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Detection of Antibodies to Human Immunodeficiency Virus (HIV) That Recognize Conformational Epitopes of Glycoproteins 160 and 41 Often Allows for Early Diagnosis of HIV Infection

  1. Jianmin Chen1,
  2. Liqiang Wang2,
  3. Jenny J.-Y. Chen2,
  4. Gautam K. Sahu1,
  5. Stephen Tyring1,4,
  6. Keith Ramsey5,
  7. Alexander J. Indrikovs2,
  8. John R. Petersen2,
  9. David Paar3 and
  10. Miles W. Cloyd1,2
  1. Departments of
  2. 1Microbiology and Immunology,
  3. 2Pathology,
  4. 3Internal Medicine, and
  5. 4Dermatology, University of Texas Medical Branch, Galveston;
  6. 5Department of Internal Medicine, University of South Alabama School of Medicine, Mobile
  1. Reprints or correspondence: Dr. Miles W. Cloyd, University of Texas Medical Branch, Dept. of Microbiology and Immunology, 301 University Blvd., Galveston, TX 77555-1070 (mcloyd{at}utmb.edu)
 
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Abstract

On the basis of human immunodeficiency virus (HIV) needlestick studies, the time to seroconversion for anti-HIV antibodies is 1–9 months (mean, ∼2–3 months). However, an earlier marker of an immune response to HIV often occurs—serum anti-HIV antibodies reactive with live HIV-infected cells, termed “early HIV antibodies.” The specificities of these antibodies are characterized by the recognition of type-specific conformational epitopes of the HIV envelope glycoprotein (gp) 160 and gp41. By use of a third-generation native HIVIIIB gp160 enzyme immunoassay (EIA), detection of HIV antibodies occurred, on average, 33 days earlier than did detection by commercial EIA and 25 days earlier than did detection by the reference antigen and reverse-transcription polymerase chain reaction (RT-PCR) assays in 3 of 5 HIV seroconversion panels. A fourth panel possessed early HIV antibodies that reacted with HIV213 but not with HIVIIIB, allowing for detection of HIV antibodies ∼3 weeks earlier than by RT-PCR or other current tests

Persons infected with human immunodeficiency virus (HIV) usually mount a humoral immune response to the virus, resulting in production of specific antibodies. Because the presence of antibody to HIV-1 is a marker for virus infection, the US Food and Drug Administration recommends anti-HIV antibody testing as a method of screening donated blood for the virus [1, 2]. Consequently, EIAs have been the principle diagnostic tool used by clinicians to detect HIV-1 infection. The frequency of false-positive results of the early EIAs resulted in the requirement that confirmatory testing, either by Western blot (WB) or fixed-cell immunofluorescence, be done for diagnosis. Although the newer EIAs are more specific [3], they fail to detect antibody in persons who are very early in the course of infection, during a “window” of time between infection with HIV-1 and the production of serum antibodies detectable by current commercial EIA/WB (seroconversion) or who are infected with certain HIV-1 clades [4 –9]. The lack of early detectability remains a persistent public health issue among recipients of blood or organ donations and raises concerns among health care workers who have been exposed to blood and body fluids from persons who test negative for HIV-1 [9 –17]

In response to the concerns related to this window, the blood banking industry is testing donated blood for the presence of both HIV antibody and p24 antigen and recently began additional testing by using polymerase chain reaction (PCR) amplification of plasma HIV RNA [18, 19]. These methods, although improved in technique, have time and cost limitations that make them impractical for large-scale screening. More important, they may not be able to identify infected persons in the weeks immediately after infection when few, if any, HIV virions (RNA copies) are present in plasma, because the small amount of virus produced within a lymph node draining a mucosal site of HIV entry is very likely “absorbed” by surrounding CD4 T cells and follicular dendritic cells (to which HIV binds with high affinity). In general, earlier studies to determine whether PCR for HIV DNA in blood cells would shorten the diagnostic window found little improvement by use of single amplification regimens [20 –26]. In addition, some studies showed that single PCR amplification regimens were unable to detect the few HIV DNA copies present in ∼106 peripheral blood lymphocytes (PBL) during the window period and that nested PCR assays were needed [27 –30]

