Characterization of Tat Antibody Responses in Chinese Individuals Infected with HIV-1

HIV-1 Tat is an important regulatory protein involved in AIDS pathogenesis. However, the immunoprofiles of anti-Tat responses remain unclear. We analysed the immunoprofiles of the anti-Tat antibody responses and the neutralizing activities. Out of 326 HIV-1-seropositive individuals, 12.9% were positive for anti-Tat antibodies. We found six different immunological profiles of anti-Tat antibody responses: full-potential response, combined response, N-specific response, C-specific response, full-length Tat-specific response and Tat-related response. These responses represent two types of anti-Tat responses: the major complete response and the alternative C-prone response. A Tat-neutralizing activity is significantly higher in anti-Tat-seropositive samples than anti-Tat-negative or healthy blood-donor samples, and significantly correlates with the anti-Tat reactivities. The data here could contribute to a better understanding of the significance of anti-Tat responses in preventing HIV pathogenesis and could be useful for designing more effective vaccines in the future.


Introduction
As one of the six accessory proteins of HIV-1, Tat is synthesized during both the early and late stages of viral replication and is critical for these processes. The HIV-1 Tat protein is encoded by two exons and can be between 86 and 101 amino acids (aa) in length, depending on the specific viral strain. The Tat protein can be divided into six functional domains [1,2]: (1) the N-terminal acidic aa region (aa 1-21), which has been linked to Tat immunosuppressive activity [3,4,5,6]; (2) the cysteine-rich region (aa 22-37), which is responsible for transactivation of transcription; (3) the ''core'' region (aa 38-48), which is highly conserved; (4) the basic region (aa 49-57), which recognizes the transactivation response element (TAR) [7]and plays important roles in both the nuclear localization of Tat [8] and the entry of extracellular Tat into bystander cells [9]; (5) the glutamine-rich region (aa 60-72), which has the highest rate of sequence variation; and (6) the C-terminal region (aa 72-86 or 72-101), which is encoded by the second exon and contains the ''RGD'' motif that allows Tat to bind integrin [10,11]. Furthermore, Tat is actively released from HIV-1-infected cells [10,11] and acts as an extra-cellular toxin [12], which plays a crucial role in HIV-1 pathogenesis, including development of HIV-associated dementia, HIV-related opportunistic infections and Kaposi's sarcoma.
Previous studies have shown that approximately 20% of infected individuals produce detectable amounts of Tat-specific antibodies, and the presence of anti-Tat antibodies is strongly correlated with slower disease progression and that no AIDS events were observed in persistently anti-Tat-seropositive subjects [13,14,15,16,17]. These results strongly suggest that Tat is a promising target for the development of both preventive and therapeutic vaccines [18,19]. However, several contrary results were also reported [20,21,22], and the detailed host anti-Tat antibody responses remains unclear. In this study, we performed anti-Tat immunoprofile analysis in 326 Chinese individuals infected with HIV-1 and defined six immunological profiles of anti-Tat antibodies responses. Our findings provide a novel source of information with respect to anti-Tat responses and Tat-neutralizing potential that should be very important for understanding the role of this response in the prevention of HIV pathogenesis and vaccine design.

Ethics statement
All aspects of the study were approved by the Ethics Committee of Beijing You An Hospital,Capital Medical University, China. Written informed consent was obtained from all participants in the study.

Vectors, bacterial strain and reagents
The prokaryotic expression plasmid pPEPTIDE2, as well as the two E. coli host strains BL21(DE3) and DH5a, were purchased from Novagen (Germany). A mouse monoclonal antibody that recognizes the N-terminus of native and recombinant HIV-1 Tat (strain HXB2) was purchased from United States Biological. HRP-LD5 consists of HRP conjugated to LD5, which is a novel evolved immunoglobulin-binding molecule (NEIBM) with a characteristic structure of consisting of alternating Finegoldia magna protein L B3 and staphylococcal protein A D domains; this structure creates synergistic double binding sites for the VH3 and Vk regions of Fab as well as to IgG Fc [23]. HRP-LD5 shows high binding affinity for IgM, IgG and IgA [24].

