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Journal of Virology, April 2007, p. 3354-3360, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02320-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Infection of Human CD4⁺ T Cells by Microbial HSP70 and the Peptide Epitope 407-426

Kaboutar Babaahmady,¹ Wulf Oehlmann,² Mahavir Singh,² and Thomas Lehner^1*

Mucosal Immunology Unit, King's College London at Guy's Hospital, London, United Kingdom,¹ Lionex Diagnostics and Therapeutics, Braunschweig, Germany²

Received 19 October 2006/ Accepted 10 January 2007

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human immunodeficiency virus type 1 (HIV-1) virions contain heat shock proteins (HSP), but these proteins have received limited attention. The objectives of this study were to establish if the microbial 70-kDa HSP exerts an inhibitory effect on the HIV-1 infection of human CD4⁺ T cells, to identify an inhibitory peptide epitope within the sequence of HSP70, and to evaluate the kinetic features of any inhibitory activity. The results of these studies suggest that microbial HSP70 exerts dose-dependent inhibition on CCR5 (R5) strains of clades B, C, and D of HIV-1 infecting human CD4⁺ T cells. The site of the HIV-1-inhibitory function was identified within the C-terminal peptide binding domain of HSP70, and the function is expressed by the peptide epitope comprising amino acids 407 to 426. The mechanism of inhibition of HIV-1 infectivity by HSP70 is blocking of the CCR5 coreceptors directly and indirectly by inducing CC chemokines and APOBEC3G. The inhibitory effect of HSP70, its C-terminal fragment, or peptide 407-426 may make HSP70 useful as a microbicidal agent. A potentiating noncognate inhibition of HIV-1 infectivity by combined treatment with HSP70 and monoclonal or polyclonal antibody to CCR5 was demonstrated. This novel strategy may be utilized in therapeutic immunization against HIV-1 infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeting the cognate human immunodeficiency virus type 1 (HIV-1) antigen is the classical way of preventing HIV-1 infection, but this approach has encountered serious problems in eliciting effective cytotoxic T lymphocytes (CTL) and neutralizing antibodies. Both CTL and neutralizing antibodies are subject to viral escape, and HIV-1 mutation may occur in the acute (30), as well as the chronic, phase of infection (4, 12, 15). Indeed, HIV-1 sequences found in a population may represent largely CTL escape variants restricted by the most prevalent HLA alleles in that population (29). Alternative preventive strategies such as the use of host antigens may have to be considered. Among a large, nonrandom selection of host proteins, HSP70 was found in the virion membrane of HIV-1; the amount was similar to that of Pol protein, and HSP70 functions as a chaperone during intracellular transport (17). HSP70 mRNA is upregulated in CD4⁺ T cells infected with HIV-1 (35), and the expression of HSP70 is significantly increased in lymphocytes from HIV-1-infected subjects (1). HSP70 elicits innate immune responses by generating chemokines and cytokines (23, 27, 38), which exert robust adjuvanticity in nonhuman primates (5). Furthermore, alloimmune responses can be enhanced by stimulating the CD40-CD40L costimulatory pathway (13) and the alternative CD40-HSP70 pathway may function in a similar way by upregulating CD40, CD80/86, HLA-II, and tumor necrosis factor alpha (TNF-) (38, 39). Recently, we and others have demonstrated that HSP70 binds directly to CCR5 expressed on the surfaces of human CD4⁺ T cells and dendritic cells (11, 41).