Immunologic studies indicate that this early window of silent infection is not really silent, and active immune responses are often elicited during that time [31 –33]. We previously found that EIA- and WB-negative early-infected persons (on the basis of HIV detection in PBL) can make antibodies to HIV that recognize only native, not denatured, HIV proteins expressed on surfaces of infected cells. This antibody was detected by use of live cell immunofluorescence assay (IFA), and it immunoprecipitated HIV gp160 from Nonidet P-40 (NP-40) lysates of HIV-infected cells [34]. The importance of such antibody responses relates to the possibility that tests that could detect these antibodies (which we operationally term “early HIV antibodies”) could be useful to screen for early infection. This could be lifesaving for recipients of blood transfusions and/or organ donations and possibly beneficial for initiation of the earliest antiviral treatment

Here, we describe studies of the nature of the antigenic specificities of early HIV antibodies and show that they react to conformational epitopes on gp160 and its cleavage product gp41. A number of companies are developing fourth-generation HIV tests [35 –40], several of which incorporate native gp160 and are able to detect the early HIV antibody

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Subjects, Materials, and Methods

Subjects and HIV-1 detectionPatients self-identified for high risk of acquiring HIV infection were recruited over a 3-year period from a dermatology clinic (37 were eventually studied). Single blood samples (10–20 mL) were collected at the first visit. Plasma separated under aseptic conditions was stored at −20°C until testing. Peripheral blood mononuclear cells (PBMC) were isolated by using lymphocyte separation medium (Organon Teknika) and rinsed once in Hanks’ balanced salt solution (HBSS). To deplete the PBMC of CD8 lymphocytes, ∼107 cells were incubated in 5 mL of tissue culture supernatant from the OKT8 hybridoma for 1 h at room temperature, followed by 2 rinses in HBSS, and then were placed in RPMI 1640 medium containing 20% “low-tox” rabbit complement at 37°C for 1 h. The PBMC then were centrifuged and replated in medium containing 15% fetal bovine serum (FBS) and 4 μg/mL phytohemagglutinin. After 3 days, the medium was changed to RPMI 1640, 15% FBS, and 40 μL/mL interleukin-2 (Peprotech). Cultures were maintained for 3–4 weeks, allowing any HIV present to spread, and 0.5-mL aliquots of cells and supernatant were harvested twice weekly. Aliquots were tested for the presence of HIV p24 antigen by using the Coulter HIV-1 antigen capture EIA. The remaining cultured PBL were collected at the end of the culturing period, rinsed, and extracted for DNA for nested HIV PCR assays

HIV DNA sequences present in the PBL DNA were determined by a nested PCR. The MZ 13/14 outer primer pairs for gag and 25 cycles of amplification were followed by the MZ 8/9 inner primer pairs and 30 cycles of amplification. The assay protocol was identical to the initial report of this assay [29]. We evaluated the amplified DNA products by agarose gel electrophoresis and ethidium bromide staining. The seroconversion panels used were from Boston Biomedica Inc. (BBI): 3 BBI panels were provided by Centers for Disease Control and Prevention (CDC), and 2 others were purchased

Live-cell IFAAn indirect microimmunofluorescence assay that used retrovirus-infected live cells as antigen has been described elsewhere [41, 42]. After immunostaining, the cells were resuspended in PBS containing 2% paraformaldehyde and analyzed in a fluorescence activated cell sorter (Becton Dickinson). The HIV-1 isolates used have been described elsewhere [34, 42, 43]. In particular, HIVAC-1, HIVC, and HIV213 were isolated from PBMC of patients with AIDS. These are T-tropic viruses and use CXCR4 as their coreceptor

HIV antibody EIA and WB assaysAll serum samples were tested for anti-HIV antibodies in the Sanofi HIV-1/HIV-2 EIA. Most were also tested by the Abbott HIVAB HIV-1/HIV-2 (rDNA) EIA in the Clinical Microbiology Laboratory (University of Texas Medical Branch [UTMB] at Galveston). This is a third-generation HIV antibody test that uses Escherichia coli– and Bacillus megaterium–expressed recombinant HIV-1 env and gag and HIV-2 env proteins as coating and detecting antigens. The sensitivity and specificity of the assay are estimated at 100% and 99.90%, respectively, with a 95% confidence interval (data provided by Abbott). The manufacturer provided no HIV strain specificity. Serum samples that tested positive were subsequently tested by WB in the UTMB Clinical Microbiology Laboratory

