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α1Proteinase Inhibitor Regulates CD4+ Lymphocyte Levels and Is Rate Limiting in HIV-1 Disease

  • Cynthia L. Bristow ,

    cyb2005@med.cornell.edu

    Affiliations Weill Cornell Medical College, New York, New York, United States of America, Institute for Human Genetics and Biochemistry, Vesenaz, Switzerland

  • Mariya A. Babayeva,

    Affiliations Weill Cornell Medical College, New York, New York, United States of America, Institute for Human Genetics and Biochemistry, Vesenaz, Switzerland

  • Michelle LaBrunda,

    Affiliation Holmes Regional Medical Center, Melbourne, Florida, United States of America

  • Michael P. Mullen,

    Affiliation Mount Sinai School of Medicine, New York, New York, United States of America

  • Ronald Winston

    Affiliation Institute for Human Genetics and Biochemistry, Vesenaz, Switzerland

α1Proteinase Inhibitor Regulates CD4+ Lymphocyte Levels and Is Rate Limiting in HIV-1 Disease

  • Cynthia L. Bristow, 
  • Mariya A. Babayeva, 
  • Michelle LaBrunda, 
  • Michael P. Mullen, 
  • Ronald Winston
PLOS
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Abstract

Background

The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLECS), it acts not as a proteinase, but as a receptor for α1proteinase inhibitor (α1PI, α1antitrypsin, SerpinA1). Binding of α1PI to HLECS forms a motogenic complex. We previously demonstrated that α1PI deficiency attends HIV-1 disease and that α1PI augmentation produces increased numbers of immunocompetent circulating CD4+ lymphocytes. Herein we investigated the mechanism underlying the α1PI deficiency that attends HIV-1 infection.

Methods and Findings

Active α1PI in HIV-1 subjects (median 17 µM, n = 35) was significantly below normal (median 36 µM, p<0.001, n = 30). In HIV-1 uninfected subjects, CD4+ lymphocytes were correlated with the combined factors α1PI, HLECS+ lymphocytes, and CXCR4+ lymphocytes (r2 = 0.91, p<0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with >220 CD4 cells/µl, CD4+ lymphocytes were correlated solely with active α1PI (r2 = 0.93, p<0.0001, n = 26). The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human α1PI. Chimpanzee α1PI differs from human α1PI by a single amino acid within the 3F5-binding epitope. Unlike human α1PI, chimpanzee α1PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4+ lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-α1PI immune complexes correlated with decreased CD4+ lymphocytes in HIV-1 subjects.

Conclusions

This report identifies an autoimmune component of HIV-1 disease that can be overcome therapeutically. Importantly, results identify an achievable vaccine modification with the novel objective to protect against AIDS as opposed to the current objective to protect against HIV-1 infection.

Introduction

Hematopoiesis in humans begins with stem cell migration from fetal liver through the periphery to the stromal area of hematopoietic tissue where cells are retained, differentiated, and released as maturing progenitor cells back into the periphery. Progenitor cells subsequently migrate to functionally unique tissues such as thymus for further steps of locally-defined differentiation. Pools of stem cells and progenitor cells are retained in hematopoietic tissue throughout life providing a microenvironment for progenitor cell renewal [1]. In human adults, hematopoiesis is dependent on the chemokine receptor CXCR4 and its ligand CXCL12 with an additional role played by cell surface human leukocyte elastase (HLECS), and these components are motogenic [2][4].

Mutations in the HLE-encoding gene ELA2 produce periodic cycling in hematopoiesis that affect monocytes and neutrophils [5], [6]. HLECS and its granule-released counterpart (HLEG) are synthesized as a single molecular protein that is trafficked to the cell surface early in ontogeny and is then redirected by C-terminal processing to the granule compartment later in ontogeny [7][9]. Generally, HLE mutations that prevent its localization to the plasma membrane cause cyclic neutropenia, while mutations that cause exclusive localization to the plasma membrane cause severe congenital neutropenia [7]. Individuals carrying a mutation in the transcriptional repressor oncogene GFI1 which targets ELA2, synthesize twice more HLE, twice fewer absolute numbers of circulating CD4+ and CD8+ lymphocytes, and 7 times more monocytic cells [10]. Thus, as opposed to the well characterized enzymatic function of HLEG, the primary functions of HLECS appear to be cell migration and hematopoiesis [2], [4], [11].

The physiologic ligand for HLECS is α1proteinase inhibitor (α1PI, α1antitrypsin, SerpinA1) which is synthesized in hematopoietic and hepatic tissue [12]. Evidence suggests that α1PI also participates in hematopoiesis, specifically thymopoiesis [13]. During thymopoiesis, a cluster of mouse genes are expressed sequentially and were previously identified to encode the T cell alloantigens, Tpre, Tthy, Tind, and Tsu [14]. The chromosomal location of these maturational markers corresponds to that of α1PI, and using monoclonal antibodies that discriminate these mouse maturational markers, the human equivalent was identified as α1PI [15].