The aims of the present investigations were to explore and characterize any inhibitory effect of microbial HSP70 on HIV-1. An examination of the effect of HSP70 on HIV infectivity revealed a dose-dependent inhibition of HIV-1 infection of activated CD4⁺ T cells. The inhibitory function of HSP70 was progressively localized, first to the C-terminal fragment (the peptide comprising amino acids [aa] 359 to 609 [p359-609]), then to the peptide binding domain (p359-494), and finally to the 20-mer peptide epitope (aa 407 to 426). The kinetics of the HIV-1-inhibitory effects of the HSP70 peptides over 24 h were comparable for HSP70, p359-609, and p359-494, though the duration of inhibition varied among the HSP70 fragments. We then explored any complementary inhibitory effect on HIV-1 by adding anti-CCR5 monoclonal or polyclonal antibodies to HSP70. Whereas HSP70 alone inhibited the R5 strains of HIV-1 in a dose-dependent way by up to a mean (± the standard error of the mean [SEM]) of 72.8% (±14.8%) and monoclonal antibody (mAb) to CCR5 inhibited HIV-1 up to 93.5% (±3.9%), combining HSP70 with mAb to CCR5 enhanced the level of inhibition to 99.7% (±0.9%).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of HSP70, its C-terminal (aa 359 to 609) and N-terminal (aa 1 to 358) fragments, and the peptide binding domain (aa 359 to 494). The recombinant Mycobacterium tuberculosis HSP70, p359-609, p1-358, and p359-494 were prepared from the Escherichia coli pop strain. DNA encoding the C-terminal peptide binding domain (aa 359 to 609 and 359 to 494) was expressed using the pJLA603 vector (34). DNA encoding the N-terminal ATPase (aa 1 to 358) domain of HSP70 was cloned and expressed in E. coli by using the PET22b vector. Cloned inserts were verified by DNA sequence analysis. In both cases, recombinant polypeptides were prepared by affinity chromatography using Ni²⁺-chelating resin and the identities of the polypeptides were confirmed by N-terminal sequence analysis (10 cycles each). The proteins were purified by using Q-Sepharose followed by ATP affinity chromatography. The heat shock protein (HSP) preparations were further treated with polymyxin B-coated beads (Sigma-Aldrich, Dorset, United Kingdom) to remove lipopolysaccharide (LPS). The LPS content of the HSP preparations was determined by using the Limulus amebocyte lysate assay (Sigma-Aldrich), which showed an LPS content of <0.006 U/µg of HSP70 or 5 pg/1 µg of the HSP preparation. Any effect of the remaining LPS contamination was excluded as described previously (38, 39). Briefly, intracellular calcium chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester] was used to examine the effect on HSP70 and its C-terminal fragment. TNF- production was inhibited in a dose-dependent manner by the calcium chelator when monocytes were stimulated with HSP70 or the two peptide binding fragments but not when they were stimulated with LPS. Proteinase K was then used, and this had an inhibitory effect on TNF- production stimulated by HSP70 and its two C-terminal fragments but did not affect LPS.

Peptide 407-426 stimulates the production of CC chemokines by dendritic cells, and p457-496 inhibits these chemokines; both of these peptides have been identified within the peptide binding epitope of HSP70 (40). The sequences of these peptides are as follows: p407-426, QPSVQIQVYQGEREIAAHNK, and p457-496, IVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEAH.

The 20-mer peptide 407-426 and a control peptide, 367-386, were synthesized by Neo MPS, Strasburg, France, and the 40-mer peptide 457-496 was synthesized by Bachem Ltd., Switzerland, all to a purity of >90% as determined by high-pressure liquid chromatography.

Preparation of the HSP70 359-609 peptide with the deletion of p407-426. In order to create a peptide spanning the amino acids 359 to 609 of HSP70 but with the deletion of amino acids 407 to 426, the following primers for three-step PCR were used: P1 (5'-GGCGGCGCATATGGAGGTGAAAGACGTTCTG-3'), P2 (5'-TAATTCAAAACTGCCCAATAGGTTGTCGGCGGTGGTGA-3'), P3 (5'-CTATTGGGCAGTTTTGAATTAACCGGCATCCCGCCGGCG-3'), and P4 (5'-GCGGCCGAAGCTTTCAGTGGTGATGGTGGTGGTGAGCCGAGCCGGGGTGGGC-3’).

