Multi-layered Gag-specific immunodominant responses contribute to improved viral control in the CRF01_AE subtype of HIV-1-infected MSM subjects

In this study, 15 subjects with the CRF01_AE subtype cluster II of primary HIV-1 infection were recruited from a large-scale, high risk MSM cohort in Liaoning, China. Blood samples and epidemiological information were acquired every 10 weeks. HIV antibody screening was tested with third-generation ELISA and rapid test, and validation was performed with western blot assay. Antibody negative samples were tested with HIV nucleic acid. Homosexual transmission modes were confirmed by epidemiological investigation for all subjects. The date of infection was estimated as 14 days prior to RNA-positive and antibody-negative results (9 subjects) or the middle time between the last antibody-negative test and the first antibody-positive test (6 subjects). The average age of the 15 subjects was 35 ± 14 years old. Peripheral blood mononuclear cells (PBMCs) from the 15 subjects were separated by Ficoll-Paque^TM Plus (GE Healthcare Bio-Sciences AB, Sweden) density gradient centrifugation and cryopreserved in liquid nitrogen. The viral set point was defined as the average viral load (at least 3 time points) from 120 days to 1 year post infection, and the mean viral set point of the 15 subjects was 4.24 log₁₀copies/ml (Table 1). The CD4⁺ T-cell counts of all the subjects were more than 200 cells/μl during the first year of infection and all the subjects were anti-retroviral naïve during the first year of infection. The signed written informed consents were got from all subjects for blood collection and the following study. The First Hospital of China Medical University Ethics Committee approved this study (2011–36).

CD4⁺ T-cell counts were determined using a FACS Calibur flow cytometer (Becton-Dickinson, USA). Plasma HIV-1 RNA levels were measured with the COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Roche, Germany). HLA typing was performed using the polymerase chain reaction sequence-specified primer (PCR-SSP) method, with two-digit specificities (One Lambda, USA).

In order to detect the CTL responses as more as possible, a set of cluster specific 18-mer peptides with 10 overlapping amino acids spanning Gag, Pol and Nef genes were synthesized by Sigma-Aldrich (U.S.). This set of peptides was designed based on the consensus sequence of 50 near full length sequences from acute CRF01_AE cluster II infected subjects which came from a HIV seronegative prospective MSM cohort,with definite laboratory and epidemiology evidences for acute infection. A total of 209 overlapping peptides (OLPs) were synthesized; screening was performed using a 14 × 16 matrix design in which the principle is to mix the Gag, Pol and Nef peptides in the second dimension rather than the first dimension (Additional file 1: Table S1). Peptides with any response in a given matrix allowed identification of the common peptide represented in the two corresponding pools. The identity of each peptide was confirmed using PBMCs from the same time point. We also synthesized optimal epitopes restricted by common HLA-I alleles (A*0201, A*11, A*24, B*13, B*27, B*40, B*51) in the Chinese population [35] (Additional file 2: Table S2). Optimal epitopes were synthesized according to sequences from CRF01_AE subtype cluster II.

IFN-γ enzyme-linked immunospot (ELISPOT) assays were performed as previously described [36]. In brief, PBMCs were plated in 96-well plates that were precoated with anti-IFN-γ monoclonal antibody at 100,000 cells per well with peptides at a final concentration of 5 μg/ml for pooled peptides, single peptides, and optimal epitopes (BD^TM ELISPOT, USA). Phytohemagglutinin (PHA) served as a positive control at a concentration of 10 μg/ml, and medium alone was the negative control. Spots were counted using the ImmunoSpot® Analyzer (Cellular Technology Ltd, USA), and the number of specific T-cells was counted by subtracting the mean negative control values. Responses were considered positive if the activity was at least three times greater than the mean number of spot-forming cells (SFCs) in the negative control as well as >50 SFCs per 10⁶ PBMCs.

Viral RNA was extracted from plasma samples obtained from MSM subjects with the QIAamp Viral RNA Mini-kit (Qiagen, UK). The Gag gene was amplified using the SuperScript Polymerase One-Step RT-PCR System (Takara, Dalian, China), followed by a second round of PCR with Gag-specific primers [36]. PCR products were gel purified with the QIAquick gel extraction kit (Qiagen, UK) and cloned with a TOPO TA cloning kit (Invitrogen, USA). The cloning PCR fragments were then sequenced with Sanger sequencing method using ABI 3730 DNA analyzer by Huada Genomics Company (China). A sequencer program was used to assemble and edit individual sequence fragments.

