changes in the lengths of various amino acids could only be observed at V4 region (Table 2). In K1314, the lengths of the amino acids and PNGS were changed in all regions except V3. Specifically, the lengths of the amino acids over the course of infection showed a decrease in V1, V4, and V5, and the number of PNGS also decreased in V1 and

Table 2.

Comparison of amino acids lengths and number of PNGS in HIV-1 env gp160 in Korean HIV-1 subjects

V4. In K2160, variations in the lengths of amino acids were observed only at V1 and V5, but the number of PNGS changed in all regions except V3.

In the analysis of the CD4- and CCR5-binding sites, most amino acids were conserved in those sites, but some changes were observed over the course of infection and the changes observed at each time point were different for each individual. Regarding changes in the CD4-binding site, D474 N was noted in subject K1004 and N279D, T283 N, and D474 N were noted in subject K1314. Regarding changes in CCR5-binding site, F317L and R440 K were noted in subject K1004, and changes in K440R were noted in subject K1314. However, no changes were observed in subject K2160 (data not shown).

3.4. Relationship between env sequence and neutralization reactivities

The results of the analysis between variations in env genes and neutralization titers (IC₅₀) showed that the lengths of the amino acids in regions V1-V5 had a significant but weakly positive correlation with the neutralization titers (r=0.279, p=0.026) in subject K1004. Also, the number of PNGS in same region was positively correlated with the neutralization titers (r=0.349, p=0.005). However, in the case of subject K1314, the lengths of the amino acids and the number of PNGS were weakly and positively correlated with the neutralization titers, but this finding was not significant. Also, correlation analysis of subject K2160 showed results that were similar to those of subject K1314 (Figure 3).

Figure 3.

Correlation between amino acid length and PNGS number in the V1–V5 regions of HIV-1-infected Korean subjects and the total reactivity against neutralizing antibodies. (A) K1004, (B) K1314, (C) K2160.