1 Department of Immunology and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy
2 Institut Pasteur du Cambodge, Phnom Penh, Cambodia
3 Institut Pasteur, Ho Chi Minh City, Vietnam
4 Department of Immunology, University of Milan, Milan, Italy
5 Unité de Biologie des Rétrovirus, Institut Pasteur, Paris, France
Correspondence
L. Lopalco
lopalco.lucia{at}hsr.it
S. E. Burastero
burastero.samuele{at}hsr.it
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ABSTRACT |
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INTRODUCTION |
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Among HIV-protective factors, a genetic polymorphism of the CCR5 coreceptor gene has been reported (Liu et al., 1996); however, this mutation cannot explain all cases of natural resistance to HIV infection (Rugeles et al., 2002
), thus suggesting that other factors may be involved. In fact, several antigen-specific responses to viral proteins described in ESN individuals could contribute to resistance to HIV infection. These include humoral immune responses, including IgA to Env (Beyrer et al., 1999
; Clerici et al., 2002
; Devito et al., 2000a
, b
; Kaul et al., 1999
; Lo Caputo et al., 2003
; Mazzoli et al., 1997
; Shacklett et al., 2002
) and anti-cell antibodies, namely anti-human leukocyte antigen (HLA) (Beretta et al., 1996
; Lopalco et al., 2000b
), anti-CD4 (Burastero et al., 1996
; Lopalco et al., 1999
) and anti-CCR5 (Ditzel et al., 1998
; Lopalco et al., 2000a
). This aspect came strikingly to light in the experimental model of vaccination in non-human primates with cellular vaccines, where a relevant carry-over of membrane molecules was included in the immunogen, which was probably responsible for the induced protection (Lehner et al., 1992
). Moreover, cellular immune responses have also been described in ESN individuals (CD8 and CD4 T cells specific to HIV epitopes) (Biasin et al., 2000
; Furci et al., 1997a
, b
; Kaul et al., 2001
; Makedonas et al., 2002
; Mazzoli et al., 1997
; Rowland-Jones & McMichael, 1995
; Shacklett et al., 2002
; Shearer & Clerici, 1996
). The study of this intriguing puzzle may pave the way to the design of anti-HIV strategies that are aimed at obtaining sterile immunity or improved viral control.
Here, we describe for the first time the characterization of several parameters of unconventional humoral immunity that were previously studied by our laboratories. These markers are specific to both cellular and HIV antigens, and were studied in three independent cohorts of ESN individuals with different exposure risks and geographical origins.
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METHODS |
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ESN individuals were defined as being seronegative by conventional ELISA (Wellcozyme HIV-1 recombinant), Western blot assays for HIV antibodies (HIV-1 blot 2.2; Diagnostic Biotechnology) and detection of HIV DNA (Amplicor; Roche) and RNA (Branch DNA; Bayer). Cut-off values for these standard assays are 50 copies of HIV DNA in 1 000 000 blood mononuclear cells and 50 copies of HIV RNA (ml plasma)1, respectively.
Sexually exposed.
Italian heterosexual couples that were discordant for HIV serostatus were enrolled in the study. In each couple, one partner was infected with HIV whereas the other was not, despite a prolonged history of penetrative sexual intercourse without condom use (and no other risk factors). Inclusion criteria required a history of penetrative sexual intercourse without a condom, at least twice per week and for at least 2 years, for the majority of the subjects. Unexposed Italian controls (NESN) were recruited among healthy blood donors. Notably, all individuals included in the Italian cohort had been living in the same geographical area (Milan metropolitan area) for at least 10 years. Controls and ESN subjects were age- and sex-matched (Table 1).
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Systemically exposed.
Vietnamese ESN subjects with a history of more than 10 years of high-risk intravenous drug use (IVDU) were included, presenting nearly 100 % prevalence of other blood-transmitted viral infections, including hepatitis B virus, hepatitis C virus and human T-lymphotropic virus 1 (Truong et al., 2003). Vietnamese healthy control subjects were recruited among Red Cross voluntary blood donors. Although the most suitable controls for Vietnamese ESN subjects would be Vietnamese individuals exposed systemically to non-HIV viruses, unfortunately this option could not be carried out. All of the study participants gave their informed consent and completed a questionnaire regarding the frequency and types of risk behaviours. Each was given counselling and information about HIV disease and safe drug or sex practices.