The motogenic activities of HLECS and α1PI involve direct or indirect interaction with Mac-1, an αMβ2 integrin [16], and members of the LDL receptor family [17], [18]. In addition, α1PI-HLECS complexes co-localize with the receptors CD4 and CXCR4 in polarized cells, an activity that promotes cell migration and facilitates HIV-1 binding and infectivity [11], [19], [20]. We and others have shown that pretreatment of cells with α1PI and other ligands of HLECS for 60 min inhibits HIV-1 binding and infectivity [21][23]. In contrast, we have also shown that pretreatment of cells with α1PI for 15 min facilitates HIV-1 binding and infectivity [19]. These opposing effects of α1PI may be due to the kinetics of α1PI–induced cell migration which begins with receptor polarization at the leading edge of the migrating cell and concludes with endocytosis of the receptor aggregate at the trailing edge of the cell [18]. After 60 min incubation with α1PI, the HIV-1 receptor aggregate has been internalized rendering cells temporally unable to bind to HIV-1 [22], [23]. Thus, it is likely that the principal influence of α1PI on HIV-1 binding and infectivity is due to its extracellular activities.

Alternative mechanisms of action have been suggested for the α1PI effect on HIV-1 infectivity including that it is both an inhibitor of and is a substrate of two proteinases, the HIV-1 aspartyl protease and the host proteinase furin, both of which participate in processing viral proteins [24]. Like other serine proteinase inhibitors, α1PI forms an irreversible, covalent-like complex with its cognate proteinase, HLEG or HLECS, thereby inhibiting elastolytic activity. The binding of α1PI to the catalytic site within HLE interrupts the electron transfer mechanism of the catalytic triad. Cleavage is not completed, and α1PI is not cleaved [25]. Interaction of α1PI with serine proteinases other than HLE, for example furin, can produce cleavage, and in this case, α1PI is acting as a substrate, not an inhibitor [26]. The evidence that α1PI is a substrate for the HIV-1 aspartyl protease implies α1PI competes with the protease's natural substrate (Gag-Pol) such that the decreased cleavage of Gag-Pol detected was due to substrate competition, rather than inhibition.

In addition to hepatocytes, α1PI is produced in bone marrow, by lymphocytic and monocytic cells in lymphoid tissue, and by the Paneth cells of the gut [27], [28]. Since α1PI therapy in our previous study produced increased CD4 numbers in PIzz as it did in HIV-1 patients, it can be interpreted that α1PI in circulation contributes to CD4 numbers [13]. Since PIzz patients have very low blood concentrations of α1PI and do not consistently exhibit below normal CD4 numbers, it can be interpreted that α1PI produced in bone marrow and lymphocytic tissue also contribute to regulating CD4 numbers.

The kinetics of T cell death and proliferation has explained, in part, the short-term depletion of the circulating pool of CD4+ T cells in HIV-1 infection [29]; however, an explanation for their long-term depletion is absent and involves both depletion of the circulating pool and depression of hematopoiesis [13], [30]. It was previously demonstrated that in HIV-1 disease, α1PI is severely deficient suggesting that insufficient α1PI could impede thymopoiesis [31]. In a longitudinal study, the number of immunocompetent CD4+ lymphocytes in HIV-1 subjects was found to increase to normal levels within 2 weeks of initiating α1PI augmentation therapy, and this suggests that α1PI participates in regulating the number of circulating CD4+ lymphocytes [13]. Herein is shown that in a cross-sectional study, α1PI correlates with the number of CD4+ lymphocytes in HIV-1 subjects. The presence of anti-HIV-1 IgG-α1PI immune complexes in HIV-1 patients is shown to cause the attendant functional deficiency in α1PI.

Results

Lymphocyte numbers are regulated by α1PI

In healthy individuals, the concentration of α1PI in serum ranges from 18–53 µM between the 5th and 95th percentiles, and 90–100% of this protein is in its active form as determined by inhibition of porcine pancreatic elastase [31]. To investigate the relationship between active serum α1PI concentration, HLECS+, and CD4+ lymphocyte numbers, blood was collected from 30 healthy HIV-1 uninfected adults, 14 males and 16 females (Table 1). Neither serum active α1PI, serum CXCL12, HLECS+ lymphocytes, or CXCR4+ lymphocytes, were independently correlated with CD4+ lymphocytes. However, using multilinear regression analysis it was found that higher CD4+ lymphocytes were significantly correlated with a combination of factors including higher serum active α1PI, lower HLECS+ lymphocyte numbers, and higher CXCR4+ lymphocyte numbers (r2 = 0.91, p<0.001). Neither serum CXCL12 (r2 = 0.21) nor CCR5+ lymphocytes (r2 = 0.56) were significantly correlated with CD4+ lymphocytes. Using multilinear regression, absolute lymphocyte numbers (T B, and NK cells) were correlated with CXCR4+ lymphocytes (r2 = 0.90, p<0.001), but not with HLECS+ lymphocytes, serum active α1PI or serum CXCL12. These results are consistent with a regulatory pathway for CD4+ lymphocyte numbers that includes active α1PIand the receptors CXCR4 and HLECS.

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Table 1. Regression analysis of CD4+ and absolute lymphocyte numbers in HIV-1 uninfected subjects.

https://doi.org/10.1371/journal.pone.0031383.t001

α1PI is correlated with CD4+ lymphocyte numbers in HIV-1 disease

In the cross-sectional study population, blood was collected from 35 HIV-1 infected adults, 33 males and 2 females. Of these 35, 26 were found to have >220 CD4 cells/µl and 9 to have <220 CD4 cells/µl at the time of blood collection. All HIV-1 infected subjects were measured for numbers of CD4+, CXCR4+, and CCR5+ lymphocytes, as well as serum concentrations of CXCL12 as well as total, active, and inactive α1PI. Of these 35 subjects, 11 had active liver disease as defined by detectable Hepatitis B or C, or elevated liver enzymes. HIV-1 infected subjects with liver disease were not different from HIV-1 infected subjects without liver disease in serum active α1PI (p = 0.95), serum total α1PI (p = 0.79), CXCR4+ lymphocytes (p = 0.63), or CCR5+ lymphocytes (p = 0.9), but exhibited significantly higher serum CXCL12 (p<0.001), HLECS+ lymphocytes (p<0.001), and CD4+ lymphocytes (p = 0.04).

