WO1997039358A1

WO1997039358A1 - In vitro prognostic test for progressors and non-progressors after hiv infection

Info

Publication number: WO1997039358A1
Application number: PCT/US1997/006136
Authority: WO
Inventors: Davide Zella; Agostino Riva; Marvin S. Reitz, Jr.
Original assignee: The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 1996-04-15
Filing date: 1997-04-14
Publication date: 1997-10-23
Also published as: AU2666997A

Abstract

Prognosis of the progression of AIDS in an asymptomatic subject who is seropositive for HIV-1, as well as determining the efficacy of therapeutic treatments of such a subject, is advanced by a prognostic test that entails contacting in vitro the peripheral blood mononuclear cells purified from the subject with interleukin-2 for a period of time sufficient for the IL-2 to stimulate the cells, and then ascertaining and comparing against normal controls the activation status of the thus-treated cells as determined by assaying for cell surface antigen markers. It has been found that this status is predictive of both progression to AIDS and efficacy of treatment.

Description

IN VITRO PROGNOSTIC TEST FOR PROGRESSORS AND NON-PROGRESSORS AFTER HIV INFECTION

FIELD OF THE INVENTION

This application relates in general to HIV infections in humans. More particularly, it relates to a prognostic in vitro test which distinguishes those HIV seropositive individuals who will progress to AIDS symptoms ("progressors") from those who may not ("non-progressors").

BACKGROUND OF THE INVENTION

The majority of human immunodeficiency virus type I (HIV-1) seropositive subjects manifest an increasing level of B and T-cell activation during their clinical history. Far from being protective, this activation appears to be detrimental for the immune system. In fact, CD4+ T-cells, one of the principal components of the immune response, can be easily infected by HIV-1 and, once activated, they efficiently support viral replication. For this reason, the massive activation response to viral infection provides HIV-1 with an increased number of target cells able, in turn, to sustain more viral production. This cycle of events results in depletion of the CD4+ T-cells and the collapse of the immune system, leading to acquired immunodeficiency syndrome (AIDS).

A minority (about 5%) of seropositive subjects do not seem to progress to AIDS, however, even years after seroconversion. These individuals show substantially reduced immune activation during HIV-1 infection compared with the majority (Pantaleo et al., N. Eng. J. Med. , 332:209 (1995)).

No common determinants, either viral or of the host immune system, have been found to date to explain these different behaviors (Baltimore, Cell, 82: 175 (1995)). One possible hypothesis is that there might be differences in the degree to which HIV-1 cooperates with the host immune system to increase its general level of activation. This hypothesis has not been tested heretofore. There currently is no suitable predictive test that distinguishes the aforementioned two types of HIV seropositive subjects (Hogervorst et al. , J. Infect. Dis. , 171:811 (1995)), reported that the level of early viral load, as determined by an assay of serum HIV-1 RNA, was 36

-2- predictive of clinical outcome. Slow-progressors had lower viral loads at early time points (i.e., 1 year post-infection) than did progressors. In a group of 11 slow- progressors, however, over 60% had high viral loads at the 5-year point, which makes the test something less than predictive. O'Brien et al. (N. Eng. J. Med. , 334:426 (1996)), have proposed a predictive test involving determination of patient's plasma levels of HIV-1 RNA and β-λ-macroglobulin, along with CD4+ response to Zidovidine therapy. The data suggested to the authors that drug-induced increases in CD4+ numbers and reduction in plasma HIV-1 RNA levels, are predictive of the clinical progression of HIV-related disease. But the report is silent as to whether the

10 test discriminates between those individuals who are non-progressors versus those who are progressors.

Clerici et al. (J. Clin. Invest. , 84: 1892 (1989)), have detected three distinct patterns of T helper cell dysfunction in asymptomatic HIV-1 seropositive 15 patients using an in vitro assay for IL-2 production in peripheral blood mononuclear cells (PBMCs) stimulated with influenza A virus, tetanus toxoid, HLA autoantigens or PHA. A time-dependent progression from a stage responsive to all four stimuli to a stage unresponsive to any stimuli (i.e., to T-cell dysfunction) is deemed by the o authors to be a valuable tool in predicting progression from asymptomatic to symptomatic. However, the progression is not correlatable to CD4+ cell numbers.

Clearly, a critical need exists for a prognostic test capable of discriminating between progressors and non-progressors among HIV-1 seropositive asymptomatic individuals. This need has been fulfilled by the in vitro prognostic test described herein.

