Conserved epitopes on HIV-1, FIV and SIV p24 proteins are recognized by HIV-1 infected subjects

Figures & data

Table 1. Description of HIV⁺ population: Demographic information and immune status

Group	Subject	Age	Gender	Race	HIV^+	CD4/μL	CD8/μL	Virus Load
LTS/ART-	SF01	42	M	White	16	1021	996	Undetectable
	SF02	44	M	White	12	528	394	Undetectable
	SF03	59	M	White	24	567	1040	5500
	SF05	49	M	White	22	483	1242	Undetectable
	SF08	48	M	White	31	529	920	3401
	SF17^,	56	M	White	11	374	1037	2000
	SF19^,	40	M	White	12	292	556	40000
	SF21	43	M	White	15	800	1375	3584
	SF23	39	M	White	10	784	1018	3160
	SF24	50	M	White	11	675	213	Undetectable
	J01	25	F	Black	11	699	935	Undetectable
	J10	35	F	Black	12	897	659	Undetectable
	J11	21	F	Black	21	564	829	932
	J14	47	M	Hispanic	25	722	1421	6790
ST/ART-	J02	50	F	Black	9mo	639	1248	710
	J03	27	F	Black/Hispanic	8mo	368	1254	25700
	J04	22	M	Black	6mo	537	1907	1740
	J05	32	M	Black	2mo	384	2202	691
	J06	28	M	White	6mo	501	1110	134000
	J07	26	F	Black	4mo	448	1306	5120
	J08	19	F	Black/Hispanic	9mo	323	932	3040
	J09	27	M	White	2mo	482	882	109168
	J12	26	M	Pacific Islander	5mo	352	688	405000
	J17	51	F	Black	2mo	375	1271	2140
ART+	SF04	55	M	White	28	540	864	Undetectable
	SF07	65	M	White	25	610	2170	Undetectable
	SF13	52	M	White	25	304	372	Undetectable
	SF16	50	M	White	8	827	1018	Undetectable
	SF18	38	M	White	4	1082	1298	1500
	SF20	53	M	White	23	76	1172	Undetectable
	SF22	48	M	White	13	1205	517	Undetectable
	J16	48	F	White	21	1250	NA^g	Undetectable
	J22	36	F	Black	15	391	490	Undetectable
	J23	50	F	Black	16	949	1261	Undetectable
	J24	41	M	Black	23	202	1192	Undetectable

^a SF prefix, HIV⁺ subject from the University of California, San Francisco. J prefix, HIV⁺ subject from the University of Florida at Jacksonville; for definition of subjects, see legend to Figure 1. The results for virus load and CD4/CD8 T-cell counts are from the 1st sample obtained from patients (yr 1).

^b M, male; F, female.

^c Duration of known HIV infection (yr).

^d Virus loads are shown as RNA copies/mL; undetectable ≤75 RNA copies/mL.

^e HIV⁺ subject who was on ART starting at or shortly after yr 2.

^f Subjects monitored for 4 yr (Figure 3).

^g NA, not available.

Figure 1. IFNγ responses to HIV, FIV and SIV p24 peptide pools. The IFNγ ELISpot responses to overlapping peptide pools of HIV p24 (Hp1-Hp18, n = 31 ; A), FIV p24 (Fp1-Fp17, n = 31 ; B), and SIV p24 (Sp1-Sp17, n = 15 ; C) are depicted as spot forming units (SFU) per 10⁶ PBMC. The HIV⁺ subjects (panel-A insert for A and B; panel-C insert) consisted of long-term survivors (LTS) without antiretroviral therapy (ART) (LTS; black bar); recently diagnosed subjects (<1 year) with short-term infection without ART (ST; gray bar); and those receiving ART with various duration of infection (ART⁺, red bar). Each bar represents a positive response by an individual subject with a threshold of >50 SFU per 10⁶ PBMC. Asterisk after the peptide pool(s) represent those with highest frequency of responders. The FIV p24 pool with the highest and the most frequent responses and its counterpart HIV-1 and SIV pools are highlighted in blue.

Figure 2. For figure legend, see page 6.

Figure 3. The persistence of IFNγ and T-cell proliferation responses to selected peptide pools. The IFNγ (A) and CD8⁺ T cell proliferation (B) responses of HIV⁺ subjects who responded at the first sample collection (t1), 2 y later (t2), and 4 y later (t3) are shown for peptide pool Fp14 (IFNγ), Hp15 (IFNγ), Hp10 (proliferation), and Fp9 (proliferation). The p-value indicates statistical differences between t1 and t2, t2 and t3, and t1 and t3. The threshold for IFNγ and proliferation responses are >50 SFU and >1% CFSE^low, respectively.