In the 26 HIV-1 infected subjects with >220 CD4 cells/µl, absolute lymphocyte counts were significantly lower in HIV-1 infected subjects than HIV-1 uninfected subjects (p = 0.03) as were CXCR4+ lymphocytes (p = 0.001), CD4+ lymphocytes (p<0.001), and active α1PI (p<0.001). On the other hand, total α1PI (p = 0.003) and inactive α1PI (p<0.001) were significantly higher in HIV-1 infected subjects. In these HIV-1 infected subjects, higher CD4+ lymphocyte numbers were correlated with higher concentration of serum active α1PI (r2 = 0.927, p<0.0001) and lower concentration of serum inactive α1PI (r2 = 0.946, p<0.0001) (Fig. 1A).

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Figure 1. Correlation between α1PI, IgG-α1PI immune complexes, and CD4+ lymphocytes in HIV-1 infected subjects.

(A) In subjects with >220 CD4 cells/µl, CD4+ lymphocyte levels correlate with active α1PI (r2 = 0.927, p<0.0001, n = 26). CD4+ lymphocyte levels also correlate with inactive α1PI, (r2 = 0.906, p<0.0001, n = 26). Subjects receiving protease inhibitor therapy are depicted by red squares. All other subjects are depicted by black circles. In the 9 subjects with <220 CD4 cells/µl, no correlation was found to exist between CD4+ lymphocyte levels and active α1PI. Non-linear regression was performed using a 3 parameter Sigmoid curve with power of test α = 0.05. In this population, all variables were found to have normality and constant variation. (B) In 8 of 35 subjects, IgG-α1PI immune complexes were detected and were correlated with CD4+ lymphocyte levels (r2 = 0.822, p = 0.05) and with inactive α1PI (r2 = 0.988, p<0.0001).

https://doi.org/10.1371/journal.pone.0031383.g001

Regression analysis revealed a sigmoidal relationship between CD4+ lymphocyte numbers and active α1PI or inactive α1PI, and this is typical of many biological relationships in which a linear relationship reaches plateau at saturation (Fig. 1A). In contrast, in HIV-1 uninfected subjects, there was no apparent sigmoidal relationship between CD4+ lymphocyte numbers and active α1PI (r2 = 0.20) or inactive α1PI (r2 = 0.22), and this is due to the multiple interacting relationships between CD4+ lymphocytes and CXCR4+ lymphocytes, CXCL12 and active α1PI.

Of these 26 subjects, 21 were additionally measured for HLECS. As in the HIV-1 uninfected population, HLECS+ lymphocyte numbers were not independently correlated with CD4+ lymphocyte numbers, but in combination with higher serum active α1PI concentration, lower HLECS+ lymphocytes were significantly correlated with CD4+ lymphocyte numbers (p = 0.01) (Table 2). In subjects with <220 CD4 cells/µl, there was no relationship between CD4+ lymphocyte numbers and active or inactive α1PI concentrations in serum (Fig. 1A), and this suggests either HIV-1 itself, or other host processes contribute to disrupting the regulation of CD4+ lymphocyte numbers, a question presently being addressed in a separate manuscript (unpublished observations). CD4+ lymphocyte numbers were not found to correlate with CXCR4+ lymphocyte numbers or CCR5+ lymphocyte numbers individually or in combination with any parameters of disease being investigated in these HIV-1 infected subjects, and this suggests that although these chemokine receptors participate during HIV-1 entry, they do not participate in the pathologic decrease in CD4+ lymphocytes.

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Table 2. Regression analysis of CD4+ and absolute lymphocyte numbers in HIV-1 infected subjects.

https://doi.org/10.1371/journal.pone.0031383.t002

It was previously demonstrated that early in disease, 89% HIV-1 infected subjects have detectable antibody that is reactive with α1PI [31]. Two monoclonal antibodies (1C1 and 3F5) which bind a conformationally determined epitope near the C5 domain of gp120 were found to also bind human α1PI [32]. To examine the possibility that antibodies reactive with α1PI might participate in the depletion of active α1PI, IgG-α1PI immune complexes were measured in all samples with sufficient residual volume. Of 22 HIV-1 infected and 21 HIV-1 uninfected individuals tested, 8 were found to be positive for detectable IgG-α1PI immune complexes, and all 8 were HIV-1 infected subjects. Significantly, IgG-α1PI immune complexes were correlated with CD4+ lymphocyte numbers (r2 = 0.822, p>0.05, n = 8) and with serum inactive α1PI concentration (r2 = 0.988, p>0.05, n = 8) (Fig. 1B).

Anti-gp120 inactivates human, but not chimpanzee α1PI

It was hypothesized that anti-gp120 mediated depletion of active α1PI might be pathognomonic for HIV-1-induced AIDS. If true, this would suggest that chimpanzee α1PI differs from human α1PI since HIV-1 infected chimpanzees survive infection and regain normal numbers of CD4+ lymphocytes [33]. Sequence comparison revealed that other than the known polymorphisms, human α1PI differs from chimpanzee α1PI by a single amino acid (aa 385) caused by a single nucleotide change (NCBI accession numbers BT019455 and XP_522938), and this variant amino acid lies in the α1PI region homologous to gp120. To determine whether this sequence difference influences the binding of anti-gp120 to α1PI, sera were compared from 18 HIV-1 uninfected humans and 20 HIV-1 uninfected chimpanzees.

