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Clinical and Vaccine Immunology, July 2009, p. 1060-1065, Vol. 16, No. 7
1071-412X/09/$08.00+0     doi:10.1128/CVI.00280-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Early Detection of Human Immunodeficiency Virus Type 1-Specific B-Lymphocyte-Derived Antibodies in a High-Risk Population

Odd Odinsen,^1* David Parker,² Frans Radebe,³ Mikey Guness,³ and David A Lewis^3,4,5

PlasmAcute AS, Bergen, Norway,¹ Novel Consulting Ltd., Dartford, United Kingdom,² Sexually Transmitted Infections Reference Centre, National Institute for Communicable Diseases, Johannesburg, South Africa,³ Department of Internal Medicine, University of the Witwatersrand, Johannesburg, South Africa,⁴ Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa⁵

Received 2 August 2008/ Returned for modification 11 September 2008/ Accepted 20 May 2009

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Diagnosis of acute human immunodeficiency virus (HIV) infection, a key driver of the HIV epidemic, remains a public health challenge. The PlasmAcute technology offers an opportunity to detect early anti-HIV antibody responses. B lymphocytes (B cells) were isolated from the blood of seronegative miners in South Africa by using the PlasmAcute method. B-cell lysates and paired sera were tested for anti-HIV-1 antibodies by two different enzyme-linked immunosorbent assays; immunoreactivity was confirmed by Western blotting. All volunteers were tested for HIV type 1 (HIV-1) viral load, p24 antigen, and CD4 count. Sera from HIV-seronegative men who had positive viral loads and were positive for p24 antigen were retested for anti-HIV antibodies after immune complex dissociation. Anti-HIV antibodies were detected in lysates from 16/259 subjects without immunoreactivity in paired sera. Four subjects, one of whom had a positive viral load initially, subsequently seroconverted. Six subjects showed transient anti-HIV-1 antibodies in the lysates and tested negative for all markers at the follow-up. Five subjects without follow-up data initially had lysate-positive/serum-negative samples, and these cases were classified as inconclusive. One subject had lysate antibodies and a detectable viral load but was seronegative at follow-up. In conclusion, lysate-derived anti-HIV-1 B-cell antibodies can be detected prior to seroconversion and earlier than or contemporary with HIV-1 RNA detection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
According to the 2006 UNAIDS report on the global AIDS epidemic, an estimated 38.6 million people worldwide were living with human immunodeficiency virus (HIV) at the end of 2005 (31). It was also estimated that 4.1 million became newly infected with HIV and that 2.8 million lost their lives to AIDS in that year. Approximately 10% of the world's population lives in sub-Saharan Africa, which is home to almost 24.5 million people with the virus, or 64% of all people living with HIV. UNAIDS estimates that 2.7 million became newly infected in this region in 2005 (31). A considerable number of new HIV infections are transmitted by recent HIV seroconverters (6, 23, 25, 26).

Early diagnosis of HIV infection is important, as it allows appropriate clinical management and counseling of patients in order to prevent further sexual transmission of HIV to partners (11, 24). It is also important in special situations, for example, screening blood for transfusion (10, 19). Access to reliable test systems for diagnosis of HIV infection at the earliest possible stage is necessary for effective prevention of transmission, early intervention, access to antiviral therapy, surveillance, and blood safety.

The enzyme-linked immunospot assay is a well-established method for the enumeration of antibody-secreting cells and study of spontaneous secretion of antibodies (4, 7, 8, 12). In this study, we utilized the PlasmAcute technology to detect anti-HIV-1 antibodies in B-cell lysates before they are detected in serum. This technology is based on the observation that B cells from peripheral blood contain functional antibodies elicited by an infectious agent or vaccine, and these antibodies can be measured before they can be detected in plasma (14, 22). This allows a narrowing of the "window period" between immunological stimulation and seroconversion. The technology involves initial capture enrichment and isolation of B cells from a peripheral-whole-blood sample by using paramagnetic polystyrene beads coated with monoclonal anti-CD19 antibodies followed by subsequent lysis and testing of the lysate in an appropriate immunoassay (22).