SUMMARY OF THE INVENTION

An approach has been discovered for prognosing the progression of AIDS in an asymptomatic subject who is seropositive for HIV-1, as well as for ° determining the efficacy of treatments of such subjects. The approach entails contacting in vitro the peripheral blood mononuclear cells ("PBMCs") purified from such subjects with interleukin-2 ("IL-2") for a period of time sufficient for IL-2 to stimulate (or activate) the cells, and then determining and comparing against normal 5 controls the activation status of the treated cells. It has been found that this status is predictive of both progression to AIDS and efficacy of treatment of the subject.

In one embodiment of the invention, the activation status of IL-2-treated lymphocytes is determined using reagents that bind to at least one of the cell surface antigens, for example, CD3, CD4, CD8, CD25, CD26, CD71, and HLA-DR. In another embodiment of the invention, the aforementioned reagents are immunoreagents.

In still another embodiment of the invention, interaction between the immunoreagents and lymphocyte surface antigens is determined by immunofluorescence and fluorescence flow cytometry.

Also, a further embodiment of the invention is an assay kit that incorporates in separate compartments IL-2 and reagents for detecting lymphocyte surface antigens.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1L, 2A-2L and 3A-3J show the results of assays evaluating CD4+ , CD3 + , and CD8+ counts in non-progressors, slow-progressors, and progressors.

Figs. 1A-1L present results of non-progressors or slow progressors. Figs. 1A-1D show the CD4+ T-cell counts in vivo in the 12 months preceding performance of the assay according to the invention. Figs. 1E-1H show the absolute CD4+-CD3+-CD8+ T-cell analyses (T=0) carried out at one time period, i.e. , November 1995. Figs. 1I-1L show the same analyses as in Figs. 1E-1H carried out at another time period, i.e., February/March, 1996. Figs. 2A-2L present results of intermediate progressors. Figs. 2A-2D show the CD4+ T-cell counts in vivo in the 12 months preceding performance of the assay according to the invention. Figs. 2E-2H show the absolute CD4+-CD3+- CD8+ T-cell analyses (T=0) carried out at one time period, i.e., November 1995. Figs. 2I-2L show the same analyses as in Figs. 2E-2H carried out at another time period, i.e. , February/March, 1996.

Figs. 3A-3I present results of progressors. Figs. 3A-3C show the CD4+ T-cell counts in vivo in the 12 months preceding performance of the assay according to the invention. Figs. 3D-3F show the absolute CD4+-CD3-r--CD8+ T- cell analyses (T=0) carried out at one time period, i.e., November 1995. Figs. 3G-3I show the same analyses as in Figs. 3D-3F carried out at another time period, i.e., February/March, 1996.

Figs. 4A-4D show a comparison between the number of CD4+ T-cells in cultures from PBMC from asymptomatic HIV-1 seropositive subjects at different stages of HIV infection. The numbers refer to patients as presented in Table 1.

FACS analysis was performed on the days indicated. As shown in Figs. 4A-4D, "RS" is recently seroconverted; "FP" is fast progressors; "TP" is typical progressors; and "LTS" is long-term survivors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

On the rationale that there might be differences between HIV- seropositive subjects in the degree to which HIV-1 cooperates with the host's immune system, thereby increasing its general level of activation, the present inventors investigated the relationship between HIV-1 replication and cellular activation in response just to minimal stimulation. This was done because potent mitogen molecules, such as PHA and anti-CD3 antibodies, could obscure any differences by unnaturally increasing to the highest degree cellular activation levels. The addition of recombinant human interleukin-2 (rhIL-2) alone has been shown to be sufficient for HIV-1 replication in vitro when exogenous virus was added to uninfected PBMCs (Kinter et al., J. Immunol. , 154:2448 (1995)). This protocol, with some modifications, was employed to study the relationship between IL-2-induced CD4+ T-cell activation and CD4+ T-cell depletion following HIV-1 infection.

Briefly, PBMCs from human donors are separated by the standard Ficoll-Hypaque centrifugation method. The procedures that follow depend upon the kind of data required. For the preferred prognostic method, PBMCs are isolated from HIV- seropositive subjects without AIDS syndrome, an appropriate number of the cells (for example, 25 - 30 million) are seeded in an appropriate culture medium such as RPMI-1640, and at appropriate times thereafter (for example, 3, 6, 9, 12 and 15 days) an effective concentration of IL-2 (typically on the order of 40 U/ml, but other concentrations can be effective) is placed in contact with the cultured cells. One or more surface markers of activation, including but not limited to at least one of cell surface markers HLA-DR, CD25, CD71, CD26, CD3, CD4 and CD8, are analyzed at the outset of incubation and at suitable intervals thereafter to determine the degree of cellular responsiveness in vitro. Increased expression of HLA-DR, CD25, and CD71 antigen markers on CD4-f- T-cells is known to correlate with the progression of AIDS, and CD4+CD26+ T-cells are known to specifically decrease during the course of the disease, even though HIV-1 appears to preferentially interact with CD4+CD26+ T-cells (Blazquez et al., J. Immunol , 9:3073 (1992)). It is preferred for prognostic purposes to assay for only three cell- surface antigens, CD3, CD4 and CD8. It is most preferred to assay for only CD4 cell surface markers. This provides all of the information required for prognostic purposes, and has the additional advantage of being less expensive than assays that use additional cell-surface markers.

