Multiple T-cell responses are associated with better control of acute HIV-1 infection

Abstract Cytotoxic T lymphocyte (CTL) responses play pivotal roles in controlling the replication of human immunodeficiency virus type 1 (HIV-1), but the correlation between CTL responses and the progression of HIV-1 infection are controversial on account of HIV immune escape mutations driven by CTL pressure were reported. The acute HIV-1-infected patients from Beijing were incorporated into our study to investigate the effects of CTL response on the progression of HIV-1 infection. A longitudinal study was performed on acute HIV-1-infected patients to clarify the kinetic of T-cell responses, the dynamic of escape mutations, as well as the correlation between effective T-cell response and the progression of HIV infection. Seven human leukocyte antigen-B51+ (HLA-B51+) individuals were screened from 105 acute HIV-1 infectors. The detailed kinetic of HLA-B51-restricted CTL responses was described through blood sampling time points including seroconversion, 3 and 6 months after HIV-1 infection in the 7 HLA-B51+ individuals, by using 16 known HLA-B51 restricted epitopes. Pol743–751 (LPPVVAKEI, LI9), Pol283–289 (TAFTIPSI, TI8), and Gag327–3459 (NANPDCKTI, NI9) were identified as 3 dominant epitopes, and ranked as starting with LI9, followed by TI8 and NI9 in the ability to induce T-cell responses. The dynamics of escape mutations in the 3 epitopes were also found with the same order as T-cell response, by using sequencing for viral clones on blood sampling at seroconversion, 3 and 6 months after HIV-1 infection. We use solid evidence to demonstrate the correlation between T-cell response and HIV-1 mutation, and postulate that multiple T-cell responses might benefit the control of HIV-1 infection, especially in acute infection phase.


Introduction
It was well accepted that cluster of differentiation 8 + (CD8 + ) T cells play a pivotal role in controlling human immunodeficiency virus type 1 (HIV-1) replication. For one thing, a temporal association is observed between the rise of virus-specific cytotoxic T lymphocyte (CTL) responses and the reduction of viremia during acute phase of HIV-1 infection, beyond that, the peak of the CTL response coincides with the drop in viral load. [1,2] For another, in the nonhuman primate models with simian immunodeficiency virus (SIV) infection, the depletion of CD8 + T cells during the acute phase of SIV infection, [3] or chronic SIV infection results in a rapid increase of viral load that is again suppressed when the SIV-specific CD8 + T cells reappear. [4] Furthermore, the escape mutations detected in the CTL epitopes reveal the evidence of immune pressure is exerted by CTL responses. [5] The first escape variants can replace the original sequence of the founder virus within days, and as a consequence of continuous immune pressure, sequential mutations at different epitopes are followed. [6] Eventually, the frequency of escape mutations correlates with the prevalence of the restricting human leukocyte antigen (HLA) class I allele at the population level. [5,7,8] While the correlation between CTL responses and the progression of HIV-1 infection became controversial, because of viral escape is also driven by CTL pressure, which is the key roadblock to the successful development of an efficient T-cell-based HIV-1 vaccine. [9] Several strategies are employed to design an effective T-cellbased HIV-1 vaccine, from immunodominant [10] and subdominant epitopes [11] to conserved [12] or mosaic [13] epitopes. Amongst these strategies, the kinetics and quality of early immune responses to HIV-1 are not very clear, while their implications for developing a successful preventive HIV-1 vaccine have become more and more valuable.
HLA-B51 is shown to be one of the protective class I alleles associated with delayed disease progression of HIV infection, especially in Asian population. [7] HLA-B51 restricted epitopes including Gag327-345 (NANPDCKTI, NI9), Pol743-751 (LPPVVAKEI, LI9), and Pol283-289 (TAFTIPSI, TI8), as well as mutations in these 3 dominant epitopes are identified in our previous study. [14] The kinetics of T-cell responses and the dynamic of escape mutations during acute phase of HIV-1 infection were demonstrated in this study to clarify the correlation between the T-cell responses, escape mutations, and disease progression.
We selected 7 HLA-B51+ individuals from acute HIV-1 infection cohort, and the kinetic of HLA-B51-restricted CTL responses was described through time points of seroconversion, 3 and 6 months after HIV-1 infection, by using 16 known HLA-B51-restricted epitopes from the Los Alamos Molecular Immunology database. Three epitopes-Pol743-751 (LI9), Pol283-289 (TI8), and Gag327-345 (NI9)-were ranked as starting with LI9 responses, followed by TI8 and NI9. The dynamics of escape mutations in these 3 epitopes were also found by using sequencing for viral clones, which were extracted and amplified from the same individuals on time points of seroconversion, 3 and 6 months of HIV-1 infection. We use solid evidence to demonstrate the correlation between T-cell response and viral mutation, and postulate that multiple effective T-cell responses (ETRs) might benefit the control of HIV-1 infection, especially in acute infection phase.