Clinical samples
Clinical samples for this study were collected from the AIDS high-risk Cohort at YouAn Hospital in Beijing, China. Informed consent was obtained from each of the participants prior to blood collection. Clinical information for each of the 326 samples was also recorded (Table S1). The cohort contained of 252 males (mean age = 33.6, SD = 8.5) and 74 females (mean age = 38.4, SD = 6.8). Mean CD4 counts/ml for the males and females were 437.4 (SD = 150.9) and 340 (SD = 283), respectively. The seropositive status of the participants was confirmed using ELISA (Diagnostic Kit for Antibody to HIV (ELISA), Shanghai Kehua Bio-Engineering Co., LTD., China) and Western blotting (HIV Blot 2.2 WB, MP Biomedicals Asia Pacific Pte. Ltd., Singapore). Control samples were obtained from 100 healthy blood donors who were confirmed to be HIV seronegative. All samples were stored at 280uC in 1.5 ml aliquots.

Synthesis of the Tat N terminus
The peptide HIV-1 HXB2 Tat 1-21 aa (sTat1-21) was produced using solid-phase synthesis by Temple University (Philadelphia, USA).

Indirect ELISA of antibodies against full-length Tat and Tat functional domain peptides
We used indirect ELISA to quantify antigen-specific antibody levels in the plasma samples using the recombinant Tat and six Tat peptides, largely as described previously [24,25]. Briefly, immunoassay plates (Nunc, Rochester, NY, USA) were coated with 1.0 mg of full-length Tat, Tat(1-48), Tat(1-86), Tat(22-100), Tat(38-100), Tat(38-61) or Tat(41-61C) recombinant proteins and sTat1-21 in 50 mM carbonate buffer (pH 9.6) and incubated at 37uC for 3 h. The plates were blocked for 1 h at 37uC with 200 ml of 4% BSA prepared in PBS-Tween 20. Next, 100 ml of a 10-fold dilution of the plasma sample were added to appropriate wells. The plates was then placed in a 37uC incubator for 1 h. After washing four times with the wash buffer (50 mM Tris, pH 8.0; 100 mM NaCl; 0.2% Tween 20), 100 ml of a 1,000-fold dilution of HRP-LD5 (1 mg/ml) was added to the strip and incubated for 45 min at 37uC. The plates were developed by the addition of 3,3',5,5'-tetramethylbenzidine and absorbance at 450 nm was read using an ELISA Reader. Coated pPEPTIDE2 protein and samples obtained from healthy blood donors were included as negative controls. Samples showing absorbances above the mean value of the control group plus 3 SD (0.06 + 0.14) were considered to be positive for Tat-reactive antibodies.

Tat-neutralization assay
The neutralizing potential of the anti-sera was evaluated by measuring their ability to inhibit the transactivation activity of native Tat (HXB2 strain) using a HEK293T cell line transfected with a plasmid encoding for the LTR of HIV-1 and secreted alkaline phosphatase (pLTR-SEAP) (kindly provided by Dr. Udaykumar Ranga) as described previously [25,27] with minor modifications.
Briefly, 96-well plates were coated with 100 ml of pPEPTIDE2 (5 mg/ml in carbonate buffer (pH 9.6) and incubated at 37uC for 3 h. The plates were blocked for 1 h at 37uC with 200 ml of 4% BSA prepared in PBS/Tween 20. The plates were then incubated with 100 ml per well of 50-fold dilution of the HIV+Tat+, HIV+Tat2 and HIV2 plasma samples in DMEM for 2 h at 37uC to deplete the non-specific antibody. Then the depleted plasma were incubated with 500 ng of Tat protein at 37uC for 30 min. The samples were then added to appropriate wells containing cells and incubated for 3 h. The supernatant was removed and 200 ml of complete medium was added to each well. The plates were incubated for 48 h and the levels of secreted alkaline phosphate (SEAP) were estimated at 48 h using a colorimetric assay (Toyobo). As a control, 96-well plates were coated with 100 ml of full-length Tat protein (5 mg/ml in carbonate buffer (pH 9.6) or protein G to deplete the Tat-specific antibody or IgG fraction of the plasma. Then the depleted plasma were used for the inhibition assay. As a positive control for Tat neutralization, we used an IgG1 monoclonal antibody, which was raised in-house against B-Tat; this antibody recognizes the N-terminal 20 amino acid residues of Tat and blocks extracellular-Tat with high efficiency.