In the first step, DNA fragments coding for aa 359 to 406 (P1 and P2) and aa 427 to 609 (P3 and P4) were amplified. The sequence of primer P2 consists of a specific region of 20 bases necessary for the amplification of the fragment in the first PCR and an additional 21 bases overlapping with the second fragment. The overlapping bases acted as primers for the annealing of the two fragments, and then the strands were completed by running five cycles of PCR. Finally, primers P1 and P4 were added to the reaction mixture and the whole fragment containing the deletion was amplified.

PCR products were purified using a gel extraction kit (QIAGEN, Hilden, Germany). As primers P1 and P4 included the restriction sites for directed cloning after digestion with endonucleases NdeI and EcoRI, the fragment was ligated to pET26b(+) (Novagen) and E. coli BL21(DE3) cells were transformed with the construct. Recombinant clones were selected on Luria-Bertani agar plates containing 50 µg/ml kanamycin. Plasmids were reisolated, and the cloned fragment was verified by sequencing. For the production of biomass, 2 liters of Luria-Bertani medium containing 50 µg/ml kanamycin was inoculated with an overnight culture with an optical density at 600 nm of 0.2 and the culture was grown at 37°C and 120 rpm. The culture was induced at an optical density at 600 nm of 0.85 with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside), and the cells were harvested 3.5 h after induction. For the purification of recombinant protein, the cells were disrupted by sonication and the crude extract was centrifuged for 20 min at 27,000 x g at 10°C. The supernatant was applied to a Ni-nitrilotriacetic acid column, and the recombinant protein was purified nearly to homogeneity by using a gradient from 0 to 500 mM imidazole. Fractions containing pure recombinant protein were pooled, and the imidazole was removed on a Sephadex G-200 column.

Assay of HIV infectivity for human CD4⁺ T cells. To test HIV-1 infectivity, we used peripheral blood mononuclear cells (PBMC) from samples of healthy blood isolated on a Ficoll-Hypaque gradient. CD4-positive cells were separated from among the PBMC by negative selection on magnetic cell-sorting columns (Miltenyi Biotec, Bisley, United Kingdom). The majority of the cells that were not bound to the columns were CD4-positive T cells; less than 5% were CD8-positive cells, and less than 1% were monocytes. Primary CD4-positive cells were activated with 10 µg/ml of phytohemagglutinin and 20 IU of interleukin-2 (IL-2; Schiaparelli Biosystems BV, Woerden, The Netherlands) in culture medium (RPMI medium with 10% fetal calf serum [Biosera], penicillin at 100 U/ml, streptomycin at 100 µg/ml, and 2 mM L-glutamine [Sigma]) for 3 days and then washed with medium and cultured in RPMI medium with 20 IU of IL-2 overnight. HIV-1 BaL (R5 strain) and LAI (X4 strain) and simian immunodeficiency virus (SIV) SIVmac251 were obtained from the National Institute for Biological Standards and Control (Potters Bar, United Kingdom). Clade C (93IN101) and D (93/UG/082) and additional clade B (92/BR/021 and JR-CSF) strains of HIV-1 R5 were obtained from the National Institutes of Health (AIDS Research and Reference Reagent Program, Rockville, MD), and the group O (clinical isolate) X4 strain of HIV-1 was kindly donated by R. Shattock. Aliquots of 0.6 x 10⁶ primary cells were infected with 10 50% tissue culture infective doses of HIV-1 in a volume of 100 µl for 2 h. Some of the infectivity studies were carried out using the PM1 cell line derived from a CD4⁺-T-cell clone (26) by R. Shattock. With PM1 cells, the virus was adjusted to infect 0.3 x 10⁶ cells and the infected cells were incubated for 2 h. The cells were washed three times with medium and cultured in triplicate at 1 x 10⁵ cells per well in 200 µl of medium with a given amount of HSP70 peptides in 96-well culture plates. Every 2 days, 100 µl of culture supernatant was replaced with 100 µl of medium supplemented with HSP70. On day 7, the culture supernatants were used to determine reverse transcriptase activity by using the Quan-T-RT assay system (Amersham Life Science, Little Chalfont, United Kingdom) and the results are presented as the means (± SEMs) of reverse transcriptase activity with 10-fold dilutions of the stock HIV-1.