Study population
A total of 6000 candidates from a homosexual cohort study of primary HIV-1-infected individuals in Beijing were monitored every 2 months for plasma HIV antibodies, HIV ribonucleic acid (RNA) levels, and clinical signs of acute infection to screen acute HIV-1-infected patients as we described in the previous study. [15] Amongst them, 105 candidates were defined as acute HIV-1infected patients and recruited in this study. Whole-blood specimens were collected every 3 months, thereafter from detection of seroconversion and used to separate plasma, peripheral blood mononuclear cells (PBMCs), and genomic deoxyribonucleic acid. Among these HLA-B51-positive individuals, patients with less than 1 ETR were segregated into Group 1, and Group 2 were patients with at least 2 ETRs.

Clinical definitions and laboratory detection
Acute HIV-1 infection was defined as the positive testing of HIV-1 RNA and negative or indeterminate testing by enzyme-linked immunosorbent assay (ELISA) and Western blot of HIV-1. [16] For detection of specific antibodies to HIV-1, standard HIV-1 ELISA (Abbott, Abbott Park, IL) and Western blot analysis (Genelabs, Redwood City, CA) kits were used. The copies of HIV-1 RNA were quantified in serum with Roche Amplicor kit (Roche Molecular Diagnostics, Indianapolis, IN). The absolute counts of CD3 + /CD4 + /CD8 À T cells (CD4 + T) and CD3 + / CD4 À CD8 + T cells (CD8 + T) were measured by using TriTEST Three-Color reagents (BD Company, FranklinLakes, NJ) and MultiSET software in the FACSCalibur Flow Cytometry System (BD Company, FranklinLakes, NJ).

HLA genotyping
Low-resolution (2-digit) HLA class I molecular typing was performed with sequence-based typing at SinoGenoMax, Chinese National Human Genome Center of Beijing, Beijing, PR China. HLA graphing was performed by HLA analysis tools from HIV molecular immunology database (http://www.hiv.lanl. gov/content/immunology/hla/hla_graph.html).

HIV-1 clone and sequencing
HIV-1 RNAs were extracted from plasma on time points of seroconversion, 3 and 6 months after HIV-1 infection, and then were cloned into pCR TM 4-TOPO ® TA vector through the TA TOPO cloning kit (Invitrogen, Carlsbad, CA). Expected 20 colonies were constructed from each sample, and HIV-1 gag and pol genes were amplified and sequenced by Beijing Institute of Genomics, Shenzhen, China.

Human IFN-gamma ELISPOT assay
A total of 200,000 PBMCs with 10 mg/mL peptide were used in a standard human interferon (IFN)-gamma enzyme-linked immunospot assay (ELISPOT) assays as described before. [14] Briefly, assays were carried out in 96-well MultiScreen filter plates (Millipore, MAIP S45, Millipore, MA) coated with 15 mg/mL anti-IFN-gamma monoclonal antibody (1-DIK; Mabtech, Sweden). A total of 5 mg/mL phytohemagglutinin (final concentration, 1 mg/mL) and RPMI1640 were used as positive and negative control, respectively. Plates were incubated for 16 hours at 37°C, 5% CO 2 . Spot enumeration was performed with ELISPOT ImmunoSpot Analyzers (Cellular Technology Limited, East Asia, Beijing, China). To quantify HIV-specific responses, mean spots of negative control were subtracted from the positive wells, and results were expressed as spot-forming units (SFUs) per 10 6 PBMCs. Responses were regarded as positive if results were at least 3 times as the mean of the negative control wells and above 50 SFUs/10 6 PBMCs.

Definition of effective T-cell response
According to our previous study, [14] epitopes Pol743-751 (LI9), Pol283-289 (TI8), and Gag327-345 (NI9) were confirmed as the protection roles in the control of HIV replication, and their mutations escapes were driven by CTL pressure. Hence, ETR was defined as a T-cell response activated by 1 epitope and without mutation at the same time.