(The data in this paper were presented in put at the joint meeting of the 17th Meeting of the International Society for Sexually Transmitted Diseases Research and the 10th World Congress of the International Society against Sexually Transmitted Infections, Seattle, WA, 29 July to 1 August 2007 [26a].)

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study site and population. This prospective study took place in a sexually transmitted infection (STI) and HIV voluntary counseling and testing clinic for male miners at a South African gold mine. The annual seroconversion rate in the HIV-negative individuals of this population was estimated to be at least 5%. These men therefore represented an appropriate target group for a study on early diagnosis of HIV infection. Miners were considered eligible for the study if they were negative for anti-HIV-1 antibodies by the rapid initial screening test (Capillus HIV-1/HIV-2 kit; Trinity Biotech PLC, Dublin, Ireland) and were able to give follow-up blood samples within 2 to 3 weeks following their initial blood draw. After obtaining informed consent, we collected blood samples for HIV serology (enzyme-linked immunosorbent assay [ELISA] and Western blotting) and determined HIV p24 antigen presence, HIV-1 viral loads, and CD4 counts. Whole-blood samples for isolation of B cells were collected in EDTA Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) and transported to the STI Reference Centre at the National Institute for Communicable Diseases for further testing at ambient temperature within the same day. Initial negative HIV-1 results determined by the Capillus kit were confirmed by retesting with two different HIV-1 ELISA tests and subsequently by HIV-1 Western blotting.

B-cell lysate preparation and testing. B cells were isolated from EDTA-blood samples by the PlasmAcute method. In brief, 500 µl of EDTA-blood was diluted in 720 µl phosphate-buffered saline (PBS) containing 1% Tween 20, 0.7% trisodium citrate dihydrate, and 2.5% citric acid (citrate buffer) in 1.5-ml Eppendorf tubes. Twelve microliters of anti-CD19-coated paramagnetic microparticles (Fluorobeads B; One Lambda, Canoga Park, CA) was added to each tube and mixed for five minutes. B cells attached to the beads were separated from the liquid phase with a magnetic stand and washed twice in 1,400 µl citrate buffer and finally once in 1,400 µl PBS containing 0.01% Tween 20 only. The cells were lysed by the addition of 50 µl of disruption buffer containing protease inhibitor cocktail consisting of 0.2% pepstatin, 0.2% leupeptin, and 0.5% aprotinin. Lysates were diluted in 400 µl of storage buffer containing protease inhibitor cocktail and 1.25% milk proteins and stored at 4°C for <24 h or at –20°C until tested. Lysates were treated the same way as plasma or serum samples when tested, i.e., according to the manufacturer's package insert. Samples that tested anti-HIV-1 antibody negative in lysate and serum but had a positive viral load and p24 antigen positivity were retested for the presence of anti-HIV-1 antibodies after dissociation of any HIV-1 antigen-antibody complexes in the serum (see below).

Anti-HIV antibody testing. Lysates and corresponding plasma samples were tested for anti-HIV-1 antibodies by two different ELISAs. In the first part of the study, Abbott Axsym HIV 1/2 (Abbott Laboratories, Abbott Park, IL) and Ortho Vitros (Ortho Clinical Diagnostics, Raritan, NJ) were used and considered ELISA 1 and ELISA 2, respectively. Due to the physical relocation of the STI Reference Centre during the course of the study, these assays were later changed to Abbott/Murex HIV-1.2.O (Abbott/Murex Biotech, Dartford, United Kingdom) and Vironostika HIV Uni-Form II Plus O (bioMérieux SA, Marcy l'Etoile, France) (for ELISA 1 and ELISA 2, respectively).

Western blot testing and interpretation. Immunoreactivities of lysate and serum were confirmed by Western blot analysis using one of two commercial Western blot assays (Genelabs Diagnostics HIV Blot 2.2 or Bio-Rad New LAV Blot).