In contrast to chimpanzee α1PI, binding of both 1C1 (data not shown) and 3F5 to human α1PI was elevated 8- to 14-fold above background in 6 repeat measurements (p<0.001) (Fig. 2A). Negative control monoclonal antibody α70 which reacts with the V3-loop of gp120 failed to bind human α1PI consistent with previous findings [31], and there was no difference between binding of α70 to human or chimpanzee sera (p = 0.6). The markedly greater affinity for 3F5 exhibited by serum α1PI in two human subjects suggests that the epitope of α1PI recognized by 3F5 may be phenotypically determined. Even when these two subjects were omitted from the comparison, the statistical difference between binding of 3F5 to human versus chimpanzee α1PI was maintained (p<0.001).

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Figure 2. Binding of anti-gp120 to human, but not chimpanzee α1PI.

(A) Monoclonal antibody 3F5 (5 µg/ml) binding to α1PI in sera from 18 HIV-1 uninfected humans and 20 HIV-1 uninfected chimpanzees was measured using ELISA. Antibody bound (A490 nm) was normalized for the active α1PI concentration in each specimen and is represented as A490nm/[α1PI (µM)]. Representative data from 6 measurements are depicted. Bars represent median values. Median 3F5 bound to human α1PI was 0.12 and to chimpanzee α1PI was 0.02. Negative control monoclonal antibody α70 (10 µg/ml) yielded A490nm = 0.02 when incubated with α1PI at concentrations varying between 3 µM and 540 µM. There was no difference in binding of α70 to human or chimpanzee sera (p>0.6). (B) IgG-α1PI immune complexes (A490 nm) were measured in sera from HIV-1 uninfected humans (n = 9), HIV-1 infected humans (n = 35), HIV-1 uninfected chimpanzees (n = 20), HIV-1 challenged chimpanzees (n = 2), rhesus monkeys pre-immunization and 2 time points post immunization (n = 12), and rhesus monkeys pre- and post-infection (n = 3). There was no significant difference in rhesus monkeys pre- and post-immunization, pre-and post-infection. Representative data of triplicate measurements are depicted. (C) Active α1PI was measured in HIV-1 uninfected humans (26 µM, n = 20), HIV-1 infected humans (18 µM, n = 35), HIV-1 uninfected chimpanzees (35 µM, n = 20), HIV-1 challenged chimpanzees (39 µM, n = 2), rhesus monkeys pre-immunization and 2 time points post immunization (36 µM, n = 12), and rhesus monkeys pre- and post-infection (43 µM, n = 3). There was no significant difference in rhesus monkeys pre- and post-immunization, pre-and post-infection. Bars represent median values. (D) Inactive α1PI was measured in HIV-1 uninfected (4 µM, n = 20) and HIV-1 infected humans (19 µM, n = 35). Bars represent median values. (E) Active α1PI levels in sera from 5 HIV-1 infected subjects after incubation with either medium (control) or with monoclonal antibody 3F5.

https://doi.org/10.1371/journal.pone.0031383.g002

None of the sera from 20 HIV-1 uninfected chimpanzees, nor sera collected from 2 chimpanzees post-HIV-1 inoculation, had evidence of detectable IgG-α1PI immune complexes (Fig. 2B). The HIV-1 inoculated chimpanzees were confirmed to be HIV-1 infected, but to have normal numbers of CD4+ lymphocytes (personal communication, Dr. P.N. Fultz) [34]. In addition, despite the presence of anti-gp120, there was no evidence of IgG-α1PI immune complexes in 12 rhesus macaques (median active α1PI = 36 µM) following immunization with simian/human immunodeficiency virus (SHIV 89.6) gp120 or gp140, or in 3 macaques infected with SHIV (median difference between pre- and post-immunization, A490nm = 0.08). Extensive in vitro ELISA and Western Blot analyses failed to demonstrate bi-molecular complexes between gp120 and α1PI which rules out the possibility that anti-gp120 association with α1PI was mediated by gp120. Further, the absence of detectable IgG-α1PI immune complexes in sera from HIV-1 infected chimpanzees suggests that gp120 and α1PI are not associated by aggregation in sera.

Consistent with previous evidence, the serum concentration of active α1PI in HIV-1 subjects (median 18 µM) was significantly below normal (median 26 µM, p<0.001) (Fig. 2C) and inactive α1PI (median 19 µM), was significantly above normal (median 4 µM, p<0.001) (Fig. 2D) [31]. In contrast to humans, active α1PI concentration in sera collected from the 2 chimpanzees post-HIV-1 inoculation (median 39 µM) were not different from normal human or chimpanzee sera (median 35 µM, p = 0.810) (Fig. 2E).

To determine whether α1PI becomes inactivated after complexing with the 3F5 anti-gp120 monoclonal antibody, 3F5 was incubated with sera samples from five healthy HIV-1 uninfected subjects. In comparison to control untreated sera, α1PI activity was significantly diminished to the same degree in all 3F5-treated sera (mean difference = 5.8±0.5 µM, p<0.001) (Fig. 2E).