Radioimmunoprecipitation (RIPA) and SDS/PAGEH9 cells (5×107), uninfected or chronically infected with HIV213 or HIVAC-1, were incubated for 4 h at 37°C in methionine-free RPMI 1640 medium containing 30 μCi/mL [35S]methionine (Translabel; Amersham). The cells were then washed and lysed in cold detergent solutions and used in overnight immunoprecipitation assays analyzed by 10% SDS-PAGE, as described elsewhere [34, 42]. In all, we used 5 different lysing solutions: (1) 0.1% octylglucoside in 50 mM Tris-HCl (pH 7.4), 30 mM NaCl, 10 mM glucose, and 1 mM EDTA; (2) 1 mM CHAPS in 50 mM Tris-HCl (pH 7.4), 0.32 M sucrose, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride; (3) 0.5% NP-40 in TNE (Tris, NaCl, and EDTA); (4) 20 mM digitonin in TNE; and (5) 0.1% deoxycholate in TNE

Since gp41 has only a few methionines and is not metabolically labeled very well by [35S]methionine, in some instances the cells were not metabolically labeled and were lysed in various detergents, and, after immunoprecipitation, the proteins precipitated were determined by WB. In brief, the cells were lysed at 2×107 cells/mL of lysis buffer (10 mM Tris [pH 7.4], 130 mM NaCl, 10 mM NaF, 10 mM NaPi, and 10 mM NaPPi) for 1 h on ice in the presence of different detergents (2% NP-40, 20 mM digitonin, 13 mM CHAPS, 0.1% deoxycholate, or 1% SDS). The lysates were precleared by incubating with normal human serum samples overnight on ice. Washed Panosorbin cells (Calbiochem) were added into the lysates to capture the antigen-antibody complexes. The precleared supernatant was then reacted with either normal, early HIV antibody–positive (WB negative), or WB-positive serum samples at 4°C overnight. Pansorbin cells were added, and captured antibody-antigen complexes were eluted by boiling in the presence of SDS. After resolving the samples by 4%–20% PAGE, proteins were transferred onto a nitrocellulose membrane. The presence of HIV envelope proteins was detected by WB by using a mixture of anti-gp41 and anti-gp120 monoclonal antibodies (MAbs)

Native gp160 EIAPurified native gp160 derived from soluble native gp160 produced from HIV-1IIIB–infected H9 cells was obtained from Advanced Biotechnologies. Native gp160 was coated onto flat-bottom PRO-BIND EIA plates (Falcon) at 50 ng/well and incubated at 37°C for 1 h and then at 4°C overnight. For EIA with denatured gp160, the same native gp160 was boiled for 3 min and cooled quickly before coating onto the plates. Detection of bound antibodies used biotinylated native gp160, followed by the addition of streptavidin conjugated with horseradish peroxidase (1:1000 dilution; BD PharMingen). Substrate (tetramethylbenzidine) was added for 5 min, and the color was read at a wavelength of 450/630 after stopping the reaction with 2 N H2SO4. Five BBI seroconversion panels were tested in the native gp160 EIA at 1:4 dilution. Over 30 serum or plasma samples from low-risk university personnel were used side by side as controls at the same dilution. Duplicates of each sample were used, and every test was repeated at least twice. The cutoff value was defined as twice the optical density (OD) value obtained with 24 random low-risk personnel samples. Results were expressed as signal-to-cutoff ratio (s:c), and an s:c ratio >1 is considered to be reactive