Detection of markers may be carried out routinely by automated flow cytometry (Ortho Diagnostic Co.) or by FACScan (Becton Dickinson) using fluorescence immunoreagents. Incubation of cells and detection of cell surface markers can also be carried out in multiple well plastic microtitre plates by routine methods of incubation and the use of detectable immunoreagents.

To mimic the in vivo situation in which the majority of the cells are presumably stimulated by IL-2 at some point after encountering HIV-1 , PBMCs from uninfected donors may be incubated with the virus for three days, IL-2 then is added to the culture medium, and cell markers are determined as described above. The assay according to the invention is valuable for helping to determine the response of CD4+ T-cells to IL-2 stimulation in vitro to evaluate the effect on a subsequent administration of IL-2 in vivo (see Example 6). Moreover, understanding the relationship among these patterns of CD4+ T-cell responsiveness, activation and HIV-1 replication is likely to help to predict the progression to AIDS in asymptomatic HIV-1 -infected subjects. Understanding the relationship among the patterns of CD4+ T-cell responsiveness, activation and HIV-1 replication can also be useful in designing therapeutic strategies to modulate the host immune system (together with down regulating viral replication). A protocol in this regard would involve determining in vitro the immune status in a HIV-1 -seropositive individual. In the case of a highly CD4+ T-cell-responsive subject (i.e., a high level of IL-2 stimulation), the therapeutic protocol can involve the administration of immunosuppressive drugs (such as glucocorticoids), down regulation of IL-2 receptor expression (i.e., by using procysteine or a receptor-specific cytotoxin) or a combination of the two strategies. Follow-up analysis of cellular response should provide information regarding the efficacy and the required duration of the therapeutic intervention(s). In summary, PBMCs from asymptomatic HIV-1 seroactive subjects were stimulated by addition of IL-2 in the absence of mitogens. Cell surface antigens were analyzed in PBMC cultures to determine surface markers of cellular activation. Such cell cultures from human subjects showed two distinct patterns of responsiveness. The CD4 + T-cells of one group were strongly activated, as judged by expression of HLA-DR, CD25, CD26 and CD71 surface antigens. CD4+ T-cells were depleted almost completely within this group after 10-15 days of culture, even though virus production was barely detectable. This group originated from progressor subjects with a low CD4+ T-cell count (below 500/mm³). In the second group, the majority of CD4+ T-cells expressed HLA-DR and CD71 markers at considerably lower levels; however, CD26 was expressed by the majority of the CD4+ T-cells and CD25 expression was transient. CD4+ T-cells were not depleted from these cultures over an 18 day period. The second group originated from non-progressor subjects who were infected with HIV-1 for more than 7 years, but whose CD4+ T-cell counts were still above 500/mm³.

It also was observed that, for prognostic purposes in the general HIV-1- infected population, non-progressors could be distinguished from progressors by estimating only CD3, CD4 and CD8 cell surface markers in PBMCs treated with IL-2 (Example 7). Thus, it was demonstrated that the degree of responsiveness to IL-2 stimulation in vitro of CD4+ T-cells, in the absence of significant HIV-1 expression, correlates with the immunological status of the HIV-1 seropositive subjects. A similar segregation into two patterns of responsiveness was seen when PBMCs from uninfected donors were exposed to HIV-1 and then treated with IL-2, suggesting that the basis for the differences observed already exists prior to virus infection. These individual differences are thought to contribute to the distinct progression patterns of CD4+ T-cell depletion found in progressor and non-progressor subjects.

Accordingly, determining the in vitro responsiveness of the CD4+ T- cells to IL-2 is useful in predicting the clinical course of the disease in the asymptomatic clinical stages, and in helping to develop combined therapies able to target HIV-1 replication through modulation of the host immune response.

The present invention is described in further detail by reference to the following examples, which are illustrative only.