The study was approved by the Ethics Committee of the San Raffaele Scientific Institute, Milan, Italy; the National Ethics Committee of Cambodia; and the Ethics Committee of Binh Trieu Hospital, Ho Chi Minh City, Vietnam.
All blood samples were collected and frozen in the country of origin of each population. In order to reduce inter-test variability, samples were thawed and analysed simultaneously for each given parameter. All of the assays were done by a person blinded to the clinical status of the subjects.
All study participants were negative for the clinical and routine laboratory parameters of systemic and organ-specific autoimmunity, including antibodies to DNA, extractable nuclear antigens, mitochondria, the Fc fragment of IgG (rheumatoid factors) and thyroglobulin.
In order to exclude the possibility that the samples we analysed were taken during seroconversion, we only used samples from individuals who remained seronegative for at least 1 year after the date of bleeding.
Anti-CCR5 antibodies assay.
The assay was performed as described previously (Lopalco et al., 2000a). Briefly, a two-step gradient was created as follows: the lower layer consisted of fetal calf serum (FCS) containing 10 % sucrose; the upper layer consisted of 80 % silicone and 20 % mineral oil (both from Sigma-Aldrich). Purified CD4 cells were incubated with appropriate dilutions of serum/plasma and/or IgG, purified and incubated with 125I-labelled MIP1
. Unbound radioactivity was separated by centrifugation on the two-step gradient in 0·3 ml tubes (Nunc). Bound radioactivity in the cell pellets was measured by a gamma counter. A specificity control, consisting of a 100-fold excess of unlabelled MIP1
, was included in all experiments. Binding of the 125I-labelled MIP1
to activated CD4 cells ranged between 1000 and 6000 c.p.m. An anti-CCR5 IgG assay was also performed by using 8x104 CXCR4- or CCR5-transfected U87 cell lines resuspended in 200 µl RPMI (Gibco Life Technologies), which were seeded and incubated for 2 days at 37 °C to obtain a cell monolayer. Serum samples taken from both ESN and NESN subjects were diluted 1/10 and incubated on the cell monolayer and 125I-labelled sheep anti-human IgG antibodies (Amersham Biosciences) were added. Unbound radioactivity was separated by centrifugation on a two-step gradient as described above (Lopalco et al., 2000a
).
Anti-CD4 antibodies assay.
The titre of IgG antibodies to human CD4 was measured by using a validated ELISA (Lopalco et al., 1999). Briefly, recombinant CD4 [obtained from the National Institutes of Health (NIH) AIDS Reagent Program, Division of AIDS, NIAID, NIH, USA] was coated at 5 µg ml1 in PBS for 18 h at 4 °C on 96-well plates (Maxisorp; Corning). Sera to be tested were diluted 1/300 in 0·05 % PBS/Tween 20 (Sigma). Each assay was run in quadruplicate. After 2 h incubation followed by washing, affinity-purified, alkaline phosphatase-conjugated goat antibody to human IgG (ICN Biomedicals) was added and the reaction was subsequently developed with para-nitrophenyl phosphate (Sigma). The amount of anti-CD4 IgG binding activity was expressed as OD405. When anti-CD4gp120 complex antibodies were to be measured, CD4 was pre-incubated with HIV BaL strain gp120 (NIH AIDS Reagent Program, Manassas, VA, USA) in equimolar amounts [2·6 µg gp120 (µg CD4)1] for 30 min on ice. All subsequent steps remained unchanged.
Measurement of inhibition of CD4 binding by homologous and heterologous antigens by cytofluorimetric analysis.