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Results

Identification of more high-risk WB-negative subjects possessing early HIV antibody in their serum samplesTo further study these antibodies, we needed serum samples from more than the 4 subjects we originally identified [34]. Patients attending a dermatology clinic were queried as to their risk for HIV infection, and 37 high-risk subjects were eventually identified over a 3-year period. None was known to be HIV positive, but all revealed high-risk histories and presented with dermatologic problems. Blood was drawn from each subject and tested for serum anti-HIV antibodies by commercial EIA (Abbott or Sanofi) and by the nondenaturing live-cell IFA. After IFA staining of the live cells with serial dilutions of a subject’s plasma, cells were examined by flow cytometry. Serum samples from 8 subjects showed reactivity in the live-cell IFA but were not reactive in WB. The serum antibody IFA titers of the 8 WB-negative patients were lower (table 1) than the titers in the serum samples of 17 patients who were WB positive (320–5000), reproducing results from our previous studies [34, 42]. None of these serum samples reacted with uninfected cells, indicating that the antibodies present were reacting to either HIV proteins or HIV-induced cellular proteins. Serum samples from 74 low-risk healthy subjects (low-risk university personnel) were similarly analyzed over many years and were always negative for both infected and uninfected target cells at dilutions of ⩾1:15 (authors’ unpublished data). Follow-up information regarding eventual seroconversion in WB was obtained for 4 of these 8 patients (R299, R328, R343, and R400), and all 4 became WB positive

Testing of patients possessing early HIV antibodies to HIVIt was crucial to determine whether the WB-negative subjects who possessed early HIV antibodies were actually HIV infected. CD8 T cell–depleted PBL cultures from these subjects were tested by p24 antigen-capture EIA over 4 weeks of culture, and 4 of 8 contained HIV p24 (table 2). DNA extracted from the PBL of the 4 p24-negative subjects at the end of the culturing period were positive by nested PCR for HIV gag sequences. As controls, 5 high-risk persons who tested negative by live-cell IFA were also tested. All were negative for HIV by p24 antigen capture EIA of supernatants of PBL cultures and by HIV-nested PCR amplification of PBL DNA (table 2). Thus, the presence of early HIV antibody in the serum samples of EIA and/or WB-negative persons tested to date correlates 100% with the presence of HIV in PBL

Specificity of early HIV antibodiesOur previous RIPA and SDS-PAGE studies showed that the antibody in the serum of 1 early infected early antibody–positive subject only reacted with HIV gp160 and not with gp120 [34]. We thus tested the serum samples of the current subjects in live-cell IFA against cell lines expressing only HIV envelope: CEM T cells that express the envelope genes from HIV213, HIVAC-1, or HIVC [44], 3 viruses we commonly use for testing for the presence of HIV antibodies [34, 42]. In cytometry results of all serum samples, some serum samples did not react with cells expressing only envelope protein, although they reacted to cells replicating whole HIV (figure 1). Table 3 summarizes the test results of 3 of the serum samples we originally reported [34] and the 8 new serum samples and shows that 8 of 11 reacted with envelope proteins. However, antibodies in some serum samples appeared to react with protein(s) other than HIV envelope. Studies to identify the proteins these antibodies recognized were initiated. However, before those studies were done, we wanted to first determine the best way to solubilize the HIV-infected cells, to retain reactivity with these antibodies, since they likely bound to conformational epitopes

Figure 1
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Figure 1

Detection of early human immunodeficiency virus (HIV) antibodies in serum samples of high-risk subjects by immunofluorescence staining of live HIV-infected H9 cells (left) or HIV env-expressing CEM cells (right). Left Live H9 T cells infected with HIV213, HIVAC-1, and HIVC at peak virus production were used as targets and stained with normal human serum (NHS), serum samples from Western blot (WB)–negative high-risk subjects (R299, R343, and R359), and serum from HIV WB-positive patient (R399) (all at 1:30 dilutions). Right HIV env-expressing cell lines (CEM-213 env plus CEM-AC-1 env) [44] were used as targets in live-cell immunofluorescence assay. Filled histogram, uninfected control cells; open histogram, HIV-infected or env-expressing cells. FITC, fluorescein isothiocyanate