Example 1

Analysis of surface cellular markers in IL-2-stimulated non-infected and in vitro HrV-1-infected PBMCs from healthy donors

Sucrose gradient purified HIV-1„_IB was added to freshly separated

PBMCs from HIV-1 seronegative healthy donors. The cultures were stimulated with

40 U/ml rhIL-2 (Boehringer) at days 3, 6, 12 and 15. The protocol followed was that of Kinter et al. (1995), supra, with some modifications. Aliquots of the cultures were analyzed by FACScan (Becton Dickinson) for the identical surface cellular markers at days 6, 12 and 18. All antibodies were from Immunotech, except anti-CD4, (Leu 3a⁺3b, Becton Dickinson). Cell count and viability were assessed by trypan blue exclusion. The study was performed on nine donors. Aliquots of lxlO⁶ cells were washed after 3 days of infection and p24 production was assessed in the supernatants at days 2, 5 and 10 after IL-2 stimulation. In the absence of exogenous IL-2, control noninfected PBMCs were considerably less activated (compared to the cells stimulated with IL-2) and the majority (above 70%) of the PBMCs died by day 10 of culture. If the same conditions were used to infect PBMCs from healthy donors with other T-lymphotropic viruses (human herpesvirus 6 or human herpesvirus 7) neither productive infection from these viruses nor depletion of CD4+ T-cells was observed.

During culture in vitro, two groups of subjects could be distinguished. In the first group, CD44- T-cells were dramatically depleted (to 4% of the total) by day 18. Analysis of cellular markers over the time in culture clearly indicated strong activation of PBMCs. This group is referred to as strongly responsive ("SR"). HLA- DR antigen was expressed on 52% of the cells, CD25 on 28%, CD26 on 80%, and CD71 on 42% at day 12. In contrast, with PBMCs from the second group of donors, the CD4 +

T-cell number was only slightly affected by the presence of the virus compared to the uninfected control. Low expression of three of four markers was observed (HLA-DR on 10% of the cells, CD25 on 5% and CD71 on 3% at day 12). However, CD26 was expressed at high levels (on 77% of the PBMCs), indicating that the cells were activated in some respects. This group was referred to as weakly responsive ("WR"). It is important to note that these differences were evident only in the presence of HIV- 1 , since uninfected control PBMCs showed similar patterns of activation in response to IL-2 stimulation.

The results showed that in vitro HIV-1 primed PBMCs from uninfected donors respond to a different extent to IL-2 stimulation. These responses (high in SR, lower in WR) correlated with HIV-1 production as measured by extracellular p24, which was significantly higher in SR (25-40 ng/10⁶ cells) compared to WR (3-10 ng/10⁶ cells). Example 2

FACS analysis of surface markers of PBMCs from seropositive subjects

Ficoll-purified PBMCs were cultured in accordance with the foregoing description in Example 1. Depending on the subject, the total viable cell number (as assessed by trypan blue exclusion) ranged between 5x10* and 6xl0³ at day 6; IxlOVml and 2x107ml at day 12; and 6x10* and 9x107ml at day 18. Aliquots of PBMCs were analyzed by double color FACS analysis. The subjects are the same as presented in Table 1.

In the first days, adherent macrophages were observed in cultures from all subjects. By day 6-8, no macrophages (CD14+) or B cells (CD19+ or CD20+) were detected in suspension and the majority (85-90%) of the cells were CD3+ as assessed by FACS analysis. PBMCs from progressor subjects became larger by day 7-8, numerous clumps were observed, and high levels of [Η]thymidine incorporation were detected, indicating strong cellular activation and replication. In contrast, PBMCs from nonprogressor subjects were small in size, clumps were rarely observed and [³H]thymidine incorporation levels were 20-50 times lower than in progressor subjects.

A strong correlation was observed between the cellular response, as determined by the above-described assay, and the immunological status of the analyzed subjects (Table 1). Specifically, in cultures of PBMCs from subjects with low CD4 + T-cell counts (below 500/mm³), fast depletion of CD4+ T-cells was observed in vitro, and these cells increasingly expressed HLA-DR and CD71 antigens over time in culture.

In contrast, in PBMC cultures from non -progressor subjects, infected for more than 7 years but with high CD4+ T-cell counts (above 500/mm³), CD4+ T-cells were still present in vitro after 18 days. During the culture period, the proportion of CD4+ T-cells expressing HLA-DR and CD71 molecules decreased. CD25 antigen was highly expressed up to day 12 and then decreased; CD26 expression was stable. HIV-1 production was low (between 50 and 150 pg p24/10⁶ cells) in the supernatants of PBMCs from all subjects. This probably was due to the low number of infected, HIV-1 producing CD4+ T-cells present in the peripheral blood compartment of seropositive persons.