To determine whether 3F5 anti-gp120 is the same antibody that produces IgG-α1PI immune complexes in HIV-1 infected subjects, α1PI activity was quantitated in 22 sera in the presence or absence of added HIV-1 virions in a blinded manner. Sera from 13 HIV-1 infected subjects with undetectable HIV RNA and >220 CD4 cells/µl, 5 of whom had detectable IgG-α1PI immune complexes, and 9 HIV-1 uninfected subjects were incubated with an inactivated simian immunodeficiency virus (SIV) chimera which expresses the HIV envelope (AT-2 SHIV) [35], [36]. The α1PI activity increased in 5 of 22 sera incubated with AT-2 SHIV, and these 5 sera were the same 5 sera from HIV-1 infected subjects that were found to have detectable IgG-α1PI immune complexes suggesting the α1PI-inactivating factor adsorbed by the virions might have been anti-gp120 (Table 3). In contrast, active α1PI was unchanged in all 9 HIV-1 uninfected sera and in all 8 of the sera from HIV-1 infected subjects lacking detectable IgG-α1PI immune complexes. HIV-1 infected subjects with IgG-α1PI immune complexes were found to have significantly lower CD4 counts (median = 210) than subjects without α1PI-IgG immune complexes (median = 327, p = 0.045).

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Table 3. HIV-1 patient serum antibodies that bind the 3F5-recognized gp120 epitope are the same antibodies that bind and inactivate α1PI.

https://doi.org/10.1371/journal.pone.0031383.t003

To demonstrate the specificity of the inactivating factor adsorbed by AT-2 SHIV, virions were pre-complexed with 3F5 monoclonal anti-gp120 prior to incubation with sera. By ELISA, it was determined that incubating AT-2 SHIV (30 µg p24) with 20 ng 3F5 in 100 µl for 45 min at 23°C results in a binding ratio of 50 ng 3F5/µg p24. After pre-complexing with 3F5, AT-2 SHIV virions were unable to remove the α1PI-inactivating factor and had no effect on α1PI activity (Table 3). These results demonstrate that 3F5 anti-gp120 in patient sera is the principal factor responsible for inactivating α1PI. Further, these results indicate that, as would be anticipated, anti-gp120 in patient sera has greater affinity for gp120 than for α1PI.

Discussion

Herein is shown that active α1PI counterbalances HLECS in regulating CD4+ lymphocyte blood levels in HIV-1 infected and uninfected subjects. In contrast to HIV-1 uninfected subjects, two important differences in the HIV-1 infected population with >220 CD4 cells/µl are notable; 1) the dependence of absolute lymphocyte numbers on CXCL12, and 2) the lack of dependence of CD4+ lymphocytes on the number of CXCR4+ lymphocytes. Since there are fewer CXCR4+ lymphocytes in HIV-1 infected subjects (73%) and even fewer CD4+ lymphocytes (49%), yet no difference in serum CXCL12 or HLECS+ lymphocytes, this suggests a flaw in the interaction between CXCR4 and CXCL12, presumably in bone marrow, that results in production of fewer CXCR4+ lymphocytes despite the presence of sufficient CXCL12. Alternatively, although the magnitude of CXCL12 in blood is small, CXCL12 might systemically influence the number of CXCR4+ lymphocytes by inducing their egress from blood into tissue. The increased CXCL12, HLECS+ lymphocytes, and CD4+ lymphocytes in the presence of active liver disease in 11 HIV-1 infected patients suggests a potential regulatory axis between the liver and hematopoietic tissue that may include additional unknown factors.

In the HIV-1 uninfected population, CD4+ lymphocyte numbers were correlated with three factors including serum active α1PI, HLECS+ and CXCR4+ lymphocyte numbers. Since HLECS and CXCR4 are known to participate in regulating hematopoiesis, this suggests α1PI, HLECS and CXCR4 regulate the number of CD4+ lymphocytes. Because CD4+ lymphocyte numbers were correlated with serum active α1PI concentration with such a high degree of significance in HIV-1 infected subjects with >220 CD4 cells/µl (r2 = 0.927, p<0.0001), it can be concluded that there is a direct regulatory link between active α1PI and CD4+ lymphocyte numbers although this doesn't show causality. Because we have previously demonstrated that α1PI augmentation therapy produced an increase in the number of circulating CD4+ lymphocytes in HIV-1 infected and uninfected subjects, it can be concluded that α1PI regulates the number of CD4+ lymphocytes in blood [13]. Since active α1PI regulates CD4+ lymphocyte numbers, yet HLECS+ and CXCR4+ lymphocyte numbers appear not to contribute to regulation in the HIV-1 infected subjects, it can be concluded that active α1PI is the rate limiting factor for regulating CD4+ lymphocyte numbers in HIV-1 infected subjects. Whether the influence of α1PI on hematopoiesis occurs in bone marrow, the thymus, or elsewhere is currently being investigated.

Three distinct activities of α1PI are performed by moieties within the carboxyl terminal of the protein: (1) inhibition of soluble HLEG mediated by active site residue Met358; disruption of this activity results in emphysema and respiratory-related infections; (2) induction of receptor polarization and cell migration mediated by α1PI residues 370 FVFLM374 [19]; evidence suggests that this domain stimulates cell motility and is responsible for the binding of HLECS- α1PI complexes to members of the LDL receptor family [37], [38]; and (3) binding of α1PI to antibodies reactive with HIV-1 gp120 [31]; functional α1PI deficiency in HIV-1 disease was previously shown to be caused primarily by IgG-α1PI immune complex formation, not by impaired synthesis, proteolysis, or oxidation [31]. IgG-α1PI immune complexes were not detected in all HIV-1 infected subjects, and this may be due to the presence of immunoglobulin subclasses other than IgG, the inability to capture α1PI bound in immune complexes with high IgG content, or their absence in some individuals [39].