In order to study the specificity for CD4 of IgG antibodies from different groups of ESN and NESN subjects, pools were prepared from people included in this study, using 25 µl of each serum. IgG fractions were isolated from pooled sera by affinity chromatography with Protein GSepharose (Amersham Biosciences) according to the manufacturer's instructions and brought to a final concentration of 1 mg ml1 in PBS. ESN sera were chosen from individuals positive for anti-CD4 antibodies (nine subjects) (Lopalco et al., 1999), whereas NESN sera were chosen from individuals with IgG to CD4 values above the 75th percentile (12 subjects). The IgG to CD4 binding activity of each fraction was evaluated by cytofluorimetric analysis with a murine cell line transfected permanently with human CD4 (3T3.T4) (NIH AIDS Reagent Program). Purified IgG was pre-adsorbed onto wild-type NIH 3T3 cells (ATCC, Manassas, VA, USA) (5x106 cells, in rotation at 4 °C for 1 h) to lower the background binding on 3T3.T4 cells. Each fraction was then incubated with soluble CD4 (5 µg ml1), Fc fragment of human IgG (10 µg ml1), human heat-shock protein 60 (HSP60) (10 µg ml1) or BSA (5 µg ml1) as a control. Subsequently, each soluble antigen-absorbed aliquot was added to the 3T3.T4 cell line (5x105 cells in a 100 µl reaction volume, 30 min on ice). Binding of IgG to 3T3.T4 cells was detected with a polyclonal, fluorescein isothiocyanate-conjugated goat antibody to human IgG (ICN Biomedicals). Analysis was performed with a FACScalibur cytofluorimeter (BD Biosciences). The control samples were 3T3.T4 cells reacted with the fluoresceinated secondary antibody only. Readings with controls were set to span fluorescence channels 110. To make the system more sensitive, for each non-adsorbed IgG fraction (positive control), a dilution was preliminarily identified if it yielded a binding activity spanning fluorescence channels 2040. Inhibition of binding was expressed as percentage mean fluorescence intensity (MFI) in the given sample compared to MFI in the positive control.
Enzyme immunoassay detection of gp41-specific antibodies.
The presence of gp41-specific IgA was evaluated by ELISA as described previously (Clerici et al., 2002). In brief, ELISA was performed by using gp41 recombinant protein for coating. IgA binding was revealed with horseradish peroxidase-conjugated rabbit polyclonal antibody to human IgA (Dako).
Statistical analysis.
Preliminary tests for normality (ShapiroWilk, KolmogorovSmirnov) consistently showed a skewed distribution of values for all parameters we measured. Thus, analysis of results was performed with non-parametric tests. Cumulative results were expressed as medians and quartiles. Subjects within the ESN groups were considered to be positive for each given variable when values fell above the 95th percentile of the distribution of values in the respective NESN group within each population. The 2 test was used to compare ESN and NESN individuals for each variable and within each population, and values of P below 0·05 were considered to be significant. The odds ratio for case-control analysis was used to calculate the risk of belonging to the ESN group when positivity for the given parameter was present; 95 % confidence limits are shown. The MantelHaenszel test was used to estimate the common odds ratio for each parameter, when considering cumulatively the values obtained from the three populations that were studied. The BreslowDay test was used to assess the homogeneity of the odds ratios in the different populations and gave non-significant results in all cases. Finally, NESN individuals of different ethnic origins were analysed in parallel (inter-cohort comparison) by comparing a single parameter from one cohort against the corresponding parameter of each other cohort. For this purpose, the KruskalWallis test was used and post hoc comparisons were adjusted with the Bonferroni procedure. Calculations were performed by using the SAS System software.