The STI Reference Centre's criteria for interpretation of an HIV-1 Western blot are similar to the Centers for Disease Control and Prevention recommendations, whereby a specimen is interpreted as being positive when there is reactivity to any two of the following bands: p24, gp41, and gp120/gp160 3). The Western blot results were also analyzed with World Health Organization criteria (whereby a specimen is interpreted as being positive when there is reactivity to at least two env-associated bands), the American Red Cross criteria (whereby the presence of at least three bands is required, one from each of the gag, pol, and env gene product groups), and the revised criteria described by Mahé et al. (whereby the specimen is considered to be reactive only when the gp160 and p31 bands are present) (20, 21, 30).

Immune complex dissociation. Serum samples from participants who had initially tested anti-HIV antibody negative for lysate and serum but had a positive HIV-1 viral load and was positive for p24 antigen were retested using procedures for immune complex dissociation as described by Troisi and Hollinger and modified by Alnaqy et al. and as performed by Miles et al. (1, 29). In brief, 400 µl of serum was added to 3.6 ml of PBS (pH 7.4) and centrifuged at 1,700 x g for 20 min at room temperature. The supernatant was mixed with an equal volume of ice-cold PBS containing 4% polyethylene glycol 6000 and 3% Tween 20 to precipitate immune complexes. The mix was incubated overnight at 4°C and recentrifuged under the same conditions. The precipitate was washed twice with 3 ml PBS containing 2.5% polyethylene glycol 6000 and 1.5% Tween 20 and resuspended in 200 µl of 3 M potassium thiocyanate to dissolve immune complexes. The solution was incubated at 37°C for 30 min and subsequently stored at 4°C. The untreated serum and the resuspended pellets were tested for anti-HIV-1 antibodies according to the package inserts of the respective ELISA tests.

CD4 counts and HIV-1 viral load testing. CD4 counts were performed using the FACSCount kit (Becton Dickinson and Co., Franklin Lakes, NJ). HIV-1 viral loads were determined using the Cobas AmpliPrep/Cobas Amplicor HIV-1 monitor test (version 1.5) assay (Roche Molecular Systems Inc., Indianapolis, IN).

Data entry and analysis. Data were entered into a Microsoft Excel spreadsheet and analyzed in terms of optical density (OD)/cutoff values and automated Western blot interpretations.

Study approvals. The study was approved by the Human Research Ethics Committee (Medical) of the University of Witwatersrand (protocol no. M02-05-20). Informed consent was obtained from participants, and the clinical research was conducted in keeping with South African good clinical practice guidelines for human subject research.

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A total of 281 volunteers were recruited for participation in the study. Sixteen individuals were excluded due to an initial false-negative Capillus rapid HIV test result. There were thus 265 initially eligible volunteers for the first visit, of whom 143 returned for a follow-up visit. Six of the 265 that tested negative for anti-HIV-1 antibodies in lysate and serum showed a positive viral load. All six sera tested reactive for HIV when retested following an immune complex disruption procedure, and these volunteers were subsequently excluded from the study, since they in principle had already seroconverted. The time span between the initial and the follow-up visit in this study varied between 11 and 253 days; 78 (55%) of these 143 volunteers reattended within the requested 2-to-3-week period, and another 17 (12%) reattended within 5 weeks of the first blood draw. Sixteen of the remaining 259 subjects with confirmed negative results from the Capillus kit (6.2%) tested positive for anti-HIV-1 antibodies in lysates at their first or follow-up visit but showed no immunoreactivity in serum. Three of the 16 volunteers (001, 003, and 267) whose initial samples were negative for all markers except lysate antibodies later seroconverted and also subsequently tested positive for an HIV-1 viral load (Tables 1 and 2). One of these 16 (PA201) was also HIV-1 viral load positive at the first visit and subsequently seroconverted (Tables 1 and 2; data not shown). Six of the 16 subjects, exemplified by 098, 244, and 259, showed only transient anti-HIV-1 reactivity in lysate and tested negative for all markers in both lysate and plasma at the following visit (Tables 1 and 2). Five initially lysate-positive/serum-negative samples were classified as inconclusive owing to a lack of follow-up samples from the volunteers.