The gp120 epitope recognized by the 1C1 and 3F5 antibodies is considered to be conformation-dependent [40]. The gp120 peptide immunogen used to raise 1C1 and 3F5 (471GGGDMRDNWRSELYKYKVVK490) [41] contains both an α-helix (aa 476–484) and linear strand (aa 485–490) (Fig. 3A,B), but other epitope determinants of the antibodies are not known. Human α1PI, which also binds 1C1 and 3F5, contains (Fig. 3C,D), the gp120-homologous sequence (369PFVFLMIDQNTKSPLFMGKVV389) that folds to form a two-stranded antiparallel β-sheet lying at the base of a cleft (4 Å deep by 20 Å long by 5 Å wide) topped by two α-helices (aa 27–44 and 259–277) in a smaller, but similar configuration as the antigen-binding cleft of MHC (10 Å deep by 25 Å long by 10 Å wide) [42]. At the end of the first of these α-helices is the N-linked mannose-containing oligosaccharide that confers structural polymorphism to α1PI (N-linkage at Asn-46) [43]. In the center of the β-sheet that lies in the cleft is Met-385 which distinguishes human from chimpanzee α1PI (equivalent residue = Val-385). Although the function of this cleft is not known, a sequence in the center of the β-sheet formation (370FVFLM374) is homologous to the fusion domain of HIV-1 gp41 [21], and this sequence has been implicated in binding to members of the LDL receptor family and in stimulating cell migration [44] [37].

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Figure 3. Corresponding conformation at the 3F5-recognized epitope in α1PI and CD4-complexed HIV-1 gp120.

HIV-1 gp120 is depicted from two perspectives (A,B) with green representing two α-helices (aa 100–115 and 476–484). The gp120 peptide immunogen used to raise 1C1 and 3F5 (aa 471–490) is located at the C-terminus of gp120, and the linear segment 486YKVV489 is depicted in red along with Met95 and the oligosaccharide-linked segment 234NGT236, all of which are within 8 Å of the conformational epitope. The gp120-homologous domain in α1PI is also located at the C-terminus of the protein, and is depicted from two perspectives (C,D) with violet representing the antiparallel β-sheet strand at the base of the cleft (aa 369–389), and green representing the α-helices that form the mouth of the cleft (aa 27–44 and 259–277). Met-385, which distinguishes human from chimpanzee α1PI, is depicted in red along with the segment 386GKVV389, the oligosaccharide, and oligosaccharide-linked segment 46NST48. The HLEG-reactive site Met-358, is depicted in yellow for orientation. Structures for human α1PI (1HP7) and CD4-complexed HIV-1 gp120 (1RZJ) from the NCBI Molecular Modeling Database (MMDB) were analyzed using Cn3D software. Small carbohydrate structures, depicted in multiple colors, were associated with 1RZJ in MMDB, and the three associated with 1HP7 were added using Adobe Photoshop.

https://doi.org/10.1371/journal.pone.0031383.g003

Within the α1PI protein, the sequence 386GKVV389 lies within 5 Å of Met-385 and Asn-46 with its N-linked oligosaccharide in a space occupying 5 Å by 5 Å by 5 Å. In the same relative orientation as in α1PI, the gp120 sequence 486YKVV489 lies within 5 Å of Met-95 and 8 Å of Asn-234 with its N-linked mannose-containing oligosaccharide [45] in a space of dimensions 5 Å by 5 Å by 8 Å. Significantly, the gp120 Asn-234 is invariant [46]. The N-linked oligosaccharide of α1PI Asn-46 confers polymorphism, and our observation that 3F5 binding to α1PI in 2 control sera is much greater than in the other 16 control sera (Fig. 3a) suggests this oligosaccharide may reside within the 3F5 epitope. Thus, these spatial analyses suggest that the 3F5 conformational epitope occupies a space approximately 5 Å by 5 Å by 8 Å which includes KVV, M, N, and the N-linked oligosaccharide. The dimensions of this proposed conformational epitope are consistent with previously characterized 8 Å by 7 Å antigens that contain oligosaccharide determinants [47].

Comparison of the amino acid sequences of human α1PI, HIV-1, HIV-2, SIV (including SIVCPZ), HTLV-1, and HTLV-2 reveals that all share homology with the hydrophobic core of the fusion domain of HIV-1 gp41 (LFLGFL), but only HIV-1 gp120 shares homology with the C-terminal domain of α1PI [31]. This observation is consistent with the species-specific differences in disease caused by the corresponding immunodeficiency viral infections. For example, neither SIV nor HTLV produce AIDS in their natural hosts [48]. SIV causes CD4 depletion in some simian species, but the progression to AIDS is disproportionately faster than in HIV-1 infection of humans. HIV-2 infection may induce AIDS, however, the frequency of AIDS incident to HIV-2 infection is 20-fold lower than for HIV-1, suggesting a variant mechanism of disease progression [49]. That antibodies reactive with α1PI are produced in response to a homologous sequence unique to HIV-1 suggests this immune reaction distinguishes HIV-1 infection from other retroviral infections. Importantly, a single amino acid differentiates chimpanzee α1PI from human α1PI, and this difference is in the HIV-1 gp120 homologous domain, perhaps explaining the lack of progression to AIDS in HIV-1 infected chimpanzees [33].