HIV-1 inhibition assays with HSP70 and its fragments and peptides. CD4⁺ T cells were infected with the HIV-1 BaL or LAI strain as described above, incubated for 2 h, washed three times with medium, and then treated with twofold dilutions of HSP70. Aliquots of 10⁵ cells/well were cultured in 96-well plates and treated with 3.1 to 50 µg/ml of HSP70 or the peptides. For further studies, an optimum concentration of 20 µg/ml of HSP70 and its fragments or peptides was used. Every 2 days, 100 µl of culture supernatant was replaced with 100 µl of medium supplemented with 20 IU of IL-2 and HSP70. The reverse transcriptase activity was assessed on day 9 as described above.

Kinetics of inhibition of HIV by HSP70. CD4⁺ T cells were separated, as described above, and treated with HSP70, p359-609, or p359-494 at 0, 1, 2, 4, 8, and 24 h after infection with HIV-1 BaL (R5) or LAI (X4). The cells were incubated with the HIV-1 (10 50% tissue culture infective doses) for 2 h, washed three times with medium, and cultured in triplicate at 1 x 10⁵ per well in 96-well culture plates. Every 2 days, 100 µl of culture supernatant was replaced with 100 µl of medium supplemented with 20 IU of IL-2 and HSP70 at 20 µg/ml. On day 7, the reverse transcriptase activity was assayed as described above.

Complementary inhibition studies. Aliquots of 2 x 10⁵ CD4⁺ T cells of the PM1 cell line were treated with HSP70 (20 µg/ml) and mAb to CCR5 (100 µl; 2D7; Pharmingen) at concentrations of 3.1, 6.25, 12.5, 25, and 50 µg/ml. In a parallel assay, the CCR5 antibodies, HSP70, and an isotype control (immunoglobulin G2a [IgG2a]) were used alone at the same concentrations as those given above for the CCR5 antibodies. The cells were then treated with HIV-1 BaL, and the reverse transcriptase activity was assayed on day 9 as described above.

In order to ascertain whether human polyclonal antibodies to CCR5 would have an effect similar to that of CCR5 mAb, we have screened normal commercial serum IgG antibodies used in immunotherapy, as these have been reported to contain CCR5 antibodies (7). We selected serum from Vigam (Bio Products Laboratory, Elstree, United Kingdom) with an anti-CCR5 titer of 1:3,200, as determined by conventional enzyme-linked immunosorbent assay.

Statistical analysis. Data were expressed as means (± SEMs), and the differences between the groups were analyzed by Student's t test. Values of P of <0.05 were considered significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of HIV infection of primary human PBMC and CD4⁺ T cells by HSP70 and its C-terminal fragments. We found a dose-dependent inhibition of up to 78.8% (±3.4%) of the infectivity of the R5 strains of HIV-1 (BaL) for the CD4⁺-T-cell line (PM1) with 50 µg/ml of HSP70, and the 50% inhibition level was reached with 6.25 µg/ml (Fig. 1A). Although some inhibition was also observed with the X4 strain of HIV-1 (LAI), 50% inhibition was not reached with any of the concentrations of HSP70 (Fig. 1A). These studies were then pursued with other clades of CCR5 and CXCR4 strains of HIV-1, and the studies demonstrated 76% inhibition by HSP70 with the R5 strain of clade C and 50% inhibition with clade D, but the X4 strain failed to show >50% inhibition (see Table 3). HSP70 derived from human CD4⁺ T cells showed no inhibition of the replication of the R5 strains or the X4 strain of HIV-1 in human CD4⁺ T cells, in contrast to the dose-dependent inhibition of the R5 strains and to a lesser extent the X4 strain of HIV-1 by microbial HSP70 (Fig. 1B). The C-terminal peptide binding fragment (p359-609) induced a level of inhibition of the R5 strains of HIV-1 very similar to that induced by the wild-type HSP70 (Fig. 1A). In contrast, the N-terminal ATPase fragment (p1-358) failed to induce any inhibition (Fig. 2B).