Early HIV antibodies reacted readily with HIV proteins solubilized in digitonin and NP-40 but poorly or not at all when solubilized in deoxycholate, CHAPS, octylglucoside, or SDSWe used RIPA of detergent lysates of HIV-infected H9 cells metabolically labeled with [35S]methionine to identify the HIV proteins to which the serum antibodies reacted. The infected cells were solubilized in digitonin, one of the least denaturing detergents and an agent often used to solubilize protein complexes, and NP-40, deoxycholate, octylglucoside, or CHAPS. Figure 2 shows the reactivity of one of the previously described patient’s (R6) serum samples [34] for proteins solubilized in the various detergents. A protein of ∼160 kDa was precipitated from infected cells solubilized in digitonin and NP-40 but not when solubilized in the other detergents. In addition, a protein of ∼55 kDa was precipitated from the digitonin lysate, but not from the other lysates. This is likely the HIV gag precursor p55gag, some of which it myristylated and partly exposed on the cell surface. This strongly indicates that the epitopes recognized by the antibody are likely conformational in nature, since they were very sensitive to denaturation. Control WB-positive serum samples showed that all 4 lysates contained other HIV proteins in addition to gp160 and p55, but gp41 was present in very small detectable quantities (authors’ unpublished data). Identical results were obtained when R6 serum samples were repeated and when serum samples from patients R23, R291, and R400 were used for immunoprecipitation (authors’ unpublished data)

Figure 2
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Figure 2

Evaluation of preservation of human immunodeficiency virus (HIV) epitopes reacting with early HIV antibodies. HIV213 and HIVAC-1-infected H9 cells were labeled with [35S]methionine and lysed in the indicated detergents, followed by radioimmunoprecipitation assay with serum from subject R6 (containing early HIV antibodies) and control normal serum (NS). The precipitates were analyzed by SDS-PAGE

To substantiate that this 160-kDa protein was an HIV protein and not a 160-kDa cellular protein induced by HIV infection, NP-40–solubilized HIV-infected H9 cell lysate was first precleared with an anti-gp120 MAb (F105) before immunoprecipitation with serum samples from subjects R6 and R23. Preclearing eliminated the 160-kDa protein from the lysate that the early HIV antibody immunoprecipitated, demonstrating that this antibody recognized HIV gp160 (authors’ unpublished data)

To definitively determine that HIV gp160, not gp120, was being precipitated, immunoprecipitation was done again with nonlabeled lysates, but WB with MAb against gp120 and gp41 was used for readout. Uninfected or HIV-infected H9 cells were solubilized in digitonin, NP-40, deoxycholate, octyglucoside, CHAPS, or SDS. These lysates were then incubated with normal serum, early HIV antibody–containing serum (R299), or an HIV WB-positive serum (R310), and the immunoprecipitates were resolved on SDS-PAGE, followed by WB analysis with mixtures of MAb HIV gp41 (Chessie 8)– and gp120 (Chessie 6)–specific antibody. Figure 3 shows the results. No proteins were precipitated from uninfected cells with R299 and WB-positive control serum samples R310. However, when mild detergents such as digitonin and NP-40 were used, maximum levels of HIV gp160 as well as gp41, but not of gp120, were precipitated with R299, whereas the WB-positive serum (R310) also precipitated gp120. When stronger detergents CHAPS and SDS were used, the majority of reactivity with R299 was lost, and deoxycholate was intermediate

Figure 3
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Figure 3

Immunoprecipitation of human immunodeficiency virus (HIV)–infected cells solubilized in different detergents and analyzed by SDS-PAGE with a Western blot (WB) readout. CEM cells uninfected or infected with HIV strains 213, AC-1, and C were lysed in various detergents and incubated overnight with normal serum (NS), early HIV antibody-positive serum (R299), or WB-positive serum (R310). Immune complexes were captured by Pansorbin, washed, and analyzed by SDS-PAGE. Proteins were then transferred to a nitrocellulose filter by WB and reacted to monoclonal antibody against gp41 and gp120

EIA that uses nondenatured gp160A third-generation EIA that used purified native, nondenatured gp160 was next developed to detect early HIV antibodies. Table 4 shows the results with this EIA and use of HIV gp160 coated either in its native form or denatured by boiling. The ODs obtained with early HIV antibody–containing serum samples of subjects R299, HR22, and R400 on native gp160 demonstrate reactivity (>1). However, when we used plates coated with denatured gp160, no serum samples were reactive. To show that both plates contained similar amount of gp160, a serum positive by WB for HIV antibody was used, and similarly high ODs were observed on both plates. This further confirms that the early HIV antibody present in early infected persons reacts with only conformational, not primary, amino acid epitopes of gp160/gp41