TABLE 1

Comparison between the clinical characteristics of the HrV-a infected subjects examined and the in vitro depletion of CD4+ T-cells

Number" Group Total years CD4+ T-cell In vitro HIV-1 number at the CD4+ positive time of analysis T cell depletion

All subjects were homosexual men. None had received any antiviral treatment in the 12 months prior to the time of the analysis with the exception of subject No. 8 who had been under AZT treatment for 2 years. None of the subjects had any symptoms of opportunistic infections in the 12 months prior to the analysis. Subject No. 5 was part of a vaccine trial study group and received soluble gpl20 before becoming seropositive.

Non-progressors were defined as being seropositive for more than 7 years and having a CD4+ T-cell count greater than 500 per mm³. Intermediate progressors had a CD4+ T-cell count between 300 and 500 CD4+ T-cell per mm³. Example 3

HΓV-I^ suppression by PBMCs from

HTV-1-infected subjects with different profiles of disease progression

HIV-1 suppression was measured by an acute viral suppression assay. Briefly, after 8-10 days in culture, PBMCs were incubated with HIV-l_MN-infected H9 cells at various effector: target ratios. After 7 days, aliquots were collected and p24 production was assessed in the supernatant fluids. The percentage of viral suppression, compared with H9-infected cells in growth medium, was determined. Subject numbers are as listed in Table 1. The calculation of the effector: target ratio was determined by FACS analysis (CD3+ and CD8+ cell percentages at the time of analysis).

Functional limiting dilution assays were performed to detect any cellular activity capable of inhibiting virus replication, in order to rule out the possibility that differences in CD4+ T-cell survival was due to differences in virus expression rather than to differences in the T-cell activation status.

The results demonstrated higher viral suppressive activity, possibly of CD3+CD8+ cells, from two of three non-progressor subjects with a high CD4+ cell count compared to progressor subjects with a low cell count. This activity seemed to be cell-associated, as supernatant fluids from the cultures failed to inhibit HIV-1 replication when assayed in the same system. Strong viral suppressive activity was also observed in one progressor subject (No. 7) with a low CD4+ cell count. This subject had received soluble gpl2O as a vaccine before serocon version. Therefore, there appeared to be no strong correlation of CD4+ decline with CD8+ antiviral activity.

Example 4

Production of cytokines by in vitro culture of PBMCs from seropositive subjects

Aliquots of cellular supernatants, from cultures treated as described in

Example 2, were assayed with appropriate ELISA kits for the production of cytokines at various days. IFN-gamma was assayed with a Biosource ELISA kit; IL-6, TNF-α, IL-10 and IL-4 were assayed by Quantikine, R & D Systems, in triplicate. Subject numbers are as listed in Table 1.

The pattern of cytokine production from the two group of cultures was measured in order to gain a further understanding for the basis of differences in CD4+ T-cell survival. IL-2 stimulated PBMCs from both groups of subjects secreting a number of cytokines, demonstrating that they were responsive to IL-2 stimulation, but the pattern of secretion was slightly different. As assessed by ELISA, IL-10 was mainly produced by PBMCs from non-progressor subjects with high CD4 + cell counts, although the small number of samples considered did not allow any statistical correlation. Interferon (IFN)-gamma, IL-6 and tumor necrosis factor (TNF)-a were produced by PBMCs from subjects of both groups, while IL-4 was not detectable in either group (not shown).

Example 5

Influence of initial CD4+ counts on the assav with asymptomatic seropositive subjects

HIV-1 infection results in a loss of CD4+ T-cells and eventually leads to AIDS. As discussed above, however, a small percentage of seropositive subjects (about 5%) remains healthy for a long time, despite the presence of replicating virus.