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FIG. 1. Dose-dependent inhibition of HIV-1. (A) Inhibition of the R5 (BaL) strain by HSP70 and p359-609 and the X4 (LAI) strain by HSP70. (B) Inhibition of the R5 and X4 strains by human HSP70 compared with that by microbial HSP70 (n, 3 or more experiments).

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TABLE 3. Dose-dependent inhibition of infectivity of R5 and X4 strains of HIV-1 by HSP70 and polyclonal antibodies to CCR5 alone and in combinationa

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FIG. 2. Effect of HSP70 (n = 6) and its C-terminal (aa 359 to 609; n = 5), N-terminal (aa 1 to 358; n = 3), and peptide binding (aa 359 to 494; n = 4) fragments on HIV-1. Shown are the levels of infectivity of HIV-1 R5 (BaL) (A) and HIV-1 X4 (LAI) (B) for CD4+ T cells separated from primary PBMC. Reverse transcriptase activity was measured and is expressed as counts per minute. *, P < 0.05. +, present; –, absent.

A systematic study was then carried out using primary CD4⁺ T cells separated from PBMC of healthy volunteers and treated with 20 µg/ml microbial HSP70 and its three fragments (Fig. 2A). This study showed significant inhibition of the R5 strains of HIV-1 by HSP70 (t = 3.57; P = 0.016) and p359-610 (t = 5.45; P = 0.005). Although p359-494 showed a similar level of inhibition, the 5% level of significance was not reached (t = 2.96; P = 0.060). The N-terminal portion of HSP70 (p1-358) consistently showed little or no effect on HIV infectivity, with reverse transcriptase activity at 4,004 ± 740 cpm before treatment and 3,490 ± 757 cpm after treatment with p1-358. Although some inhibition of the X4 strain of HIV-1 was found by treating primary CD4⁺ T cells with HSP70 and its fragments, the 5% level of significance was reached only with the intact HSP70 (Fig. 2B).

Kinetics of inhibition of HIV by HSP70. The next point of investigation was to find out whether HSP70 inhibits HIV infectivity for CD4⁺ T cells only if applied at the same time as the virus or whether HSP70 has any effect on HIV transmission if applied later. Microbial HSP70 significantly inhibited the R5 strains of HIV up to 24 h after the CD4⁺ T cells were exposed to HIV (Fig. 3A). p359-610 also significantly inhibited HIV infectivity, but only up to 4 h after viral challenge. Infectivity studies with the X4 strain of HIV (LAI) showed similar kinetics, but the 5% level of significance was reached only with the wild-type HSP70 at up to 4 h after the HIV challenge (Fig. 3B). Thus, the inhibition of the R5 strains of HIV was most potent when HSP70 was applied at the same time as the HIV (P < 0.02) or after 1, 4, and 8 h (P < 0.01), and significant inhibition was diminished when HSP70 was added later than 8 h after HIV challenge. However, while the infectivity of both groups of HIV strains gradually rose to the level for untreated cells, even 24 h after the infected cells were treated with the HSP70 preparations there was significant inhibition of HIV by the wild-type HSP70 (P < 0.05). The peptide binding domain (p359-494) was then used to inhibit the two groups of strains of HIV-1 over a period of 24 h. Significant inhibition of the R5 strains after treatment with p359-494 was observed at 2 and 4 h but only at 4 h for the X4 strain of HIV-1 (Table 1).

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FIG. 3. Effects of sequential treatment from 0 to 24 h with HSP70 (n = 7) and its C-terminal fragment (aa 359 to 609; n = 5). Shown are the levels of infectivity of HIV-1 R5 (BaL) (A) and HIV-1 X4 (LAI) (B) for CD4+ T cells separated from primary PBMC. *, P < 0.05. Reverse transcriptase was assayed on day 7, and reverse transcriptase activity is expressed as counts per minute.

