Conceived and designed the experiments: MP CB CLG. Performed the experiments: MP CC CA AL VSM BM JLV EG. Analyzed the data: MP CC AT EY CB CLG. Contributed reagents/materials/analysis tools: BM LR JLV JS. Wrote the paper: MP EY AT CB CLG.
The authors have declared that no competing interests exist.
To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient.
We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs.
The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC.
Long term non progressor elite controllers (LTNP-EC) constitute a subset of Human Immunodeficiency Virus (HIV-1) infected naïve individuals whose viral load is below 50 copies/ml for more than 10 years of infection
Viral factors, host genetics and immune responses have been associated with the control of HIV-1 replication and lack or slow disease progression. In some studies, mutations or deletions in HIV-1 proteins, like Nef
Host genetic factors have also been associated with viral control in LTNPs. Genetic polymorphisms in the coding and the promoter regions of the
Several studies have addressed whether ECs have broadly neutralizing antibody (Nab) responses that could account for their ability to control their virus
In previous analysis of HIV-1 superinfection (SI), infection with a second virus during the course of an established infection was generally associated with loss of viral control and abrupt decline in CD4+ T cells counts
We identified a case of HIV-1 SI in a LTNP-EC that was able to control both viruses and maintain undetectable viral loads for >20 years
The Ethical Committee of the Hospital German Trias i Pujol approved the current investigation and the patient gave its informed consent for the study.
Samples from a homosexual man, who maintained undetectable viral loads below 50 copies/ml, occasional blips, and high levels of CD4+ T cells for more than 20 years were analyzed (
CD4+ T cell counts and viral load are represented in the Y axis against time in the X axis. The first sample was taken 78 months after the first documented HIV-1 positive test diagnosis. Blue arrow shows the first sample available, close to the estimated moment of SI (1995). In yellow are represented samples taken to perform quasispecies analysis and gp160 amplification. Serum samples used for neutralization analysis (
Genetic polymorphisms were chosen based on genome-wide association studies, or selected from the literature according to the quality of their supporting evidence. These included the
Peripheral blood mononuclear cells (PBMC) were separated by phycoll-hypaque centrifugation. Proviral DNA was obtained from 1×107 cells by a standard phenol-extraction method. Proviral HIV DNA was amplified in the gp160 region in
For the virus selection, the C2-V5 region of gp160
Maximum Likelihood tree was performed with the help of the MEGA program. Symbols represent sequences taken in different year: 1995 □, 1996 ▾, 1998 ◊, 2001♦,
Four complete gp160 amplicons from the patient, one from LTNP 64 and three amplicons obtained for a patient with rapid progression were used for the generation of chimeric viruses and gp160 from NL4.3 reference virus was also included. The full-length infectious molecular clones were constructed by replacing the gp160
In order to generate the chimeric viruses, 10 µg of the recombinant plasmids were transfected into 3×106 293 T cells using calcium chloride protocol
The gp160 amino acid sequence of viruses a1, a2 a3 and b were aligned using the Bioedit Program. The subject' sequences were automatically compared to all available sequences of subtype B (702 sequences) from the Los Alamos HIV Sequence Data base using the Quickalign tool (
Co receptor usage was determined with the PSSM prediction tool (
Virus titration was performed in duplicate in TZM-bl cells. Five serial ten-fold dilutions of viral stocks were assayed. After 48 h, cells were stained for β-galactosidase activity as described
U87.CD4.CCR5 and U87.CD4.CXCR4 cells were cultured in DMEM supplemented media with 15% fetal bovine serum plus 300 µg/ml G418 (Sigma-Aldrich) and 1 µg/ml puromycin (Sigma-Aldrich). 50×103 U87.CD4.CCR5 or CXCR4 cells per well were seeded in a 24 well plate and infected with 1 ng of p24 of the different viruses. Replicative capacity was evaluated quantifying the p24 production in the culture supernatant after 3, 7, 10 and 14 days post infection.