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RESULTS |
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Predictive power of HIV exposure by humoral immune-response analysis in different groups of ESN subjects
The presence of IgG to CCR5, IgG to CD4 and IgA to gp41 was previously correlated with protection in Caucasian ESN individuals (Burastero et al., 1996; Clerici et al., 2002
; Lopalco et al., 2000a
). Here, we simultaneously evaluated these three parameters in parallel in Asian and Caucasian populations. The relevance of the initial steps in viruscell interaction has recently been highlighted by the possibility of interfering with several viral processes through protective immune responses against strictly conformation-dependent epitopes that are exposed during viral docking and entry. Thus, we also evaluated antibodies to gp120-dependent CD4 epitopes as further potential immune correlates of HIV exposure in all studied populations. Cell-associated self-antigens that are involved in HIV entry (CD4, CCR5) and strictly virus-specific epitopes (gp41, gp120CD4 complex) were evaluated separately. Firstly, in order to compare the distribution of levels of each single parameter in the NESN control populations, the KruskalWallis test was used to compare single parameters against the corresponding parameter of each other group and post hoc comparisons were adjusted with the Bonferroni procedure. Non-significant differences were found between the two Asian cohorts for all considered parameters. In contrast, highly significant differences were found when comparing the NESN subjects from Italy with their counterparts from both Vietnam and Cambodia, with the exception of anti-CCR5 antibodies, which were relatively homogeneous in the three populations. In particular, extremely significant differences were found when considering IgG to CD4 and to gp120CD4 complex, as well as IgA to gp41 (Table 2
). These results prompted us to evaluate the levels of natural antibodies in the different populations, in order to explore the possibility that polyreactive antibodies could affect the single tests that we used differently (see below). Secondly, in order to verify whether these humoral parameters can be considered as specific predictive markers of HIV exposure, we calculated the odds ratio, which measures the magnitude of divergence between HIV-exposed versus non-exposed subjects, for each variable.
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In Italian ESN subjects, the odds ratio of IgA to gp41 antibodies was extremely significant (P<0·0001) and that of anti-CD4gp120 complex antibodies was significant (P=0·0143). As for anti-cell antibodies, both the odds ratios of IgG to CCR5 and of IgG to CD4 were significant (P=0·0021 and 0·0078, respectively) (Table 3).
In order to assess the homogeneity of the odds ratios in the different populations, the BreslowDay test was preliminarily performed, which indicated that the common odds ratio could be extrapolated from the cumulative analysis of each parameter in all studied populations. Strikingly, the significance of the predictive power of each parameter was higher than those observed in each single population (Table 3). In particular, anti-virus antibodies were extremely significant predictors of HIV exposure (P<0·0001).
In order to express the data in a graphical way, we used box-and-whisker plots. In this representation, the box indicates the lower and upper quartiles and the central line represents the median. The points at the ends of the whiskers' indicate extreme values. Single dots in ESN graphs indicate values above the extreme value of the respective NESN group (Fig. 1). Moreover, we arbitrarily extrapolated the proportion of ESN individuals positive for the presence of antibodies to each marker as the ratio between the number of individuals with values above the extreme NESN value in the corresponding population and the number of ESN subjects that were analysed.
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Different levels of IgG to CD4 antibodies were displayed in NESN populations of Asian origin compared to Italians, as demonstrated previously by the KruskalWallis test (Table 2, Fig. 1b
).
This high background probably affected the comparison with the corresponding ESN individuals, although the proportion of ESN subjects with values higher than the NESN extreme values was similar (range, 2·47·8 %) (Fig. 1b). IgG to CD4gp120 complex, similarly to anti-CD4 antibodies, was higher in the NESN populations of Asian origin (Table 2
, Fig. 1c
). However, for this parameter, higher proportions of ESN subjects with values above the respective NESN extreme values were found (range, 13·117·6 %). Notably, the mean value of the proportion of ESN subjects with antibodies to CD4gp120 complex was about three times higher (15·9 %) than that of ESN subjects with antibodies to CD4 (5·5 %).
For IgA to gp41 in ESN subjects, the proportion of positive subjects ranged from 17 to 57·3 % (Fig. 1d). Notably, Vietnamese IVDU subjects, who were exposed systemically to HIV, showed lower level of IgA antibodies than the two populations of sexually exposed individuals. This result is evident in the odds-ratio test, which was barely significant in Vietnamese ESN subjects (P=0·0453), compared to the two sexually exposed groups (P<0·0001 in both Italian and Cambodian ESN subjects) (Table 3
).