View this table: [in this window] [in a new window]

TABLE 1. Lysate, serum, and plasma test results for 11 subjects at two consecutive visitsa

View this table: [in this window] [in a new window]

TABLE 2. Western blot patterns for seroconverters and transiently reactive samplesa

One individual (246) showed an absence of immunoreactivity in both lysate and serum at the first visit but was positive for anti-HIV-1 antibodies in lysate at the second visit (Tables 1 and 2). This volunteer also had a positive HIV-1 viral load at the same follow-up visit.

In summary, 5 (3.5%) of the 143 miners, without anti-HIV antibodies in serum, demonstrated anti-HIV-1 reactivity in B-cell lysates contemporary with or before detection of positive HIV-1 viral load. HIV-1 seroconversion was documented in four of these five miners.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Diagnosis of acute HIV infections in clinical practice remains a public health challenge, and improvement in existing tools to diagnose HIV infections at the very earliest stage remains a technological priority. Empirical evidence based on mathematical modeling suggests that the most important biological factor driving the HIV epidemic may be the high HIV-1 viral load in genital fluids at the time of acute HIV infection (13, 26). This is particularly important in the African context, as acute primary HIV is frequently detected (24). Early diagnosis of acute HIV infections may enable public health interventions, transmission reduction counseling, and access to antiretroviral therapy for those with coexisting immunosuppression.

The present study demonstrates that the PlasmAcute technology is able to detect anti-HIV-1 antibodies prior to seroconversion and that this is possible earlier than or contemporary with detection of a positive HIV-1 viral load. Lysate samples from the four seroconverters, for which ELISA results for lysate were positive but ELISA results for serum were negative, also showed reactivity in the Western blot analysis, supporting the validity of the ELISA results (Table 2). The Western blot analysis also confirmed the lack of reactivity in the corresponding paired serum samples. Only one of these four participants' samples was simultaneously positive for HIV-1 RNA. This suggests that early B-cell-associated antibodies can serve as a valuable tool to aid screening for acute HIV infection (2).

Both enzyme-linked immunospot assay and cell sorting analysis have shown that B-cell response is transient, peaks at approximately 7 days postchallenge, and is barely detectable 14 days postchallenge. Similarly, specific antibodies are detectable in B-cell lysates in the same time frame. Secreted antibodies are detected in overwhelming amounts in the plasma around days 10 to 14, when the level of specific antibodies in the total B-cell population is below detection even with sensitive ELISAs. Circulating B cells normally have a half-life of 3 to 5 days, and ELISAs cease detecting specific antibodies in the total population when the relative proportion of antigen-specific B cells falls below a threshold level of approximately 1%. More-sensitive research techniques may detect antigen-specific B cells at a level of approximately 0.1% in immunoprotected subjects (9).

The six volunteers in whom the B-cell lysates showed transient anti-HIV-1 reactivity without subsequent evidence of seroconversion are particularly interesting. This observation could be due to exposure to HIV-1 without progression to viremia and seroconversion. The volunteers were miners from a known population at high risk of contracting STIs and HIV. In contrast, no transiently reactive lysates were detected among 8,000 blood donors attending the South African National Blood Service blood bank (South Africa) in 1995 (O. Odinsen and D. Parker, unpublished data). Most blood donors in South Africa are non-black Africans, whereas almost all miners are black Africans. Further investigations looking at B-cell lysates from black African blood donors at low risk of contracting HIV versus those from the black African miners might reveal whether the observed transient results are due to ethnicity issues and genetic differences.

The phenomenon of transient antibody formation has been reported previously in the context of influenza and hepatitis B vaccination research (14, 22). In support of transient B-cell immune responses to HIV exposure, Tenenbaum et al. showed transient anti-HIV-1 seroreactivity in several hemophiliac patients after exposure to HIV-contaminated clotting factor concentrates (28). Data from a Nairobi sex worker cohort has also provided evidence for nontransient B- and T-cell immune responses among HIV-1-resistant women (15, 16, 17, 18).