PBMC obtained from two different uninfected donors, were grown in RPMI 1640 (Bio-Whittaker) supplemented with 10% fetal bovine serum (Gibco) plus 1% antibiotics (Bio-Whittaker), and were activated with phytohaemagglutinin 2 µg/ml (Sigma-Aldrich) for three days before infection. In order to compare the replicative capacity of the variants present before and after SI, 1×107 PBMC cultured in RPMI supplemented with 2.5 ng/ml of recombinant human interleukin-2 rhIL-2 (Bender Medsystems), were infected in two independent experiments, by spin inoculation with equivalent amounts of viruses (1 ng)
ELISPOT assay was performed as previously described
Cryopreserved PBMCs (105 cells) from 1995 and 2004 samples of LTNP-EC were incubated with the matrix peptides, at a 14 µg/µl concentration, in a 96-well plate (Millipore, Barcelona, Spain) pre-coated with anti-human interferon–gamma monoclonal antibody (Mabtech, Sweden). Cells with medium only were used as negative controls and cells with phytohaemagglutinin were used as positive controls. PBMCs were cultured overnight at 37°C, 5% CO2 atmosphere and then washed six times with PBS. Plates were then incubated for 1 hour at room temperature with the biotinylated anti-INF monoclonal antibody (Mabtech, Sweden) followed by 6 washes and 1 hour incubation with the streptavidin-coupled alkaline phosphatase (Mabtech, Sweden). After washing the plate, nitroblue tetrazolium and 5-bromo-4-chloro-3indolul phosphate (Bio-Rad, Barcelona, Spain) was added for color development. After a short incubation, the reaction was stopped by washing the plate with water. The INF production was detected as blue spots, counted using an ELISPOT reader (CTL, Germany). Results are expressed as spot-forming cells (SFC) per million inputs PBMC. Responses were considered positive if they exceeded i) 50 SFC/106 PBMC per well, ii) the mean of negative wells plus 3 standard deviations and iii) three times the mean of the negative well
Sera samples taken at different times (1995, 1996/01, 1996/07 and 1998) were tested with a panel of six recombinant viruses (VI191 subtype A, 92BR025 subtype C, 92UG024 subtype D, CM244 subtype E, AC10 and NL4.3 subtype B) obtained as previously described
To study the LTNP-EC host genetic markers potentially associated with control of disease progression, we assessed the most validated genetic markers
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HHC/HHF*2 |
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wt/wt |
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wt/mut |
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A*0301 | A*0201 |
B*4402 | B*3501 |
Cw*0501 | Cw*0401 |
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DRB1*1301 | DRB1*1501 |
DRB3*0202 | DRB5*0101 |
DQB1*0603 | DQB1*0602 |
In order to study the virological factors potentially associated with viral control, we cloned the gp160 sequence of
The chimeric viruses derived from the patient were functional but gave low titers in TZM-bl cells ranging from 1.1±0.2×102 to 2.2±1.2×103 TCID/ml (
Viral replication was measured by the p24 production, showed in logarithmic scale in the Y axis, in U87.CD4.CCR5 in panel A) and PBMC cells in panel B). In X axis are represented days of culture.
TZM-bl |
CCR5 |
CXCR4 |
PBMC |
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1.1±0.2×102 | − | − | − |
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6.4±1.3×103 | + | nd | − |
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6.5±4.8×102 | − | − | − |
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2.2±1.2×103 | + | nd | − |
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5.3±3×104 | +++ | nd | + |
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6.5±1×103 | + | nd | + |
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5.3±3×102 | ++ | nd | + |
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1.4±0.2×105 | ++ | nd | + |
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2.0±0.4×103 | − | ++ | + |
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2.3±0×103 | − | +++ | ++ |
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6.4±1.3×105 | − | ++ | + |
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1.3±0×104 | ++++ | nd | ++ |
Tissue culture Infectious Dose 50% (TCI D/ml).
*Positive replication is considered when an increase in p24 production was observed in p24 production between the third and 14 days in culture for cells U87.CD4.CCR5 or CXCR4 or 17 days for PBMCs.
+ represents 2 to 100 fold increase, ++ 102–103 fold increase, 103–104 fold increase and ++++ >105. nd- not done.
The replicative capacity of the recombinant variants from the LTNP-EC was studied in heterologous PBMC that represent more physiologic conditions (
In order to investigate the mutations or defects that could explain the lack of replication of a1, a2, a3 and b1 viruses, the complete amino-acid sequence of the gp160 region was determined (
Amino acid sequences of viruses a1, a2, a3 and b1 are shown. Underlined residues marked the N-glycosylation sites. Empty vertical arrows signal CD4+ binding residues. Cysteines in the variable loops in gp120 are marked with asterisks. Shaded areas correspond to the signal peptide, variable regions in gp120, fusion peptide, homology regions and lentivirus lytic peptide (LLP1-2) region in gp41. In green are shown the two positions in common for a2 and b1 viruses and that are different from the non-replicating viruses.