Table 2. Fp9/Fp9–3, Fp14/Fp14–3, and their counterpart aa sequences and CMI responses

^aPeptide pools are not hyphenated (e.g., Fp9) while the individual large peptides have a hyphen followed by a number (e.g. Fp9–3) indicating the number of the overlapping individual peptide starting from amino-end.

^bAlignments denote identical amino acids (aa) as (*), aa with most similarity based on charge, polarity, acid/base, and hydrophilicity/hydrophobicity as (:), those with some similarity as (.), and each gap with a (-). The internal gaps are due to best alignment and the external gaps are due the length of the selected peptide or peptide pool. The percentage of aa sequence similarity and identity was determined from these alignment criteria.

^cNote that Sp14 pool is a single 13mer peptide Sp14–1 (TDAAVKNWMTQTL). As a result, Sp14–1c, the counterpart SIV Sp14–1 peptide for HIV Hp15–1 peptide, is a 10mer and did not include the first 3 aa (AEQ). Whereas Sp14–1b is a 10 mer with glutamine (Q) added to amino-end and threonine (T) deleted from the carboxyl-end rather than a sequence of Sp14–1c.

Figure 4. IFNγ and CD8⁺ T-cell proliferation responses to Fp9, Fp14, and Hp15 peptide epitopes. Peptide pool Fp14 consists of 4 overlapping 13–15mer peptides spanning the amino- to carboxyl-terminus (Fp14–1, Fp14–2, Fp14–3, Fp14–4), while pools Hp15 (Hp15–1, Hp15–2, Hp15–3) and Fp9 (Fp9–1, Fp9–2, Fp9–3) each consist of 3 overlapping 13–15mer peptides. IFNγ responses to individual Fp14 peptides (A; n=9 ), Hp15 peptides (B; n=9 ) and CD8⁺ T-cell proliferation responses to individual Fp9 peptides (C; n=7 ) are shown along with responses to their corresponding peptide pools. Only responders to pools Fp14 (A), Hp15 (B), and Fp9 (C) were tested. The responders consisted of long-term survivors (LTS) without ART (LTS; black bar); recently diagnosed subjects (<1 year) with short-term infection without ART (ST; gray bar); and those on ART with various duration of infection (ART⁺, red bar). Each bar with (*) in panels A and B are from the same 3 LTS.

Table 3. Sequence conservation in _EC p24 epitopes of Hp15/Fp14/Sp14 pools

^aSubtype specific sequences used to generate peptides.

^bAlignments are compared with HIV UCD-1 and denote identical amino acids (aa) as (*), aa with most similarity based on charge, polarity, acid/base, and hydrophilicity/hydrophobicity as (:), those with some similarity as (.). The percentage of aa sequence similarity and identity was determined from these alignment criteria.

Figure 5. IFNγ and T-cell proliferation responses to 9–13mers of FIV peptides Fp9–3, Fp14–3, and Fp14–4. Six 9–12mer peptides for the Fp9 pool (A1, B1, C1) and 7 9–13mer peptides for the Fp14 pool (A2, B2, C2) described in Table 3 were tested for their ability to induce CD8⁺ T-cell proliferation (A), CD4⁺ T-cell proliferation (B), and IFNγ production (C) and compared to results with the Fp9 and Fp14 peptide pools. Thirteen–15mer peptides Fp9–3, Fp14–3, and Fp14–4; and mitogen (PHA) were included as controls. A total of 9 HIV⁺ responders to the Fp9 pool (A1, B1, C1) and 10 HIV⁺ responders to the Fp14 pool (A2, B2, C2) were tested up to 4 y after the beginning of the study. Two to 4 of the responders were lost to follow up. Their proliferation and IFNγ results to the 13–15mer peptides Fp9–3, Fp14–3, and Fp14–4, and are denoted with an (*) for each individual missing.

Table 4. 9–13mer T-cell epitope mapping of Fp9 and Fp14 sequences using responders to Fp9 or Fp14

^a The 13–15mer peptide designations used in the peptide pools and their corresponding aa sequences are in bold.

^b Number of amino acids.

^c The responder frequencies to the large peptides were derived from yr 2 and included only responders to either the Fp9 or Fp14 peptide pool via CFSE proliferation. The responder frequencies to the small 9–13mer peptides were obtained at yr 4 and included only responders to either the Fp9 or Fp14 peptide pool via both proliferation and IFNγ.

^d The two highest frequency of responders to small peptides by both CD8⁺ and CD4⁺ T cells are highlighted in bold.

^e Proliferation results are from small number of subjects tested (n = 4) but ICS results are from five subjects (Figure 6).

Table 5. HLA supertypes of the responders to key short and long peptides

^a Four subjects who responded to the designated peptide were HLA class I typed, and their HLA alleles were compared to the HLA supertype(s) predicted for the designated peptide using the NetCTL 1.2 and NetMHC 3.2 algorithms. The HLA A and HLA B allotypes for the subjects are shown as HLA supertypes.