It may be hypothesized that, in cases where the immune system is receiving insufficient antigenic stimuli to maintain the antibody response, deterioration and apoptosis of B cells will lead to the release of incompletely assembled antibodies and subunits into the bloodstream. The limited amount of antibodies released will be diluted throughout the peripheral blood system and will remain undetectable with conventional serological methods. These incomplete immunoglobulins and related fragments will have a half-life in blood (28 min) shorter than that of fully mature immunoglobulin molecules (330 min) and are likely to disappear quickly from circulation (5). This could possibly explain why the follow-up plasma samples from volunteers with transiently positive lysates failed to show anti-HIV-1 reactivity at the follow-up.

This study's observations support the hypothesis that transiently antibody-reactive B-cell lysates may represent exposure to HIV-1 without subsequent progression to infection and seroconversion, for which further investigations are required. The detection of transient positive ELISA reactivity in lysates from miners belonging to a population at high risk of HIV infection, however, does appear to limit the usefulness of the PlasmAcute technology for accurate diagnosis of acute HIV infection. The data reported show that transiently lysate-positive samples were detected from 6 of the 10 participants with follow-up samples available for analysis. Further research is needed to better understand the nature of transient reactions, and efforts will be required to improve the PlasmAcute technology for any future diagnostic role in diagnosing early HIV infections in high-risk populations. At present, this technology offers at best a screening methodology for diagnosis of early HIV infections but will still require confirmation with either HIV nucleic acid amplification testing or else repeat routine serological ELISA testing after a sufficient time period. The detection of transiently lysate-positive individuals in high-risk populations by the PlasmAcute technology may potentially identify those individuals who are highly exposed but not yet infected by HIV. These are key individuals for whom behavioral interventions should be urgently initiated in order to prevent future seroconversions. The ability to analyze newly synthesized B-cell antibodies (in the presence of preexisting plasma anti-HIV antibodies) may also be useful when evaluating therapeutic HIV vaccine responses, as demonstrated in recent work on influenza vaccine-induced antibodies (27, 32).

In conclusion, the PlasmAcute technology is able to diagnose acute HIV infection at the same time as RNA amplification assays and, in some cases, slightly before. The phenomenon of transiently positive ELISA results for lysate limits the PlasmAcute technology as a diagnostic tool for use with high-risk patients. With further evaluation, the technology may prove to be a useful tool in screening patients' sera and blood donations for early HIV infection, potentially identifying individuals at high risk for seroconversion, and evaluating therapeutic HIV vaccine responses.

 
We are grateful to Hendrik Koornhof, former acting head of the STI Reference Centre at the National Institute for Communicable Diseases, and Lars Haaheim, University of Bergen, for their assistance and enthusiasm in setting up this study. We also thank Sr. Thoko Nhlapo, who assisted with participant recruitment and enrollment at the mine clinic, as well as Adrian Puren and his staff, who assisted with anti-HIV antibody, CD4, and HIV-1 viral load analyses.

This study was financially supported by PlasmAcute AS (Bergen, Norway) and a grant from the Norwegian Research Council (project no. 156669/210).

O.O. and D.P. both have associations with PlasmAcute AS (Bergen, Norway). O.O. is a former employee of PlasmAcute AS and holds shares in Novel Diagnostics ASA (Bergen, Norway), which is the sole owner of PlasmAcute AS. D.P. is a consultant for PlasmAcute AS and also holds shares in Novel Diagnostics ASA. D.A.L. has, until recently, received research funding from PlasmAcute AS for another study involving assessment of the PlasmAcute technology among women at high risk of infection. F.R. and M.G. have no conflict of interests.

 
* Corresponding author. Mailing address: PlasmAcute AS, Sognsveien 32, 0851 Oslo, Norway. Phone: 47 91 37 22 39. Fax: 47 55 54 38 98. E-mail: odd.odinsen{at}getmail.no

Published ahead of print on 27 May 2009.

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Clinical and Vaccine Immunology, July 2009, p. 1060-1065, Vol. 16, No. 7
1071-412X/09/$08.00+0     doi:10.1128/CVI.00280-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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