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TABLE 1. Effect of sequential treatment with HSP70a

The effect of peptide epitopes 407-426 and 457-496 on HIV-1 infection of CD4⁺ T cells. Peptide mapping of the peptide binding domains of HSP70 (aa 359 to 494) has identified two peptides with opposing functions (40). Peptide 407-426 upregulated CCL5 (RANTES), as well as TNF- and IL-12 production, whereas p457-496 inhibited these functions of human monocyte-derived dendritic cells. Since the stimulation of CD4⁺ T cells with HSP70 also generates CCL5, CCL3, and CCL4, the question arose whether the HSP70-derived peptides might affect the HIV infection of CD4⁺ T cells. The results suggest that treatment with p407-426 inhibits the replication of the R5 strains of HIV-1 in the human CD4⁺-T-cell line (PM1) in a dose-dependent manner by up to 77.2% (±9.9%), in contrast to p457-496 or p367-386, which had no effect on HIV replication (Fig. 4). This result was comparable with that for p359-609, which inhibited HIV-1 up to 78.7% (±11.7%), though this peptide was more effective at lower doses. The specificity of inhibition was further demonstrated by deleting p407-426 from the C-terminal fragment of HSP70 (p359-609), which completely lost its HIV-1-inhibitory effect (Fig. 4). A parallel experiment with the X4 strain of HIV-1 showed negligible effects from these peptides (data not shown).

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FIG. 4. Dose-dependent inhibition of the R5 strains of HIV-1 (BaL) in CD4+ T cells by p407-426 compared with inhibition by p457-496 (n = 3), p359-609 (n = 3), p359-609 from which aa 407 to 426 were deleted (407-426; n = 2), and the control p367-386.

Inhibition similar to that of the PM1 cells was found with SIV replication in C8166 cells treated with p407-426 but not with p457-496, p367-386, or albumin (Table 2). A significant difference in the level of inhibition of SIV was found with peptides 407-426 (84.5% ± 2.3%) and 457-496 (35.2% ± 13.2%; t = 3.06; P = 0.037; n = 4).

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TABLE 2. Dose-dependent inhibition of SIVmac251 by HSP70 and the peptide epitope 407-426 in CD4+ T-cell line (C8166)

Complementary inhibition of HIV-1 infectivity by HSP70 and mAb to CCR5. The possibility was then examined that treating CD4⁺ T cells with a constant amount of microbial HSP70 (20 µg/ml) and increasing concentrations of mAbs to CCR5 might potentiate the inhibition of the R5 strains of HIV-1. HSP70 alone inhibited HIV-1 up to 72.8% (±14.8%), and CCR5 mAb inhibited HIV-1 up to 93.5% (±3.9%), but combining the two potentiated the HIV-1-inhibitory effect up to 98.7% (±0.9%) (Fig. 5A). Indeed, concentrations of CCR5 mAb as low as 3.1 µg/ml when added to HSP70 showed >50% inhibition, whereas a fourfold increase of the mAb to CCR5 alone was required to yield comparable inhibition of HIV-1 (Fig. 5A). Isotype controls showed negligible inhibition. A similar study with selected normal human serum IgG containing CCR5 antibodies showed again maximum dose-dependent inhibition of HIV-1 (clade B), and >50% inhibition was achieved with an eightfold-lower concentration of CCR5 antibodies when the antibodies were combined with HSP70 than when the antibodies were used alone (Fig. 5B). Similar results were obtained with clades C and D and another clade B strain of R5 HIV-1 (Table 3) but not with the X4 strain group O (Fig. 5B and Table 3). These experiments demonstrate the principle that while CCR5 antibodies or HSP70 alone elicits significant inhibition, a potentiating effect between HSP70 and monoclonal or polyclonal antibodies to CCR5 may enhance the inhibitory effect on the infectivity of the R5 strains of HIV-1.