Statistical analyses
Statistical analyses were performed using SPSS 17.0 and SAS 9.3. All experiments were performed three times, and the values obtained from three replicate samples were averaged for each experiment. Data are presented as the median and quartiles. Statistical significance was tested using Nemenyi or Wilcoxon nonparametric test. Differences between measurements were considered to be significant at p-values of less than 0.05. The correlation was assessed by Spearman correlation coefficient.

Anti-Tat antibodies in Chinese individuals infected with HIV-1
We collected 326 HIV-1-infected samples from a clinical cohort of Youan hospital in Beijing, China. Plasma samples from 100 healthy blood donors were included as controls. We established full-length, recombinant, subtype B Tat protein based ELISA assay to screen out Tat-seropositive samples. This assay is able to detect all of the antibody isotypes using HRP-LD5 as conjuate [23,24]. Out of 326 samples tested, only 42 (12.9%) were positive for anti-Tat antibodies, and most of these (31/42 or 73.8%) showed only weak reactivity (Fig. 2a, 3a). No anti-Tat positive samples were detected in the blood-donor sample. In contrast, gp41 showed strong antigenicity: all 326 samples reacted with gp41, and most of these exhibited either strong or moderate binding reactivity (Fig. 2a).
Other than the obviously different antigenicity, the N and C antigens showed good complementarity for anti-Tat detection. The reactivity rates of the N and C antigens with the Tatseropositive samples reached 93%, which was much higher than that observed with the N (59.5%) or C antigens (69.0%).

Characterization of Tat-antibody-response profiles in HIV-1-infected individuals
As described above, both the C and N antigens showed complementary but different reactivity patterns; based on these differences, the anti-Tat responses could be easily classified into one of the following five profile classes (Fig. 3a).
Profile 1) full potential response: Three of the 42 Tatseropositive samples fell into this category, which was characterized by reactivity, usually strong or moderate, against all of the N and C antigens. All the plasma samples from this profile reacted with full-length Tat at a strong or moderate level.
Profile 2) combined response: Twelve of 42 Tat-seropositive samples fell into this category, which was characterized by reactivity against both N and C antigens. This profile could be further divided into two distinct reaction types: (1) N-preferred reaction, which reacted with both the Tat(1-48) and Tat(1-86) (and possibly more) N antigens as well as with at least one of the C antigens, usually at strong or moderate level. Six of seven plasma samples of this type reacted with full-length Tat at a strong or moderate level. (2) Common reaction, which reacted with one or two of C antigens and only the Tat(1-48) of N antigens at weak or moderate level. All five plasma samples of this type only weakly reacted with full-length Tat.
Profile 3) N-specific response: Ten of 42 Tat-seropositive samples represented the response of this profile which was only characteristically against one, Tat(1-48), or more, Tat(1-86), of the N antigens usually at weak level. The plasma samples with this profile reacted with full-length Tat usually at weak level.
Profile 4) C-specific response: Fourteen of 42 Tat-seropositive samples fell into this category, which was only reactive against the C antigens. This profile, could be further divided into two distinct reaction types: (1) full C reaction, which reacted with all four C antigens, mostly at moderate levels; three of the four plasma samples of this type reacted with full-length Tat at weak level. (2) Common reaction, which reacted with one or more, but not all, of the C antigens at weak level. All ten plasma samples of this type reacted with full-length Tat at weak level.
Profile 5) full-length Tat-specific response: Only three of 42 Tatseropositive samples fell into this response profile, which was characterized by weak reactivity against full-length Tat, but no reactivity against the N and C antigens.
Considering the nonimmunodominant nature of Tat, we screened out 6 samples with the highest anti-Tat OD values from 100 HIV-seropositive and anti-Tat-seronegative samples and further assessed their reactivities with the N and C antigens, and we uncovered yet another response profile. Profile 6) Tat-related response: Six of the 100 Tat-seronegative samples fell into this category, which was characterized by reactivity against C antigens at weak level (Fig. 3a). It was also very interesting to find that five of these six samples reacted with Tat(22-100).