Molecular mimicry by viruses accommodates non-disruptive incorporation into its natural host thereby facilitating reproduction and avoiding immune clearance. Cross-species virus infection produces a situation in which molecular mimicry is less efficient thereby leading to an inflammatory response and immune recognition. Such situations can secondarily produce autoimmunity. Considering that the proteinases and proteinase inhibitors that maintain homeostasis are species-specific, the variable disease sequelae caused by SIV infection in variant monkey species could be due to the interaction of SIV or anti-SIV with a variety of homologous proteinases and proteinase inhibitors involved in homeostasis, for example, coagulation and complement proteins. Similarly, the variation in onset and course of HIV-1 and HIV-2 disease in man could be caused by differences in the interaction of HIV-1, HIV-2, anti-HIV-1, or anti-HIV-2 with various blood components involved in homeostasis. The binding of HIV-1-specific antibodies to human proteins is well-established, for example, Mac-1 [50], cardiolipin [51], inter-α-trypsin inhibitor [52], and MHC Class II [53]. In one study, 49 of 150 HIV-1 infected subjects were found to have autoantibodies reactive with conjugated fatty acids [54] Evidence presented here and elsewhere suggests that inactivation of α1PI by anti-gp120 is the fundamental reason CD4+ lymphocytes fail to be replenished and that α1PI augmentation therapy can overcome this autoimmune phenomenon and re-establish normal levels of CD4+ lymphocytes [13].

The ability of HIV-1 virions to liberate α1PI from anti-gp120 antibodies is not surprising since HIV-1, not α1PI, is the immunogen. The competitive advantage of HIV-1 for binding to anti-gp120 provides a unique method for therapeutically liberating α1PI by blocking autoimmune antibodies. In this manner, humans might co-exist with HIV-1, as chimpanzees do, without developing AIDS. Theoretically, modification of candidate HIV-1 vaccines so that antibodies that react with human proteins are avoided altogether provides a means to develop a vaccine that protects against AIDS as opposed to the current approach to develop a vaccine that protects against HIV-1 infection.

Materials and Methods

Ethics Statement

Collection of blood from research subjects was approved by the institutional review board of Cabrini Medical Center. Written informed consent was obtained from all subjects prior to participation in these studies. Residual sera from chimpanzees were purchased or approved for use by the LEMSIP IACUC.

Human subjects

After obtaining informed consent, blood was collected from 30 HIV-1 seronegative, healthy adults, 14 males and 16 females, and from 39 HIV-1 seropositive adults attending clinic, 37 males and 2 females. Two of the HIV-1 uninfected individuals were healthy adults with the inherited version of α1PI deficiency (PIZZ) and exhibited 41% and 42% CD4+ lymphocytes (reference range = 34%–58%). HIV-1 infected individuals with malignancies (n = 4) were omitted from analyses of CD4+ lymphocyte levels and are being evaluated separately. Of the remaining 35 HIV-1 infected individuals included in the study, 11 had evidence of active liver disease.

Rhesus serum (Macaca mulatta)

Adult monkeys were immunized with SHIV or were infected with SHIV 89.6 as described in a previous report [55]. Residual sera from 12 immunized and 3 infected monkeys were obtained from Dr. D.C. Montefiori with approval from the Duke University Medical Center IACUC.

Chimpanzee serum (Pan troglodytes)

Residual sera from 20 uninfected HIV-1 adult chimpanzees, 10 males and 10 females, were purchased from YERKES Regional Primate Center of Emory University. Residual sera from 2 chimpanzees collected pre- and 42 months post-HIV-1 challenge were obtained from LEMSIP of NYU Medical Center after obtaining approval from the LEMSIP IACUC [34], [56]. One chimpanzee was inoculated IV with cell-free HIV-DH12 [57]. The other chimpanzee was inoculated twice via the cervical os with HIV-infected peripheral blood cells from a chimpanzee infected with HIV LAI/IIIB and once IV with cell-free HIV-LAI/IIIB [34]. Both chimpanzees were previously confirmed to be infected and to have normal CD4+ lymphocyte levels [34].

Flow cytometric analysis

Surface staining for markers on cells was performed by incubating whole blood for 15 min at 23°C with anti-CD4-FITC, anti-CXCR4-PE, anti-CCR5-PE, isotype controls (BD Biosciences, San Diego, CA). Cells were subsequently stained to detect HLECS by incubating whole blood for an additional 15 min at 23°C with rabbit anti-HLE (Biodesign, Kennebunkport, ME) or negative control rabbit IgG (Chemicon, Temecula, CA) which had been conjugated to Alexa Fluor 647 (Molecular Probes, Eugene, OR). After lysing red blood cells and washing, stained cells were fixed. At least 10,000 cells from each sample were acquired using a FACSCalibur flow cytometer. Markers on cells in the lymphocyte gate were quantitated, and CD4+ cells in the lymphocyte gate were compared with measurements obtained from an outside contractor to validate gating. In all cases CD4+ lymphocyte numbers were within 95% agreement between the two laboratories. Cell staining was analyzed using CellQuest (BD Biosciences) or FlowJo software (Tree Star, Inc., Ashland, OR).