^b The most common supertypes between subjects and the HLA algorithm predictions are shown. The bolded supertype represents the most common supertypes among the subjects.

^c All results were from responders to either Fp9 or Fp14 peptide pools. The results for individual 9–11mer peptides were derived from Figure 6. Those for 13–15mer peptides (Fp9-1, Fp14-1, Hp15-1) were derived from Figures 5 and/or 7. The average positive values are considered low when the frequency of response is <15% CFSE or <125 SFU, and high when >30 CSFE or >300 SFU. The cytotoxin result is considered high when four or five subjects express one or more cytotoxins in the CD8⁺ T cells.

Figure 6. Stimulation of cytotoxins by CTL epitopes on Fp9, Fp14, and Hp15 peptide pools, and their individual peptides. The ICS analyses for perforin (Perf, ○), granzyme A (GrzA, □), and granzyme B (GrzB, Δ) are shown for CD8⁺ T cells (A, D) and CD4⁺ T cells (B, C) from 5 HIV⁺ responders with or without ART. Two LTS/ART⁻, one LTS/ART⁺, one ST/ART⁺, and one ST/ART⁻ were first evaluated (A, B). Peptides tested included Fp9, Fp14, and/or Hp15 peptide pools or large 13–15mer peptides (A, B). Responses from 5 HIV⁺ responders of short-term HIV-infected subjects not on ART (ST/ART⁻) were tested using small 9–13mer peptides within Fp9–3, Fp14–1, Fp14–3, Fp14–4, and Sp14–1 (C, D). Note that Sp14 pool is a single 13mer peptide Sp14–1. Counterpart peptides are shown with blue-dashed box for Fp14–1a/Hp15–1c/Sp14–1c, purple-dashed box for Fp14–1b/Hp15–1a/Sp14–1b, and red-dashed box for Fp14–3/4f/Hp15–2/3a/Sp14–1a. The peptides tested with one less subject are denoted by (**), while their number of subjects and number of responses are denoted by (*). Each separate symbol color represents one subject, and each color-coded subject is shown with his/her infection status. The threshold for T cells expressing cytotoxin is set at >0.1% CD4⁺ or CD8⁺ T cells.

Figure 7. Direct effect of Fp9–3, Hp15–1, and Hp15–3 peptides on in vitro HIV-1 infection. The direct effect of FIV peptides Fp9–3 and FRT3–4 (light gray bar), HIV-1 peptides Hp15–1 and Hp15–3 (dark gray bar), and virus positive control (+ control, black bar) on HIV-1 infection of PBMC from healthy uninfected subjects is shown as average end-point dilution titer with standard deviation. Staphylococcal enterotoxin A control (SEA, white bar) is included as a T-cell mitogen control to serve as positive control for FIV enhancement. FIV reverse transcriptase peptide FRT3–4 previously reported to be non-reactive to T cells from HIV⁻ subjects is included as a negative control.⁴⁸ Statistically significant difference between the average result of each peptide and the average virus control are shown with (*) for P < 0.02 and (**) for p < 0.001.

Figure 8. Summary of functional epitope analyses. Results from 3 functional analyses are summarized according to frequency of HIV⁺ (lanes 1–4) or HIV⁻ (lane 5) responders and are shown as (+) for frequency of >25 % responders, (±) for 19–25% responders, (−) for <19% responders, or as (*) for not available. Each lane, abridged in the insert, shows the ability of the peptide pool or peptide to stimulate an IFNγ response (lane 1), CD8⁺ T-cell proliferation (lane 2), and CD4⁺ T-cell proliferation (lane 3). Lane 4 denotes the ability to induce cytotoxin(s) in only CD8⁺ T cells (+) or in both CD8⁺ and CD4⁺ T cells (++) by 4 or more HIV⁺ subjects when at least 9 subjects tested. In lane 5, the positive result (+) indicates substantial frequency of proliferation responses from HIV⁻ subjects and may indicate a safety concern; whereas a negative result (−) indicates no substantial HIV⁻ response to the peptide pool or peptide. Positive response of HIV⁻ control had CD8⁺ T-cell proliferation response in 30–42% of HIV⁻ subjects. The large 13–15mer peptides with the best CMI responses without stimulation in HIV⁻ subjects are shown with dashed boxes, while the best small peptides are shown with solid boxes. Therefore, Hp15–1, Hp15–2/3a, Fp14–4, Fp14–1b, Fp14–3/4e, and Fp14–3/4f peptides appear to contain the best potential epitopes to target for use as HIV immunogens.

Sanou MP, Roff SR, Mennella A, Sleasman JW, Rathore MH, Yamamoto JK, Levy JA. Evolutionarily conserved epitopes on human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus reverse transcriptases detected by HIV-1-infected subjects. J Virol 2013; 87, 10004-15; PMID:23824804; http://dx.doi.org/10.1128/JVI.00359-13

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