Studies on the specificity of the anti-CD4 response by purified IgG
In order to evaluate the possibility that different levels of cross-reactive antibodies in different populations could correlate with different levels of binding to the antigens that we included in our tests, dedicated assays were designed. Firstly, the IgG fraction of antibody was isolated from sera, in order to exclude interference by the highly cross-reactive IgM isotype. Secondly, competition assays were performed with antigens that were representative of self/non-self cross-reactive molecules (Burastero et al., 1988; Casali et al., 1987
). Results of three separate experiments of inhibition with soluble antigens are shown in Fig. 2
. Although the analysis is merely qualitative, it appears that IgG of individuals from both Cambodia and Vietnam were efficiently displaced from binding to CD4, not only by the homologous soluble molecule, but also by the heterologous antigens that we tested. This applied to both ESN and NESN subjects of these groups. In contrast, IgG of ESN and NESN individuals from Italy was displaced from binding by pre-incubation with soluble CD4, but not with soluble Fc of HSP60. Similar results were also observed with thyroglobulin, another autoantigen that is recognized by natural polyreactive antibodies (data not shown) (Nakamura et al., 1988
). A representative result is shown in Fig. 3
(control line). The CD4 binding of IgG purified from ESN individuals from Italy (Fig. 3
, left panel) was reduced from an MFI of 32·6 to an MFI of 11·8 (63·8 %) by pre-incubation with soluble CD4, but remained unchanged following pre-incubation with the heterologous antigen HSP60 (MFI, 31·0) (5 %). In contrast, the CD4 binding of IgG from individuals from Cambodia (Fig. 3
, right panel; MFI, 84·1) was similarly reduced by incubation with both soluble CD4 (MFI, 32·8) (61 %) and HSP60 (MFI, 33·8) (59·8 %).
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DISCUSSION |
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Different genetic backgrounds and different antigenic experience due to distinct environmental conditions can significantly modulate immune responses to microbes (Bentwich et al., 1995). Thus, we decided to analyse humoral markers of HIV exposure that were previously identified in Caucasian ESN subjects in a large cohort of Italians and, in parallel, in two cohorts of Asian origin (Cambodian and Vietnamese subjects). Moreover, individuals with different routes of exposure to HIV were included, as Italians and Cambodians were sexually exposed, whereas Vietnamese were exposed by IVDU.
We made inter-group comparisons between NESN individuals in order to evaluate whether populations living in geographical areas with distinct lifestyles and social-health policies displayed different levels of the humoral immune parameters that we analysed, independently of specific exposure to HIV. This might be correlated, among other factors, with different levels of natural polyreactive antibodies. Indeed, all considered parameters were higher in both Asian populations than in the Italian group (P<0·0001), with the exception of autoantibodies to CCR5, which were homogeneously distributed (Table 1) and tended to be higher in Italians than in Asians, although they did not reach significance. It is tempting to speculate that the well-known reverse correlation between autoimmunity (and allergy) and overall frequency of infections, which has been observed in both humans and animal models (Yazdanbakhsh et al., 2002
), might explain this result. Notably, IgG to CCR5 was detected with an assay that was specific for a single epitope, whereas all other assays used whole molecules and were therefore more susceptible to interference by natural polyreactive antibodies. Indeed, we confirmed this hypothesis by analysing a selected panel of sera. We found that an autoantigen, such as HSP60 (an example of an antigen recognized by natural polyreactive antibodies) (Pashov et al., 2002
; Potter et al., 2000
), could displace IgG from Cambodian and Vietnamese individuals from binding to membrane-bound CD4 as efficiently as soluble CD4 itself. Also, the Fc fragment of human IgG, which is recognized by natural serological rheumatoid factor activity (Burastero et al., 1988
), could efficiently compete with CD4 binding; similar results were observed with thyroglobulin, as described previously (Nakamura et al., 1988
). In contrast, binding activity to membrane-bound CD4 by IgG tp CD4 purified from Italians was specifically outcompeted by soluble CD4, but not by the heterologous antigens (Fig. 2
).