Table 1
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Table 1

Serum antibody titers of Western blot–negative subjects

Table 2
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Table 2

Human immunodeficiency virus (HIV) detection in peripheral blood lymphocytes (PBL) of high-risk Western blot–negative, live-cell immunofluorescence assay (IFA)–positive subjects

Table 3
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Table 3

Summary of live-cell immunofluorescence assays (IFAs) of EIA and/or Western blot (WB)–negative serum samples

Table 4
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Table 4

EIA results by using native or denatured human immunodeficiency virus gp160–coated plates

Early HIV antibodies can be present for weeks before seroconversion in current EIA, antigen, and reverse-transcription (RT)–PCR assaysThe above results and those of our previous studies [34] clearly show that antibodies reacting with conformational epitopes of cell surface-expressed HIV gp41 can be present in the serum samples of persons with early HIV infection prior to antibodies that react in denatured antigen (EIA and WB assays). An important remaining question is how long before seropositivity in the latter tests is the early antibody present?

To address this question, we tested 5 BBI seroconversion panels in our new third-generation native HIV IIIB gp160 EIA. Results are shown in table 5. The initial blood sample of panel PRB 931 was negative in our test, but the serum obtained 2 days later was positive, as were later blood samples. However, not until day 15 did both the Coulter HIV antigen and Roche RT-PCR tests start to become positive. Thirteen days later, a commercial EIA (Abbott HIV-1/2) showed positivity. Thus, early antibodies were present in this panel 13 days before positivity in the Coulter HIV antigen and Roche RT-PCR tests and ∼26 days earlier than positivity for antibodies reactive in commercial denatured antigen EIAs

Table 5
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Table 5

Comparisons of native gp160 EIA IIB with the most sensitive commercial human immunodeficiency virus (HIV) antibody, antigen, RNA, and Western blot (WB) assays in 5 seroconversion panels

In the second panel, PRB923 (table 5), the first blood sample and subsequent blood samples up to day 30 were positive in our test, but the antibody titers continually decreased. On day 35, the native gp160 EIA became negative, and the plasma became positive for HIV RNA by the Roche RT-PCR test. One explanation is that the continual decreases in early HIV antibody over time were due to slowly increasing virus loads, which likely absorbed the antibodies and kept them from reacting in the assay. Thus, scoring for early antibodies allowed for detection of HIV infection ∼35 days earlier than scoring for HIV RNA by the Roche virus load test, 37 days earlier than the most sensitive HIV antigen test, and 47 days earlier than the Abbott HIV-1/2 antibody test in this panel

In the third panel, PRB932 (table 5), the first blood sample was positive in our test 27 days earlier than in both the Roche RNA and the Abbott HIV-1/2 antibody test, but the second blood sample 3 days later was negative while the third, fifth, and sixth bleeds were positive. Other studies have shown that this seroconverter panel alternates positive and negative for antibodies, similar to our findings, and this could be due to bursts of virus, which are absorbing out the antibodies. However, in 2 additional panels PRB 925 and PRB 935 (table 5), the early HIV antibody to conformational epitopes of gp160 could not be detected in the native HIV IIIB gp160 EIA any earlier than the other tests. In fact, in panel PRB 935, PCR positivity for RNA was seen on day 24, and antigen was observed on day 28, times when no antibodies against HIV IIIB native gp160 were detected

To determine whether these panels had early HIV antibodies that would react with native gp160/gp41 from other HIV strains, we tested these panels by live-cell IFA (table 6) and found that panel PRB 935 had low-titer antibodies that reacted with HIV 213, predominantly, but not with HIV IIIB. Panel PRB 925 did not have early antibody that reacted with any of the 5 HIV strains tested. Table 7 summarizes the results from all 5 panels in the native envelope antibody assays (native IIIB gp160 EIA or live-cell IFA) in comparison with standard reference HIV antibody, antigen, and RNA assays. In summary, native assays detected HIV infection 13–44, 13–47, and 26–47 days earlier than the best HIV RNA, antigen, and antibody assays, respectively, in 4 of 5 seroconverter panels