Subtype B Consensus | Patient viruses | |||||||||||||||
a1 | a2 | a3 | b1 | |||||||||||||
pos |
REGION | aa | % | (n° seq) | mut | % | (n° seq) | mut | % | (n° seq) | mut | % | (n° seq) | mut | % | (n° seq) |
34 | C1 |
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87,4 | 614 |
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3,3 | 22 |
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1,3 | 8 | ||||||
59 | C1 |
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94,7 | 665 |
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0,0 | 0,0 | |||||||||
63 | C1 |
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87,3 | 613 |
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2,1 | 14,0 | |||||||||
105 | C1 |
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88,6 | 622 |
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10,4 | 72,0 | |||||||||
113 | C1 |
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98,0 | 687 |
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1,4 | 10 |
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1,4 | 10 |
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1,4 | 10 | |||
125 | C1 |
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97,7 | 686 |
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0,4 | 3,0 | |||||||||
128 | C1 |
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95,3 | 669 |
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1,3 | 9,0 | |||||||||
177 | V2 |
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86,5 | 607 |
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7,6 | 53,0 | |||||||||
200 | C2 |
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98,7 | 693 |
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1,1 | 7 | |||||||||
203 | C2 |
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98,7 | 693 |
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0,1 | 1,0 | |||||||||
204 | C2 |
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97,8 | 687 |
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1,2 | 9,0 | |||||||||
237 | C2 |
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99,0 | 693 |
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287 | C2 |
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89,6 | 629 |
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10,0 | 70,0 | |||||||||
318 | V3 |
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86,2 | 605 |
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3,0 | 21,0 | |||||||||
384 | C3 |
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97,7 | 686 |
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1,7 | 12,0 | |||||||||
392 | V4 |
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91,0 | 639 |
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2,1 | 15,0 | |||||||||
434 | C4 |
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96,0 | 674 |
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2,8 | 20,0 | |||||||||
492 | C5 |
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94,6 | 664 |
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1,1 | 8,0 | |||||||||
494 | C5 |
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93,9 | 659 |
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4,0 | 28,0 |
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4,0 | 28,0 | ||||||
580 | HR1 |
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89,0 | 625 |
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7,4 | 52 |
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7,4 | 52 |
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3,1 | 22,0 | |||
593 | HR1 |
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96,6 | 678 |
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3,2 | 24,0 | |||||||||
605 | HR1 |
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93,8 | 659 |
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3,9 | 27,0 | |||||||||
611 | HR1 |
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99,0 | 698 |
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616 | HR1 |
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96,6 | 678 |
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1,4 | 10 |
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1,4 | 10 | ||||||
617 | HR1 |
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86,8 | 609 |
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12,0 | 85 |
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12,0 | 85,0 | ||||||
704 | MSD |
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89,0 | 625 |
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6,0 | 42,0 | |||||||||
735 |
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89,7 | 630 |
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9,5 | 67,0 | ||||||||||
752 |
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88,0 | 616 |
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10,0 | 71,0 | ||||||||||
769 | LLP2 |
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92,0 | 646 |
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7,6 | 53 | |||||||||
807 | LLP1-2 |
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94,6 | 664 |
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5,4 | 38,0 | |||||||||
808 | LLP1-2 |
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90,0 | 632 |
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3,3 | 23 |
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3,3 | 23 |
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3,3 | 23 |
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6,1 | 43,0 |
825 | LLP2 |
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99,0 | 692 |
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856 | LLP1 |
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86,6 | 608 |
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8,7 | 61 |
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8,7 | 61 |
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8,7 | 61 |
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8,7 | 61,0 |
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*Position relative to HXB2 sequence (accession number K03455).
Unusual residues in conserved positions (more than 85% conservation) are shown.
Those residues present below 99% in the los Alamos Data Base are underlined.
To estimate the role of the cellular immune response in the control of viral replication, we evaluated CTL responses of the LTNP-EC in the first sample available after SI (1995 sample). The LTNP-EC responded to a total of 27 peptides (OLP). Of the 27 responses, 6 (22.2%) targeted Gag protein, 3 (11.1%) targeted Nef, 2 (7.04%) Protease, 11 (40%) Reverse Transcriptase, 3 (11.1%) Integrase and 2 (7.04%) Gp120 (
The analysis was performed in the first sample available after SI (▪) and on a second one taken 8.8 years later (▪). The results are expressed as spot-forming cells (SFC) per million inputs PBMC in Y axis. The X axis shows the overlapping peptides in viral proteins along the genome eliciting a positive response.