Higher and frequent exposure to numerous infectious agents in poor countries, such as Cambodia and Vietnam, is probably inducing a general immune activation, as has been described in Africa (Rizzardini et al., 1996, 1998
). An increase of polyreactive antibodies consequent to immune activation may thus underlie the differences observed between Asian and Italian populations. Further studies are needed to test the level of immune activation in the Asian groups (e.g. by measuring parameters of T-lymphocyte and complement activation, cytokine production, etc.).
In order to verify whether the humoral parameters that we studied can be considered as specific predictive markers of HIV exposure, we calculated the odds ratio, which measures the magnitude of divergence between HIV-exposed versus non-exposed subjects for each variable.
On one side, a cumulative analysis of each parameter was performed in all studied populations (BreslowDay test followed by CochranMantelHaenszel statistics), which allowed a strengthening of the power of predictivity of the markers (Table 3). On the other, qualitative analysis of each marker by box-and-whisker plots further allowed us to visualize the pattern of distribution of values within each ESN versus NESN group (Fig. 1
). Taken together, both approaches demonstrate unequivocally that ESN subjects from cohorts recruited in distinct geographical areas, implying striking environmental and genetic differences, do display immunological signs of HIV exposure, compared to their NESN counterparts.
Notably, anti-virus antibodies (IgA to gp41 and IgG to gp120-dependent CD4 epitopes) were more powerful predictors of HIV exposure than anti-cell antibodies (IgG to CD4 and CCR5) (Table 3). This observation suggests that this humoral immune response is strictly dependent on actual exposure to HIV. This may be due to: (i) an extremely low level of ongoing virus replication, as recently reported in a few ESN individuals (Zhu et al., 2003
); (ii) a previous event where single rounds of HIV replication took place, followed by complete viral clearance; or (iii) previous exposure to viral debris, in amounts and with modalities compatible with the elicitation of HIV-specific antibodies. On the other hand, non-HIV-specific events could also contribute to the induction and modulation of anti-cell humoral immune responses towards membrane molecules that are involved in HIV docking and entry. Indeed, our results also show that anti-cell antibodies must be considered as predictive markers of HIV exposure (Ditzel et al., 1998
; Lehner et al., 1992
, 1999
; Lopalco et al., 2000a
).
We show here for the first time that the fine specificity of anti-CD4 antibodies in ESN individuals includes epitopes whose expression on CD4 molecules is triggered by binding to gp120. This is consistent with the exclusive mechanism for generation of the anti-CD4 response that was described previously in ESN subjects (Furci et al., 1997a), which was strikingly different from that observed in HIV-positive individuals with anti-CD4 antibodies (Salemi et al., 1995
).
Moreover, although one study failed to detect the presence of HIV-specific mucosal IgA (Dorrell et al., 2000), several other reports confirmed this observation in populations with different genetic backgrounds, including Kenyans, Thai commercial sex workers and an Italian SC cohort (Beyrer et al., 1999
; Clerici et al., 2002
; Devito et al., 2000a
, b
; Kaul et al., 1999
; Lo Caputo et al., 2003
; Mazzoli et al., 1997
; Shacklett et al., 2002
). Taken together, these data confirm the heterogeneity of mechanisms leading to ESN status and stress the relevance of the route of exposure.
In conclusion, both anti-cell and anti-HIV antibodies are constant hallmarks in individuals who are exposed continuously to HIV infection, but remain persistently seronegative when evaluated with standard assays. We believe that we are providing important confirmation of the presence of these potentially protective antibodies in ESN individuals. Moreover, the present work is the first evidence that race and route of virus exposure may differentially affect the antibody specificity that is associated with ESN status. Finally, we show evidence of the nature of polyreactive antibodies and their role in generating and measuring the antibodies evaluated here. Overall, this work increases our understanding of innate and acquired immune responses that might protect against HIV-1.
The study of unconventional immunity in ESN subjects needs to be further fostered, in order to identify potential strategies to sterile immunity or HIV-infection control. Our work also indicates that special caution is to be utilized to overcome the problems that are encountered when non-standard assays for detecting viral or cellular targets of cellular and humoral immune responses are applied to people with different ethnic origins and/or antigenic experience.
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ACKNOWLEDGEMENTS |
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Received 3 September 2004;
accepted 1 November 2004.