Table 6
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Table 6

Live-cell immunofluorescence assay results of 2 seroconverter panels

Table 7
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Table 7

Comparison of the performance of native gp160 EIAIIIb, live-cell immunofluorescence assay, human immunodeficiency virus (HIV)–1/2 third-generation plus EIA, p24 antigen detection, HIV-1 RNA reverse-transcription polymerase chain reaction (RT-PCR), and Western blot (WB) in Boston Biomedica Inc. (BBI) seroconversion panels

We next tested for early HIV antibody in another clinical setting. Serum samples from an HIV needlestick case provided by the CDC were tested by live-cell IFA and our native gp160 EIA. Serum taken 3 weeks after the exposure was negative by commercial EIA but positive in both the native gp160 EIA and live-cell IFA (table 8). By 4 months after exposure, the serum was positive in all tests (table 8). Again, this case further demonstrates that a test for early HIV antibody allows for earlier diagnosis of infection

Table 8
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Table 8

Timing of early seroconversion in a health care worker exposed to human immunodeficiency virus (HIV)–positive blood via needlestick

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Discussion

The results of this study extend our initial findings that showed the existence of early HIV antibody in early HIV-infected, EIA-negative, WB-negative patients [34]. These antibodies are believed to be unique in that they are directed to conformational epitopes, and hence are nonreactive in the conventional denatured antigen EIAs or WBs that use complete or fragments of bacterially expressed recombinant HIV proteins or synthetic polypeptides. The epitopes are likely located discontinuously, and unraveling of the folded conformation of gp41 or gp160 results in loss of reactivity. It is not surprising that antibodies to conformational epitopes of cell surface–expressed gp41 may be the first antibodies induced after natural infection. These are what the immune system will be first exposed to, and conformational epitopes are often strong immunogens [45 –47]

Studies quantitating HIV RNA in tissues of HIV-infected subjects show that 90% of all HIV in a person is attached to the surface of follicular dendritic cells in the lymphoid tissues [48]. These cells are the major initiators of immune responses and do not always process antigen, but expose it in its native form [49, 50]. Thus, these native proteins would stimulate B cells to produce antibodies directed to conformational epitopes. Therefore, it can easily be envisioned why a strong conformational immunogen on gp41 would induce the first antibody response. There is precedent for this observation, since antibody responses to natural murine retroviral infections involve such antibodies as a major component of the immune response [51 –53]. Thus, antibodies to conformational epitopes may be very important as early diagnostic markers

There are numerous early data plus the more definitive needlestick and blood transfusion data that show that there is an interval of time (termed the immunologically silent window) after HIV infection in which persons do not test positive by serologic tests that use denatured antigens (EIA and WB). This silent period may range from 1 to many months [4, 13, 54 –65], during which persons transmit HIV through blood transfusion, sexual activity, or organ donation [4, 10, 15, 16, 66 –69]. Even with availability of third-generation EIAs, case reports of transmission still appear [70, 71]. A number of studies have attempted to shorten the silent period by use of PCR [20 –26, 72], but, in general, routine single-amplification regimen PCR for HIV DNA does not detect infection much earlier than the newest EIAs [73]. The new RT-PCR assays for HIV RNA in plasma, however, appear to shorten the window somewhat

Recently, several fourth-generation EIAs [35 –40] were developed to detect HIV antigen and antibody simultaneously. One such test, VIDAS HIV DUO Ultra, uses native IIIB gp160 as one of the coating antigens and detects infection in 10 BBI seroconversion panels ∼12 days earlier, on average, than the current antibody assays [35]. However, only 2 of 10 panels had earlier detection in this fourth-generation test than in an antigen-only test. An earlier version of this test, VIDAS HIV DUO, which contains the same antigens as DUO ultra except that it lacks native gp160, detected infection 4 days earlier, on average, than the third-generation antibody assay in 5 of 12 BBI seroconversion panels tested. These results strongly suggest that the antibody to the native gp160 component within the DUO Ultra fourth-generation format provides the earliest detection of HIV infection, rather than the antigen assay component. Of particular interest is that, within this antibody assay, full-length native gp160 was added as part of the coating antigens, in addition to gp41 and gp36 polypeptides, which are also used in third-generation antibody assays. It seems likely that the native gp160 added in the antibody assay component provides the ability to detect HIV infection earlier than the third-generation antibody assays. Our native gp160 assay, which showed further improvement over the VIDAS HIV DUO Ultra, may be due to the increased sensitivity of a pure native gp160 format