Characterization of the Tat-neutralization potential of the different response profiles
Forty-eight samples from these six profiles, twelve anti-Tatnegative HIV samples and 18 healthy blood-donor samples were evaluated for extracellular Tat-neutralization activity. The percentage of SEAP-expression inhibition for each group is presented in Fig. 4a. Anti-Tat-positive samples showed significantly higher Tat-neutralizing activities comparing with anti-Tat-negative and blood-donor samples (Fig. 4a). Among the six immunological profiles, the N-preferred reaction in combined response showed significant Tat-neutralizing activity (Fig. 4b), which was significantly higher compared with the HIV-1-seropositive and anti-Tatseronegative group (HIV+Tat-) and healthy blood-donor plasma (HIV-) group. We choose ten samples with higher antibody reactivity and neutralizing activity to further assess the neutralization activity after depleting the anti-Tat antibodies or IgG fractions of the plasma. These samples lost entire and most neutralization activity after depleting the anti-Tat antibodies or IgG fraction with about 20% inhibition of transactivation ability, which is similar to the percent inhibition from HIV+Tat-and HIV-plasma samples (Table 1). These demonstrated that anti-Tat antibodies are specifically responsible for the neutralization activity and IgG fraction contributes to most of this neutralization activity.
Statistical analyses revealed that the neutralizing activity of the group that exhibited strong binding reactivity (OD values above 1.0) to full-length Tat was significantly higher than the group that exhibited weak binding reactivity (OD values between 0.2-0,3) (Fig. 5a). Correlation analyses between the antibody reactivity of each antigen and Tat-neutralizing activity were carried out for the 48 samples from these six profiles. We found that the reactivity with Tat(1-86), Tat(1-48), full-length Tat, Tat(38-61), Tat(38-100) and Tat  was significantly correlated with Tat-neutralizing activity (Fig. 5b).