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FIG. 5. Inhibition of HIV-1. (A) Dose-dependent inhibition of the infectivity of the R5 strains of HIV-1 (BaL) for human CD4+ T cells (PM1 cell line) by HSP70 (20 µg/ml) and mAb to CCR5 alone and in combination. (B) Inhibition of R5 (BaL) and X4 (group O clinical isolate) strains of HIV-1 with HSP70 and polyclonal CCR5 antibodies alone and in combination. Ab, antibody.

Top Abstract Introduction Materials and Methods Results Discussion References

 
HSP70 and other HSPs have received limited attention, despite their presence in HIV virions (17). HSP70 appears to be essential for virion assembly and may interfere in early events of infection, virion uncoating (8), and targeting of the HIV-1 preintegration complex (2). The ATPase activity of virion-associated HSP70 is required to maintain the integrity of HIV-1 virions (18). Furthermore, HSP70 binds directly to CCR5 (11, 41), blocking HIV-1 access to its coreceptor, and elicits CC chemokines which also bind CCR5, thereby enhancing and prolonging the inhibitory effect of HSP70. Here we demonstrate that HSP70 inhibits the R5 strains of HIV clades B, C, and D 50% and to a limited extent (<40%) inhibits the X4 strain of HIV-1. The inhibitory effect of HSP70 was localized to its C-terminal fragment (aa 359 to 610) and the peptide binding domain (aa 359 to 494). The cytokine- and CC chemokine-stimulating epitope p407-426 (40) also showed a dose-dependent inhibition of HIV-1 and SIV, but not the CC chemokine-inhibitory epitope p457-496 or the control p367-386, all of which are derived from the sequence of HSP70. The specificity of inhibition of HIV-1 replication by p407-426 was further demonstrated by deleting p407-426 from the C-terminal fragment. The dose-dependent inhibition elicited by p359-609 was completely lost through the deletion of p407-426. However, while p407-426 induced inhibition similar to that induced by p359-609 at high concentrations, the latter peptide showed greater inhibition than the peptide epitope at lower concentrations.

Similar results were found with SIV replication in C8166 cells, in which SIV replication was inhibited by the wild-type HSP70 or the peptide epitope 407-426 but not by the other peptides. This finding is consistent with SIVmac251's utilizing CCR5 receptors, as do the R5 strains of HIV-1. Although any residual contamination with LPS has been excluded (see Materials and Methods), a further control has been the lack of inhibition of HIV-1 replication elicited by the N-terminal fragment of HSP70 (aa 1 to 358). Finally, the HIV-1-inhibitory peptide epitope 407-426, derived from the sequence of HSP70, has no LPS and reproduces the function of the intact HSP70.

If HSP70 is added to the T cells at the same time as HIV, all three HSP70 preparations induce maximum inhibition of HIV replication. However, if HSP70 is added sequentially after HIV infection has been initiated, inhibition of the R5 strains is progressively decreased, though significant inhibition is still maintained for up to 24 h with HSP70 but only for up to 8 h with p359-610. The kinetics of inhibition of the X4 strain of HIV are similar, but only the wild-type HSP70 significantly inhibits the virus for up to 4 h postinfection. Thus, if HSP70 were to be used as a virucidal agent, it should retain some antiviral effect up to 24 h after exposure to HIV-1.

CCR5 is the major coreceptor for R5 strains of HIV-1 and SIV, and antibodies to CCR5 have been identified in xenoimmunized macaques (19); HIV-1-exposed, uninfected women (25); and alloimmunized women (37). Here we show that the treatment of activated CD4⁺ T cells with HSP70 (20 µg/ml) and increasing concentrations of CCR5 monoclonal or normal human polyclonal serum antibodies potentiates the HIV-1-inhibitory effect, both in the level of inhibition and in the requirement for four- to eightfold-lower concentrations of IgG to elicit >50% inhibition. This complementary effect was demonstrated with the R5 strains of clades B, C, and D but not with the X4 strain of HIV-1. Although it is not clear how these in vitro results may translate in vivo, there is clear evidence that a decrease in viral load to less than 1,700 HIV-1 RNA copies is a major factor in the control of HIV-1 transmission (14, 16).