Binsbergen et al. [37, 38], showed that their fourth-generation test, HIV UFII Ag/Ab, which also contains IIIB gp160, could detect HIV infection 1–2 weeks earlier than antibody EIAs, but not earlier than antigen tests. They argued that this earlier detection was due to the antigen detection part of this assay, but stand-alone antigen assays are more sensitive for detecting antigen than the combined antibody-antigen fourth-generation format. They tested the 5 panels that we tested and did not detect the early HIV antibody that we detected. Thus, we suspect that their fourth-generation test incorporates denatured gp160 or has too little to detect the early antibody

gp160 from a single HIV strain will not be the only antigen needed to detect all early infections, since these antibodies have some HIV strain specificity. The use of native env antigens to assess antibody specificities in serum samples of WB-positive subjects revealed that much of the env-specific antibodies were directed to type-specific epitopes [42]. In this study, we found that antibodies in 1 seroconverter panel (PRB935) did not react to native gp160 from HIVIIIB but reacted to HIV213 (table 6). It is also evident that not all early antibody reacts to envelope, as demonstrated in figure 1 and table 3. Our RIPA of metabolically labeled infected cells shows that p55 was also precipitated with some early antibodies, and, since a myristylated form of this gag precursor molecule is cell-surface expressed, it may be important to also use native p55gag as an EIA antigen. All current diagnostic EIAs use denatured gag proteins and gp41. In addition, since we observed a dip in titer just before or at the time that viral RNA became detectable in plasma, it is likely that virus will “absorb out” the early HIV antibodies

We know that the majority of the conformation-dependent early antibodies are neutralizing (authors’ unpublished data). At the earliest stage of HIV infection, these antibodies may bind to the viruses and kill them through the complement pathway. Weeks later when extensive virus spread has occurred, not enough early antibodies may be available for neutralization, and the viruses become detectable. This, and the fact that much of the virus produced in early infected lymph nodes is likely adsorbed to follicular dendritic cells or CD4 lymphocytes and does not get into the blood may explain why early antibodies are detectable earlier than plasma HIV RNA

By use of the BBI seroconversion panels, we showed that native gp160 assays could detect HIV infection ∼2–4 weeks earlier than both the HIV RNA and antigen assays and 4–6 weeks earlier than the antibody assays in some, but not all, cases. Furthermore, the current HIV tests, even the fourth-generation combined antibody/antigen format, probably should include native gp160s as antibody-detecting antigens to detect earliest HIV infection. This will allow for treatment of HIV infection at the earliest stage possible

Several other markers of an immune response to HIV also appear to be good indicators of early infection. In addition to our study, which showed the existence of early conformation-specific anti-gp41 antibodies, T cells reactive to HIV peptides were present in high-risk, EIA-negative persons [31, 74]. In addition, in some early infected EIA-negative patients, B cells, which make anti-HIV antibodies, can be expanded from peripheral blood in vitro [33]. Antibodies to HIV Nef and p17 proteins have are present before seroconversion in EIA [75, 76]. Consequently, several studies indicate that immune responses occur early after infection and these do not necessarily involve induction of antibodies that react to linear epitopes of HIV proteins

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Acknowledgments

We thank Donica Westerheide and Cheryl Ross, for technical assistance; Denise Cardo and Elise Beltrami (Centers for Disease Control and Prevention [CDC], Atlanta), for serum samples from a needlestick case; and Joanne Mei (CDC), for 3 Boston Biomedica Inc. seroconverter panels

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Footnotes

  • ↵All subjects gave written informed consent

  • Received February 7, 2002.
  • Revision received April 16, 2002.
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