One of the most intriguing and still unexplained differences in the two groups of subjects involves the apparent control of viral replication by the host immune system, although the immune parameters that afford a measure of control over the virus are not clear. Despite a vigorous immune response and a large numbers of HIV- 1 specific cytotoxic T-cells, the majority of the seropositive subjects progress to AIDS, and strong activation of the host immune system appears to correlate with rapid progression of the disease. In contrast, the immune system of non-progressor subjects is generally less activated compared to the progressor group, and shows strong CD8- mediated virus suppressive activity. However, reduced HIV-1 replication in the absence of enhanced CD8+ cellular and viral suppressive activity was recently observed in a few non-progressor subjects, suggesting that still unknown host immune determinants are involved in the control of viral replication in these subjects. As activation of CD4+ T-cells is essential for orchestrating an effective immune response, we analyzed the expression of activation markers on these cells after in vitro IL-2 stimulation in two groups of asymptomatic HIV-1 seropositive subjects, one with low CD4+ cell count (below 500/mm³) and the other with high CD4+ cell count (below 500/mm³). In our system, HIV-1 -primed, in vitro IL-2- stimulated PBMCs showed two different patterns of expression of activation markers, and these patterns correlated with the immunological status of the subjects as judged by the CD4+ T-cell counts. If subjects had a CD4+ cell count below 500/mm³ (progressor), their CD4+ T-cells responded to IL-2 by strong activation in the culture, as demonstrated by the expression of all four cellular activation markers analyzed (CD25, CD26, CD71 and HLA-DR). A marked depletion of CD4+ cells was observed after 12 to 15 days of culture. The low amount of p24 found in the supernatant indicates that mechanism(s) other than infection and viral expression alone must be involved in the depletion. A more complex picture was observed in culture of PBMCs from non- progressor subjects with CD4+ cell count above 500/mm³. Low expression of some markers (HLA-DR and CD71) on CD4+ T-cells indicated low activation of these cells. Nevertheless, high transient expression of CD25 and high longer term expression of CD26 antigens, together with cytokine production, indicated that there was some cellular activation and response to IL-2 stimulation in these cultures. The number of CD4+ T-cells was unchanged during the culture period. Frequently, the CD8+ T-cells from these subjects were generally somewhat more efficient in inhibiting the viral replication in a permanently infected cell line, compared with the CD8+ T-cells from the subjects with low CD4+ cell counts.

These data suggest that asymptomatic HIV-1 seropositive progressor and non-progressor subjects (with low or high CD4+ counts, respectively) can be discriminated according to their different patterns of CD4 + T-cell response to IL-2 stimulation in vitro.

Example 6

IL-2 administration to HrV-1-seropositive patients

As a result of IL-2 administration to HIV-1 seropositive patients, immunological improvements were observed in 6 out of 10 subjects with CD4+ T-cell count above 200/mm³; however, increased amounts of HIV-1 RNA levels in plasma was observed in the other 4 patients. In addition, in 15 subjects with CD4+ T-cell below 200/mm³, the same IL-2 therapy was associated with substantially toxic effects.

Example 7

Application of inventive in vitro assay to a second cohort of asymptomatic, HIV-1 -seropositive subjects using only three cell surface markers

The in vitro assay protocol described in Example 1 was used for these analyses, which were carried out on a cohort of patients different from that used in the previous examples. Three groups were studied: non- (or slow-) progressors (Figs. 1A-1L); intermediate progressors (Figs. 2A-2L); and progressors (Figs. 3A-3I). In each group, the CD4+ T-cell counts in vivo in the previous 12 months were determined before the in vitro tests were applied (i.e. , Figs. 1A-1D; 2A-2D; 3A-3C).

The tests were performed at a first time period (in November, 1995) applying the in vitro test and analyzing the CD4 + , CD3+ and CD8+ T-cell counts in the PBMC samples (Figs. 1E-1H; 2E-2H; 3D-3F). The same tests were again carried out at another time (in February/March, 1996) (Figs. 1I-1L; 2I-2L; 3G-3I).

The data indicate that in healthy, uninfected donors, absolute CD4+ T- cell number increased in culture, while the absolute T-cell number for CD84- did not increase. PBMCs of subjects infected for a long period of time ( > 5-7 years) and with a high number of CD4+ T-cells in vivo (long term non-progressors) had a response to IL-2 similar to that of the control group.

Two other responses were observed in cultures of PBMCs from other subjects at different stages of the disease, but unrelated to the CD4+ T-cell count of the subject in vivo: (1) absolute CD4+ and CD8+ T-cell numbers did not increase in cultures; or

(2) absolute CD4+ T-cell number did not increase in cultures, but absolute CD8+ T-cell number did. This group is considered to contain the progressors. Without being bound by theory, it is hypothesized that stage (1) represents a functional defect in the response of CD4+ T-cells, while stage (2) represents a response of CD8+ T-cells to the viral infection of the MHC-1 presenting cells (macrophages) in the in vitro conditions. Based on the present in vitro assay, it will be possible to detect the defect(s) of CD4+ T-cells and the response of CD8+ T-cells, before the AIDS symptoms appear.

On the basis of these results, it is possible to obtain the necessary prognostic information simply by determining only three cell surface markers, namely, CD4+CD3+ and CD8+CD3+ , in, for example, an automated flow cytometry instrument using immunoreagents.