HSP70 stimulation elicits a multifunctional mechanism of protection against HIV-1 or SIV, demonstrated in vitro with human cells and in vivo with macaques (5, 6, 22, 23). This mechanism is independent of HIV or SIV priming and thus constitutes noncognate immunity to these viruses. The mechanism of protection involves the three CC chemokines (CCL3, CCL4, and CCL5) which are elicited in vitro and in vivo by the stimulation of human or simian CD4⁺ T cells and dendritic cells with HSP70 (21, 39). These chemokines inhibit preentry HIV-1 binding to CCR5 by the R5 strains of HIV-1 and by most SIV strains (9, 24); CCL3 may also inhibit postentry HIV-1 (3). Production of the nonspecific cell-free antiviral factor (28) has also been demonstrated in vivo to be increased by HSP70 and alloimmunization of CD8⁺ T cells (24, 37). Recently, a potent intracellular innate anti-HIV-1 factor, APOBEC3G, which inhibits both R5 and X4 strains of HIV-1, was identified (33). APOBEC3G is upregulated by stimulating CD4⁺ T cells and dendritic cells with HSP70 (31), and this upregulation may account for the HSP70-induced inhibition observed with the X4 strain of HIV-1. Thus, while the R5 strains of HIV-1 are significantly inhibited by HSP70, with both preentry (HSP70 and CC chemokines) and postentry (APOBEC3G) inhibition of the virus, the X4 strain is inhibited only by APOBEC3G, which may account for the lower level of inhibition of the latter strain.

Quite apart from the antiviral activity of HSP70, it performs a number of immunological functions, which boost immunity and may be significant in therapeutic immunization. The sites corresponding to the chemokine, cytokine, and human-dendritic-cell-maturation functions of microbial HSP70 have been identified within the peptide binding domain of microbial HSP70 (aa 359 to 494) and located in the epitope p407-426, which elicits these functions (39, 40). HSP70 and especially its C-terminal fragment (aa 359 to 609) induce IL-12, TNF-, and nitric oxide (40), as well as IL-1ß and IL-6 (27). Furthermore, HSP70 is capable of the presentation of exogenous antigen by the class I (cross-priming) and class II pathways and it can provide CD4⁺-T-cell help for CD8⁺-CTL memory (32).

Previous investigations of HIV-1 or SIV vaccination were confined to CD8⁺ CTL, CD4⁺-T-cell helpers, and neutralizing antibodies, as these were considered to be the most desirable correlates of vaccine-induced protection. This strategy has been questioned (5, 20, 24, 36) and recently highlighted by a multifunctional analysis using four cytokines and CCL4 generated by CD4⁺ or CD8⁺ T cells (10) which failed to demonstrate a consistent pattern or a correlation with gamma interferon. The immunization of macaques with HSP70 linked to HIV gp120, SIV p27, and CCR5 peptides generated gamma interferon and IL-2, in addition to CCL3, CCL4, and serum and vaginal IgG and IgA antibodies (5). A significant number of the immunized animals vaginally challenged with simian-human immunodeficiency virus 89.6P cleared the virus to undetectable levels. Multifunctional responses of HIV-1 noncognate immunity are consistent with the proposed complementary hypothesis regarding diverse components of immunity. HSP70 and its C-terminal fragment or p407-426 might be utilized as microbicidal agents or as agents in therapeutic immunization either separately or combined with CCR5 antibodies.

 
This work was supported by European Union grant no. LSHP-CT-2003-503558.

 
* Corresponding author. Mailing address: Guy's Hospital, Guy's Tower Floor 28, St. Thomas' Street, London SE1 9RT, England. Phone: 44 207 188 3072. Fax: 44 207 188 4375. E-mail: Thomas.lehner{at}kcl.ac.uk.

Published ahead of print on 24 January 2007.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, April 2007, p. 3354-3360, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02320-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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