Data analysis and graphical presentation were performed with IBM SPSS version 20.0 software and GraphPad Prism version 5.0 software. Statistical analysis of significance was performed using either the Wilcoxon signed-rank test or Friedmen’s two-way analysis of variance by ranks followed by pairwise multiple comparisons for comparing characteristics of HIV-1-specific CTL responses and using the Mann-Whitney test for comparing the viral set points between different groups. Spearman rank correlation was used to assess relationships between viral set points and HIV-specific T-cell responses. A p value less than 0.05 was considered statistically significant.

To clarify the characteristics of CTL responses during the first year of infection, Gag-, Pol- and Nef-specific CTL responses were evaluated longitudinally in 15 MSM subjects with the HIV-1 CRF01_AE subtype at 3 months and 1 year post infection. There were no significant differences at either time point among Gag-, Pol- and Nef-specific CTL responses in terms of magnitude and breadth (p > 0.05). The magnitude and breadth of Gag, Pol and Nef responses increased over time, with significant differences in the magnitude of Gag and Pol (p < 0.05 for both) and breadth of Pol and Nef (p < 0.05 for both) (Fig. 1a, b).

We adjusted for the length of amino acids by dividing the amino acids number of the corresponding protein and multiplying by 100, and found that Gag and Nef responses were the mainly targets during the first year of infection. The adjusted breadth of Gag was significantly broader than that of Pol at 1 year post infection (p < 0.05, Fig. 1d). Similarly, the adjusted breadth of Nef was significantly broader than that of Pol at 3 months and 1 year post infection (p < 0.05 and p < 0.001, respectively, Fig. 1d). In addition, the adjusted magnitude of Gag and Nef were significantly higher than that of Pol at 1 year post infection respectively (p < 0.05 and p < 0.01, respectively, Fig. 1c). There were no significant differences among Gag, Pol and Nef in adjusted magnitude at 3 months post infection. Different protein regions presenting different T-cell responses may be associated with variations in viral control.

To clarify the relationship between CTL responses and viral control in the CRF01_AE subtype, we analyzed the effects of magnitude and breadth of Gag-, Pol- and Nef-specific CTL responses at 3 months and 1 year post infection on viral set point. Neither magnitude nor breadth of Gag-, Pol- or Nef-specific responses were correlated with viral set point during the first year of infection (p > 0.05, Fig. 2a–f and Additional file 3: Figure S1a–S1f), although the magnitude of Nef at 1 year post infection was significantly positively correlated with viral set point (p = 0.01, r = 0.642, Fig. 2f).

Several studies have suggested that relative magnitude and breadth, which were defined in this study as the proportion of magnitude and number of reactive peptides in a specific protein to the total virus-specific magnitude and the total number of recognized peptides, respectively, are more sensitive markers of viral control than magnitude and breadth [16, 37]. Thus, we further analyzed the effect of relative magnitude and relative breadth of Gag-, Pol- and Nef-specific CTL responses on viral set point during the first year of infection. We observed a significantly negative correlation between the relative magnitude of Gag and viral set point at 3 months (p = 0.002, r = −0.726, Fig. 2g) and 1 year (p = 0.025, r = −0.574, Fig. 2j) post infection. However, no correlation was observed between the relative magnitude of Pol-specific responses and viral set point during the first year of infection (p > 0.05, Fig. 2h–k). In contrast, the relative magnitude of Nef at 1 year post infection was significantly positively correlated with viral set point (p = 0.004, r = 0.697, Fig. 2l). In addition, no correlation was observed between the relative breadth of Gag-, Pol- and Nef-specific responses and viral set point at 3 months or 1 year post infection (Additional file 3: Figure S1g-S1l). This suggests that the higher relative magnitude of Gag presented during the first year of infection leads to improved viral control.