Example 8

Comparison between the number of CD4+ T-cells in cultures of PBMCs from asymptomatic HIV-1 seropositive subjects at different stages of HIΥ infection/disease

HIV-1 seropositive subjects at different stages of disease were examined. All of the subjects were asymptomatic for disease. The subjects were divided into four categories according to their CD4+ T-cell count in vivo and the time after seroconversion. The four categories were: 1) recent seroconverters (RS), defined as seropositive for less than 20 months; 2) fast progressors (FP) , defined as infected for longer than 20 months but less than 5 years with a CD4+ count of < 300/mm³ and expected to progress to AIDS before a median time of 10 years; 3) typical progressors (TP), defined as infected for more than 7 years with a CD4+ count below 350/mm³ and expected to progress to AIDS in a median time of 10 years; and 4) long-term survivors (LTS), defined as seropositive for more than 8 years with a CD4+ count above 500/mm³, for whom the time of progression to AIDS is not predictable.

The majority of the subjects were not undergoing antiviral therapy at the time of the analysis. Table 2 presents the clinical characteristics of the subjects in this study, including the plasma viral load, as well as the amount of virus that

PBMC cultures were producing. CD4-I- T-cell number was determined as described for Figs. 1-3. Figs. 4A-D represent a horizontal analysis of the proliferative response of CD4+ T-cells in the HIV-1 seropositive individuals whose clinical characteristics are described in Table 2.

The HIV-1 seropositive subjects of Table 2 were from the Clinical Malattie Infettive, Policlinico S. Matteo, Pavia, Italy. All of the subjects were free of AIDS-defining clinical symptoms and were under no antiviral treatments at the time of the analysis. Lymphocyte count and T-cell subsets were determined on whole blood using laser-based flow cytometry and OKT4A (anti-CD4), OKT8 (anti-CD8) and OKT3 (anti-CD3) monoclonal antibodies (Orthodiagnostic Systems). Informed consent was obtained from all the subjects involved in these studies.

Viral load was measured as conventionally known in the art. Definitions of abbreviations used in Table 1 are as follows: HS: homosexual; IVDU: intravenous drug user; MHE: multiple heterosexual exposures; RS: recently seroconverted; FP: fast progressor; TP: typical progressor; LTS: long-term survivors; ND: not done.

TΛBLE 2

Clinical characteristics of the HIV-1 seropositive subjects examined

*+: below 5000 copies/ml; + + : between 5000 and 50000 copies/ml; + + +: above 50000 copies/ml. HIV- 1 RNA copy number in the supernatants was assessed at day 15 of culture.

HZV detected 5 years ago.

AZT treatment started 5 months before the analysis.

4 years ago AZT was taken for 12 months.

4 years ago AZT was taken for 6 months; 2.5 years ago AZT+IFNα were taken for 6 months; 1.5 years ago AZT+IFNβ were taken for 6 months. ^•: HCV positive. ^f: 5.5 years ago AZT was taken for 2 years; 1 year ago AZT+DDI were taken for 1 month; 8 months ago AZT+DDI were taken for 2 months. *: AZT+DDI treatment were started 6 months before the analysis.

A CD4+ T-cell proliferative response was observed in healthy, seronegative individuals. Such a response is considered to indicate a normal functioning of the immune system and is used as a control. From Figs. 4A-4D, it can be observed that a proliferative response, i.e., the response that the test of the invention measures as a predictor of the AIDS disease progression, varies according to the different categories of HIV-1 seropositive subjects examined. Subjects with an immune system less compromised (LTS) had a better proliferative response than subjects with an immune system already compromised (RP and TP). This difference in response was seen in subjects analyzed early after seroconversion (RS). The response was not wholly correlated with viral load, although subjects with high viral load tended to have a lack of proliferative response. Thus, it may be surmised that subjects with a lack in a CD4+ T-cell response will be likely to progress faster toward AIDS disease.

To better demonstrate this issue, HIV-1 seropositive subjects are followed from the beginning of the infection, until they develop symptoms of AIDS disease; as appreciated by the skilled practitioner, the development of AIDS symptoms can take a number of years. Notwithstanding, the present invention of measuring the immunological functionality of CD4+ T-cells can serve as a predictor of the development of the AIDS disease; the horizontal analysis presented in Figs. 4A-4D strongly suggests that the test is predictive. In addition, the predictive value of the in vitro tests of the present invention can also be correlated with conventional viral load tests, such as assessed by PCR and branch DNA to measure the of number of viral nucleic acid or virions in the plasma.

The references, texts, abstracts and the like presented herein are hereby incorporated by reference in their entirety to more fully explain the state of the art to which the invention pertains.

Claims

WHAT IS CLAIMED IS:

1. A method for prognosing the progression to acquired immunodeficiency syndrome (AIDS) of an asymptomatic individual who is seropositive for HIV-1 , or for determining the efficacy of treatment of said individual, comprising the steps of:

(a) isolating peripheral blood mononuclear cells from peripheral blood of said subject;

(b) incubating said peripheral blood mononuclear cells with an amount of IL-2 effective to activate said peripheral blood mononuclear cells; and

(c) determining the activation status of said peripheral blood mononuclear cells, wherein said activation status is predictive of progression to AIDS or effectiveness of treatment.