Discussion
In this study, we define for the first time host's anti-Tat responses in Chinese patients infected with HIV-1. Consistent with the previous findings that Tat is intrinsically nonimmunodominant in nature [13,14,15,16,17], our results also verified this conclusion (Fig. 2). This nonimmunodominant property is consistent with the results of structural studies which revealed that HIV Tat is an intrinsically unstructured protein, or unfolded protein lack of secondary structures and high structures [28,29,30,31], and which could be likely to help avoid elicting the host's anti-Tat immunity.
In this study, the N antigens and C antigens clearly showed obviously different antigenicity (Fig. 2b, 3). Moreover, there were still some differences in antigenicity between different N antigens or different C antigens (Fig. 2b, 3). Based on these differences, we characterized six immunological profiles: full potential response, combined response, N-specific response, C-specific response, fulllength Tat-specific response and Tat-related response. Interestingly, samples from the full-potential response and N-preferred reaction type in the combined response profile presented strong or moderate reactions to the N, C and full-length Tat antigens and also showed the highest neutralizing activity (Fig. 3, 4). These sample types only accounted for 23.8% of total Tat-seropositive samples, and they represent the strongest reactive groups. In contrast, all other samples (except for the samples of full C reaction type in C specific response profile) almostly present weak reactions to N, C and full-length Tat antigens, and showed weak neutralizing activity (Fig. 3, 4). Based on these findings and the fact that Tat is intrinsically nonimmunodominant, we hypothesize that the full-potential response profile and N-preferred reaction in combined response profile represent the major complete anti-Tat antibody response, which was elicited recently by a large amount of transiently released Tat produced on the occasion of vigorous replication of the newly immuno-escaped HIV strains, and target all, or at least multiple, N and C epitopes of Tat. It is possible that the other response profiles (except for the full C reaction type and part of the common reaction type in C-specific response profile) could represent various degraded forms of this complete antibody response. Follow up of these Tat-seropositive individuals will help to verify this hypothesis.
It was also interesting to find that the samples from the full C reaction in C-specific response profile showed moderate reactivity to all four C antigens but weak reactivity to full-length Tat (Fig. 3), as well as showed significant neutralizing activity (Fig. 4). This result could suggest that there were few individuals (,9.5%) who elicited an alternative antibody response that is more specific to C antigens (C-prone response) other than the major complete anti-Tat antibody response. Some samples of the common reaction type in C-specific response profile might represent the degraded form of this C-prone response. The possibility that these subjects are genetically predisposed to the C-prone response needs to be evaluated in future studies.
Very interesting, Tat(1-21) had previously been shown to be a dominant linear epitope. In addition to the Tat(1-21) domain, the Tat(1-48) antigen contains an additional cysteine-rich domain (CRD), which was recently identified as an immunodominant Bcell epitope [27] and could account for the enhanced antigenicity of Tat(1-48). Unexpectedly, Tat(1-86), which contained at least one more known epitope (the BD epitope) in addition to those in Tat(1-48), showed weaker antigenicity compared with Tat(1-48). This indicates that the 49-86 aa in Tat(1-86) does not provide additional reactive epitopes but does affect the conformation of the reactive epitopes found in the Tat(1-48) fragment, which provides new supportive evidence for the conformational nature of the Tat antigen [32].
Theoretically, Tat-neutralization potential, as opposed to anti-Tat reactivity, could represent well the anti-Tat protective role. Our study shows that strong Tat-neutralization potential is related to N-preferred reaction in combined response immunoprofiles which represent newly elicited complete anti-Tat antibody responses (Fig. 4b). Also, Tat-neutralization potential was showed to be significantly related to the reactivity of the specific antigens Tat(1-86), Tat(38-61), full-length Tat, Tat(1-48), Tat(1-21) and Tat(38-100). Among N antigens, the reactivity of Tat(1-48) and Tat(1-86) significantly related to Tat-neutralization potential. Considering that the reactivity of Tat(1-48) and Tat(1-86) is highly correlated with that of full-length Tat (Fig. 3b), and the previous findings that B-clade Tat(1-86) is the mostly wellcharacterized active form of Tat, it is reasonable to conclude that the principle protective epitopes of full-length Tat should be in the N-terminal 1-48 aa. Among four C antigens, only the reactivity of Tat(38-61) and Tat(38-100) significantly related to Tat-neutralization potential (Fig. 4b). It was intriguing to find that the reactivity of Tat(38-100) showed a highest correlation to that of Tat(38-61) with R value of 0.833 among all C antigens. Considering that all four of the C antigens contained the 38-61 aa domain, Tat(38-61) and Tat(38-100) carried no or the least additional amino acids which could affect the conformation of Tat(38-61), it is reasonable to hypothesize that Tat(38-61) contains an another important protective epitope. These findings are likely to be important for understanding of protective anti-Tat antibody responses as well as for future vaccine designs.
In conclusion, in this study, we define for the first time six different immunoprofiles of anti-Tat responses in Chinese patients infected with HIV-1, which represent two types of anti-Tat antibody responses: the major complete response and the alternative C-prone response. Tat-neutralizing potential was demonstrated to be significantly related to specific immunoprofiles and to the reactivity of specific antigens. The findings presented here could significantly contribute to our understanding of anti-Tat responses in preventing Tat-mediated HIV pathogenesis and aid in future vaccine designs.

Supporting Information
Table S1 Baseline characteristics of the study participants. (DOC)