A higher relative magnitude of Gag is likely associated with improved viral control, indicating an immunodominant response, which is defined as one of the strongest responses for each subject at each time point in this study, targeting the Gag protein may also be related to better viral control. We analyzed the relationship between immunodominant response and viral set point for each subject to clarify the effect of Gag immunodominant response on viral control. Different subjects possessed shifting immunodominance hierarchies over time due to interactions between host and virus. Based on different characteristics of immunodominance and viral set point, we classified the 15 subjects into 3 groups. Group 1 was characterized by having multi-layered immunodominant responses targeting Gag during the first year of infection, which was defined as having Gag immunodominant responses targeting the same epitope or different epitopes at both 3 month and 1 year post infection to force persistent immune pressure on virus. 5 subjects belonging to group 1 showed lower viral set points, ranging from 2.84 to 3.75 log₁₀copies/ml. Of the 5 subjects, subject 2 had an immunodominant response targeting the KK10 epitope restricted by HLA-B27 at 3 months and 1 year post infection. However, the other 4 subjects (subject 1, 3, 4 and 5) had immunodominant responses targeting different Gag epitopes at 3 months. The immunodominance hierarchies subsequently shifted over time, and the subjects developed another layer of immunodominant responses targeting new Gag epitopes at later stages of infection (Fig. 3a–e). Group 2 was characterized by having immunodominant responses targeting different proteins during the first year of infection, the viral set points of the 6 subjects belonging to group 2 ranged from 4.29 to 4.62 log₁₀copies/ml. Of the 6 subjects, subject 12 had Gag immunodominant responses at 3 months, but not at 1 year post infection (Fig. 3f). Subject 6, 9 and 11 had Pol immunodominant responses at 3 months post infection, and subject 11 had a persistent Pol immunodominant response during the first year of infection (Fig. 3g–i). Subject 7 and 10 had Nef immunodominant responses at 3 months, but not at 1 year post infection (Fig. 3j, k). Group 3 was characterized by having persistent immunodominant responses targeting Nef during the first year of infection, 4 subjects belonging to group 3 showed the highest viral set points among these three groups, ranging from 4.43 to 5.38 log₁₀copies/ml (Fig. 3l–o). None of the subjects in group 2 nor group 3 had multi-layered immunodominant responses targeting Gag during the first year of infection.

We further compared the viral set points from different subjects according to Gag immunodominance and found that the viral set point in group 1 was significantly lower than in group 2 and group 3 (3.33 log₁₀copies/ml VS 4.69 log₁₀copies/ml, p = 0.002), meaning that the viral set points in subjects who had multi-layered immunodominant responses targeting Gag were significantly lower than in subjects without the same response during the first year of infection. This suggests that multi-layered immunodominant responses targeting Gag during the first year of infection were correlated with viral control.

The observation that multi-layered Gag-specific immunodominant responses during the first year of infection were correlated with viral control suggested that Gag immunodominant responses may exert strong immune pressure on the virus. To study epitope mutation under the pressure of Gag immunodominant responses, we analyzed Gag cloning sequences during the first year of infection. Epitope and flanking region mutations were defined as amino acid mutations with a ≥50 % change in cloning sequences at 1 year compared to the cloning sequences at 3 months. We observed a large proportion of mutated Gag immunodominant epitopes and their flanking regions over time. The AV9 epitope in subject 4 and the IL10 epitope in subject 12 had mutations at 1 year post infection. In addition, the GI11 epitope in subject 5 had flanking region mutations at 1 year post infection. OLP-45 in subject 1 and OLP-10 in subject 3 had flanking region mutations at 1 year post infection based on putative epitopes within the 2 overlapping peptides. Only the KK10 epitope in subject 2 had no mutations and maintained an immunodominant response at 1 year post infection (Table 2). Epitope and flanking region mutations could hamper CTL responses [6, 38]. With epitope and flanking region mutations, the magnitude of AV9, GI11, OLP-45 and IL10 epitopes were all reduced at 1 year post infection, and only OLP-10 in subject 3 was slightly increased (Fig. 3). Under CTL pressure, 5 out of 6 Gag immunodominant epitopes or their flanking regions mutated over time, suggesting that Gag immunodominant responses at 3 months exerted strong pressure on the virus.