2. The method according to claim 1 , wherein said activation status is determined by assaying for one or more cell surface antigens on said peripheral blood mononuclear cells.

3. The method according to claim 2, wherein said cell surface antigens are selected from the group consisting of CD3, CD4, CD8, CD25, CD26, CD71 and HLA-DR.

4. The method according to claim 2, wherein said cell surface antigens are CD3, CD4 and CD8.

5. The method according to claim 3, wherein said cell surface antigen is CD4.

6. The method according to claim 2, wherein said assaying comprises flow cytometry or fluorescence activated flow cytometry.

7. The method according to claim 1, further comprising the step of (d) assessing the CD4+ T-cell number of said peripheral blood mononuclear cell population in culture.

8. The method according to claim 7, wherein said IL-2 activation status is correlated with said CD4+ T-cell number.

9. The method according to claim 8, wherein a low CD4+ T-cell number, a high level of IL-2 activation and a depletion of CD4+ T-cells in the peripheral blood mononuclear cell population over time in culture indicates progression of disease caused by HIV-1 infection.

10. The method according to claim 9, wherein expression of HLA- DR and CD71 cell surface antigens further conelates with disease progression.

11. The method according to claim 8, wherein a high CD4+ T-cell number, a low level of IL-2 activation and virtual stability of CD4+ T-cell numbers in the peripheral blood mononuclear cell population over time in culture indicates non- progression of HIV-1 infection.

12. The method according to claim 11 , wherein a decrease in HLA- DR and CD71 cell surface antigen expression, a decrease followed by an increase in CD25 cell surface antigen expression and a stable expression of CD26 further correlate with disease non-progression.

13. The method according to claim 9, further comprising carrying out a therapeutic protocol comprising the administration of immunosuppressive drugs, downregulation of IL-2 receptor expression, or a combination thereof, to alter the pattern of CD4+ T-cell responsiveness.

14. A kit for carrying out the method according to claim 1 , comprising, in separate compartments, IL-2 and at least one reagent for carrying out step (c).

15. A method for determining the progression to acquired immunodeficiency syndrome (AIDS) of an asymptomatic individual who is seropositive for HIV-1 or for determining the efficacy of treatment of said individual, comprising:

(b) culturing said peripheral blood mononuclear cells with IL-2 in an amount effective to stimulate said peripheral blood mononuclear cells; and

(c) determining the activation status of said peripheral blood mononuclear cells by assaying for the presence of cell surface antigens on said peripheral blood mononuclear cells, wherein said activation status is predictive of progression to AIDS or effectiveness of treatment.

16. The method according to claim 15, wherein said cell surface antigens are selected from the group consisting of CD3, CD4, CD8, CD25, CD26, CD71 and HLA-DR.

17. The method according to claim 15, wherein said cell surface antigens are CD3, CD4 and CD8.

18. The method according to claim 16, wherein said cell surface antigen is CD4.

19. The method according to claim 15, further comprising: (d) determining the CD4+ T-cell number of said cultured peripheral blood mononuclear cell population.

20. The method according to claim 19, wherein said IL-2 activation status is correlated with said CD4+ T-cell number.

21. The method according to claim 20, wherein a low CD4+ T-cell number, a high level of IL-2 activation and a depletion of CD4+ T-cells in the peripheral blood mononuclear cell population over time in culture indicates progression of disease caused by HIV-1 infection.

22. The method according to claim 21 , wherein expression of HLA- DR and CD71 cell surface antigens further correlates with disease progression.

23. The method according to claim 20, wherein a high CD4+ T-cell number, a low level of IL-2 activation and virtual stability of CD4 + T-cell numbers in the peripheral blood mononuclear cell population over time in culture indicates non- progression of HIV-1 infection.

24. The method according to claim 23, wherein a decrease in HLA-

DR and CD71 cell surface antigen expression, a decrease followed by an increase in CD25 cell surface antigen expression and a stable expression of CD26 over time in culture further correlate with disease non-progression.

25. The method according to claim 21, further comprising carrying out a therapeutic protocol comprising the administration of immunosuppressive drugs, downregulation of IL-2 receptor expression, or a combination thereof, to alter the pattern of CD4+ T-cell responsiveness.

26. A kit for carrying out the method according to claim 15, comprising, in separate compartments, IL-2 and at least one reagent for carrying out step (c).