Ethics
The study was approved by the Institutional Review Board of Beijing You'An Hospital. The ethics committee approved the relating screening, inspection, and data collection of the patients, and all subjects signed a written informed consent form. All works were undertaken following the provisions of the Declaration of Helsinki.   (Table 1).

Dynamic of escape mutations driven by dominant epitopes
Based on our previous study, we found that the escape mutations of epitopes-Pol743-751 (LI9), Pol283-289 (TI8), and Gag327-345 (NI9)-were associated with HLA-B51-specific CTL response. [14] Then we identified the dynamics of escape mutations in these 3 epitopes by using sequencing for viral clones, which extracted and amplified from the same individuals at seroconversion, 3 and 6 months of HIV-1 infection. As shown in Table 2 and Fig. 2B, epitopes LI9 mutated first at seroconversion, the mutation rate was from 8.57%, increased to 21.06% at the 3 months, and to 58.57% at the 6 months of HIV-1 infection; epitope TI8 escape mutation rate was found from 2.86% at seroconversion, increased to 14.13% at 3 months and 54.49% at the 6 months of HIV-1 infection. Epitope NI9 mutated later than LI9 and TI8, only 7.13% of variations were identified at 6 months of acute HIV-1 infection.

Correlation between multiple effective T-cell responses and disease progression
We evaluated the status of ETR at seroconversion, the third month, and the sixth month after HIV infection according to corresponding T-cell response and viral mutation. And then, the correlation between ETR status and the disease progression was performed by analyzing the differences between various ETR in CD4 counts and plasma viral load. A significant difference was found in HIV viral load between Group 1 (ETR 1, 4.616 ± 0.143) and Group 2 (ETR ≥ 2, 3.715 ± 0.156, P = 0.0005, Fig. 3A). Moreover, CD4 + T-cell count in Group 2 (636.9 ± 90.63 cells/mL) was much higher than that in Group 1 (360.5 ± 39.6 cells/mL, P = 0.0174, Fig. 3B). In addition, the change of CD4 cell counts was further confirmed from seroconversion to 6 months after HIV-1 infection. At 3 months, the CD4 cell counts of patients in Group 2 (ETR ≥ 2) was significantly higher than those patients in Group 1 (ETR 1) (P = 0.0046, Fig. 4).

Discussion
It has been demonstrated that HLA-B51 is one of the protective class I alleles associated with delayed disease progression of HIV infection. [7,14] We previously found that the HLA-B51-restricted epitopes, Gag327-345 (NANPDCKTI, NI9), Pol743-751 (LPPVVAKEI, LI9), and Pol283-289 (TAFTIPSI, TI8), as well   We also employed clone sequencing to reveal the dynamic of escape mutations in dominant epitopes. Interestingly, the same rank of change was found comparing with T-cell response. Epitopes LI9 and TI8 mutated first at seroconversion, and the   Table 2 The rate of mutations in epitope LI9, TI8, and NI9 at seroconversion, 3 and 6 months of acute human immunodeficiency virus type 1 infection.    [5] but also confirmed our previous proposal in chronic HIV-1 infection cohort study [14] in which HLA-B51restricted epitopes mutated regularly, epitope LI9 mutated first, followed by epitope TI8, and then epitope NI9. In our previous study, we found that the viral load of HLA-B51positive individuals is lower than those of HLA-B51-negative individuals. [14] But here, the viral load of HLA-B51-positive individuals was not significant with those of HLA-B51negative individuals, which might be caused by the HLA-B51positive individuals being only 7. In here, the most important finding of this study was that we integrated T-cell response and escape mutations together to evaluate the ETR, which was correlated with the disease progression. A clear phenomenon was found that those patients with more than 2 ETRs had the lower HIV viral load and the higher CD4 + T-cell count, comparing with those with less than 1 ETR. This finding strongly exposed the importance of multiple effective T cell to the control of progression in acute HIV-1 infection, which could be used as a new strategy in the selection of T-cell vaccine to HIV infection.

Period of infection
In conclusion, our data explicitly showed that the kinetic of dominant epitopes restricted by HLA-B51 during the first half-year of acute HIV-1 infection, the dynamic of escape mutations driven by dominant epitopes, and the correlation between multiple ETRs and disease progression. This finding not only confirmed our previous study about the majority of viral mutation under CTL driving pressure, but also expanded the role of ETR to control the disease progression.