LTNP-EC HLA | PROTEIN | SEQUENCE | MAGNITUDE CTL (SFC/106 PBMC) 1995 sample | MAGNITUDE CTL (SFC/106 PBMC) 2004 sample | |
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EKIRLRPGG |
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LR AEQA SQ |
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QVPLRPMTYKAAVD |
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KK |
Sequence of the OLP (GAG-3, GAG-42, NEF-11 and RT-16) are shown in bold.
Optimal CLT epitopes are underlined. Underlined and in cursive residues represent the mutations in optimal residues found in LTNP-EC virus.
We also compared the CTL responses of the first sample available after SI (1995) with a second one taken 8.8 years later (sample 2004). In this second sample the study subject had a total breadth of 23 responses, of which 13 (56.5%) were already detected at the first time point. In this case, the total magnitude was 26.080 SFC/million PBMC (
Mean CTL Magnitude | Mean Breadth of Responses | |
(EC Percentile) | (EC Percentile) | |
SI Patient 1995 sample | 35.725 | 27 |
Peru Cohort | 5.202 (99) | 15 (85) |
Barcelona Controllers | 19.345 (88) | 23 (69) |
SI Patient 2004 sample | 26.080 | 23 |
Peru Cohort | 5.202 (98) | 15 (80) |
Barcelona Controllers | 19.345 (71) | 23 (67) |
In order to compare these cellular immune responses to reactivities in HIV controllers and non-controllers, we compared the data from the SI LTNP-EC with data from a cohort of 250 treatment naïve Peruvian patients (
The neutralization capacity of the LTNP-EC plasma samples was assayed against a mini-panel of six recombinant viruses with envelopes from different subtypes and tropisms (see
Numbers indicate the percentage ± SD of infectivity after neutralization (100% infectivity is 0% neutralization).
Infectivity is indicated as follows: ▪<10% ▪ 10–30% ▪ 30–50% □ >50% infectivity.
Number of neutralized viruses | % of samples with 50% neutralization |
0 | 6.05 |
1 | 24.19 |
2 | 20.16 |
3 | 14.52 |
4 | 18.55 |
5 | 11.69 |
6 | 4.84 |
In this work we described a very uncommon case of SI in an HIV-1 LTNP-EC. This study subject, who maintained the EC status for 20 years even after SI, showed natural control of the two viruses in the absence of protective HLA class I alleles. The investigation of the viral and immune factors associated with viral control identified a strong and sustained broad CTL and high neutralization response, beneficial HLA class II alleles as well as the presence of deleterious viruses as potential factors contributing for the observed clinical outcome.
Host genetic factors, like co-receptors polymorphisms and HLA I alleles, have been strongly associated with long term virological control
Due to the extremely low viral load and to the selection of the fittest variant when the co-culture method is applied
It is well known that provirus from patients may include many defective viruses, i.e. viruses that are not able to replicate
The presence of viruses with limited replicative capacity before and after SI could be explained by two hypotheses: i) the study subject was infected twice by viruses with a very low replicative capacity. Although this option could not be rejected, it seems unlikely because it is known that there is wide spectrum of phenotypic characteristics among primary isolates
Accumulation in the proviral quasispecies of impaired viruses with truncated
In the reported SI cases in HIV-1 infections, viral control is generally lost after SI even in EC patients
Previous work shows that the impact of T cell responses on control of viral replication cannot be explained by quantification of the magnitude and breadth of this response
The neutralizing antibody response of this LTNP-EC was analyzed measuring heterologous neutralization against a panel of reference strains. The observation that neutralizing antibodies are capable of preventing infection in animal models
In summary, this study associates the ability to contain two divergent HIV infections with the maintenance of a strong HIV-1-specific CD8+ T cell response, beneficial HLA class II alleles as well as a good humoral neutralization response in a SI HIV-1 LTNP-EC. In addition, representative variants from the primo and SI viruses showed impaired replicative capacity in U87-CCR5 and PBMCs cells.
Whether the strong cellular and humoral immune responses were indeed mediating viral control and how other SI LTNP-EC contain their secondary viruses will require the identification of larger numbers of dually or super-infected LTNP-EC and close monitoring of these subjects from early infections time points. Although the latter may be complicated as secondary infections may go clinically unnoticed, such studies may warrant the effort as they could be highly informative for HIV vaccine immunogen development.