Quantitation of serum CXCL12, α1PI, α2M, and anti-α1PI

Serum CXCL12 was measured in duplicate using an ELISA kit according to the manufacturer (R&D Systems, Minneapolis, MN). Total serum α1PI protein was determined in 8 serial serum dilutions by ELISA using previously described methods with the modification that serum dilution buffers contained 10 mM EDTA [58]. Active and inactive fractions of α1PI in once-thawed sera were detected using inhibition of porcine pancreatic elastase (PPE, Sigma, St. Louis, MO) as previously described [58] with the modification that end-point, rather than kinetic, analysis was measured [58]. IgG-α1PI immune complexes were measured in triplicate in phosphate buffered saline pH 7.2 by incubating sera in wells of a microtiter plate pre-coated with chicken anti-human α1PI IgG (OEM Concepts, Toms River, NJ) as previously described [31], and captured α1PI-complexed IgG was detected using peroxidase-conjugated rabbit anti-human IgG (Sigma), a reagent that was confirmed to react with chimpanzee and rhesus macaque IgG. Serum from HIV-1 uninfected subjects that had been collected into tubes containing clot activating additive were excluded from immune complex analysis because of buffer incompatibility.

Binding of anti-gp120 antibody to α1PI

Human or chimpanzee sera from HIV-1 uninfected subjects were diluted 1∶10 in 200 µl phosphate buffered saline containing, 5% fish gelatin and 10 mM EDTA to which was added 10 µl containing 1 µg murine monoclonal antibodies 1C1 (Repligen, Inc., Cambridge, MA) or 3F5 (hybridoma culture supernatant, 0085-P3F5-D5-F8, a generous gift from Dr. Larry Arthur, NCI-Frederick). Monoclonal 1C1 and 3F5 were raised against a peptide immunogen from the HIV-1 gp120 C5 domain (aa 471–490, GGGDMRDNWRSELYKYKVVK) [40] and bind a conformational epitope [32]. Alternatively, sera were incubated with 10 µl containing 2 µg negative control antibody Clone α70 (ICN Biochemicals, Aurora, OH) reactive with the V3 loop of HIV-1 gp120. Immune complexes that were formed by adding anti-gp120 monoclonal antibodies to human or chimpanzee sera were captured by incubating sera in wells of a microtiter plate pre-coated with chicken anti-human α1PI IgG (OEM Concepts). Binding was detected using horse radish peroxidase-conjugated rabbit anti-mouse IgG (Sigma) followed by substrate, orthophenylene diamine HCl.

Inactivated virus adsorption of antibodies from α1PI

The AT-2-inactivated SHIV preparation used in this study was generously provided by Jeff Lifson, Julian Bess, and Larry Arthur of the AIDS Vaccine Program (SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD, USA). Chemically inactivated non-infectious virus with conformationally and functionally intact envelope glycoproteins was produced by treatment with AT-2 as described [35], [36]. The SHIV89.6 virus was produced from the CEM X 174 (T1) cell line [35]. Virus content of purified concentrated preparations was determined with an antigen capture immunoassay for capsid protein (AIDS Vaccine Program). Virus stocks were diluted in 1% BSA (Intergen, New York, NY, USA) in Dulbecco's PBS and stored as aliquots (3 µg capsid protein/ml) at −80°C until use.

To determine the adsorption of anti-gp120 antibodies from α1PI, AT-2 SHIV (3 µg/ml) or dilution buffer (Dulbecco's PBS +1% BSA) were added to an equal volume of serum and incubated for 30 min at 23°C prior to measuring α1PI activity. To demonstrate the specificity of antibody adsorption, AT-2 SHIV virions (5.5 µg/120 µl) were pre-incubated with 3F5 monoclonal anti-gp120 (10.5 µg/500 µl) or dilution buffer (500 µl) for 60 min at 23°C prior to with serum. Unbound 3F5 was removed by centrifugation of AT-2 SHIV virions at 14,000 g for two hrs, and virions were resuspended in 120 µl Tris-buffered saline, pH7.8, containing 10 mM EDTA.

Statistical Analysis

Least squares linear and multiple linear regression were performed using SigmaPlot. Unless other stated, Measurements are presented as mean ± standard deviation for absolute lymphocyte counts, CD4+, CXCR4+, CCR5+, and HLECS+ lymphocytes, as well as serum concentrations of CXCL12, total α1PI, active α1PI, inactive α1PI, IgG-α1PI immune complexes, and 3F5 anti-gp120 binding to human and chimpanzee α1PI. Absolute lymphocyte counts, CXCR4+, and CCR5+ lymphocytes were normally distributed, and means were compared. HLECS+ lymphocytes, and serum concentrations of CXCL12, active α1PI, inactive α1PI, IgG-α1PI immune complexes, and 3F5 anti-gp120 binding to human and chimpanzee α1PI were not normally distributed, and medians were compared. CD4+ lymphocytes were normally distributed in the HIV-1 uninfected subjects, but not normally distributed in the HIV-1 infected subjects, and medians were compared. Means were compared using Student's t-test, and medians were compared using the Mann-Whitney Rank Sum Test.

Acknowledgments

We wish to thank J. Bess, Drs. L. Arthur and J. Lifson for providing monoclonal anti-gp120 and inactivated virus; J. Matthews for his assistance in obtaining informed consent and specimens; Dr. T.C. Rodman for providing chimpanzee sera; Dr. D.C. Montefiore for providing rhesus macaque sera; Dr. P.N. Fultz for providing HIV-inoculated chimpanzee disease parameters; A. Long and R. Tamayev for technical assistance; Dr. M. Murtiashaw for manuscript advice; Dr. S.K. Sullivan for analytical discussion.

Author Contributions

Conceived and designed the experiments: CB. Performed the experiments: MB ML CB. Analyzed the data: CB. Wrote the paper: CB. Monitored patient participation: MM ML. Directed the project: RW.

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