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      An improved microtiter assay for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

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          Abstract

          Background

          The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay.

          Methods

          In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor(s), the tested sera or plasma was removed by a washout procedure after initial 3–6 h culture in the assay.

          Result

          This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method.

          Conclusion

          The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.

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          Most cited references16

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          Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes.

          J Schmitz, M Kuroda, S Santra … (1999)
          Clinical evidence suggests that cellular immunity is involved in controlling human immunodeficiency virus-1 (HIV-1) replication. An animal model of acquired immune deficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to show that virus replication is not controlled in monkeys depleted of CD8+ lymphocytes during primary SIV infection. Eliminating CD8+ lymphocytes from monkeys during chronic SIV infection resulted in a rapid and marked increase in viremia that was again suppressed coincident with the reappearance of SIV-specific CD8+ T cells. These results confirm the importance of cell-mediated immunity in controlling HIV-1 infection and support the exploration of vaccination approaches for preventing infection that will elicit these immune responses.
          Article 0 comments Cited 305 times – based on 0 reviews     Review now
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            Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection.

            L Musey, J. Hughes, T Schacker … (1997)
            Early in human immunodeficiency virus type 1 (HIV-1) infection there is a decline in viral replication that has been attributed to host immunity, but the components of this response, particularly the ability of cytotoxic T lymphocytes to control viral burden and influence the outcome of disease, are poorly understood. We prospectively studied 33 patients with primary HIV-1 infection for HIV-specific activated cytotoxic T lymphocytes and memory cytotoxic T lymphocytes and compared these lymphocyte responses with changes in viral load and clinical status over the subsequent 18 to 24 months. Soon after infection, activated HIV-specific cytotoxic T lymphocytes, mediated primarily by CD8+ cells, were detected in 17 of 23 patients (74 percent). Memory cytotoxic T lymphocytes were found in 6 of 6 patients tested (100 percent) during the first three months of infection and in 17 of 21 patients (81 percent) tested during the first six months. The frequencies of memory cytotoxic T lymphocytes varied markedly over time, but overall they declined over the first 6 to 8 months and then stabilized over the next 12 to 18 months. The patients with higher frequencies of Env-specific memory cytotoxic T lymphocytes had a median level of plasma HIV-1 RNA about one third that of the patients with lower frequencies, (median number of RNA copies per milliliter, 22,000 vs. 62,000; P=0.006). Patients with low frequencies of Env-specific memory cytotoxic T lymphocytes (or none) in early infection had a more rapid decline to less than 300 CD4+ cells per cubic millimeter (P = 0.05). In early HIV-1 infection, the induction of memory cytotoxic T lymphocytes, particularly those specific for Env, helps control viral replication and is associated with slower declines in CD4+ cell counts. Host cytolytic effector responses appear to delay the progression of HIV-1 disease.
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              Passive transfer studies to elucidate the role of antibody-mediated protection against HIV-1.

              John R. Mascola (2002)
              In order to understand immune correlates of protection and to develop effective immunization strategies, it is important to know if antibodies can confer protection against HIV-1 infection or disease. The recent development of the pathogenic simian/human immunodeficiency virus (SHIV)-macaque model based on env genes from primary HIV-1 isolates permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, we and others initially showed that passively infused antibody can protect against an intravenous SHIV-challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. We, therefore, adapted the SHIV89.6PD model to allow evaluation of anti-HIV-1 antibodies against vaginal challenge. In order to make comparisons to our prior intravenous challenge study, we used the same SHIV89.6PD stock and antibodies. Our data show that antibodies can confer protection against vaginal exposure to a pathogenic SHIV; if virus transmission occurs, their presence can ameliorate the subsequent pathogenic manifestations of virus infection. Compared to our previous intravenous challenge study, greater protection was achieved after vaginal challenge. Because the highest level of protection occurred when the most potent combinations of antibodies were used, the data confirm that in vitro neutralization assays on peripheral blood mononuclear cells (PBMC) targets cells are a relevant measure of protective antibody activity.
              Article 0 comments Cited 22 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): BMC Infect Dis
                Title: BMC Infectious Diseases
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1471-2334
                Publication date Collection: 2003
                Publication date (Electronic): 23 December 2003
                Volume: 3
                Page: 30
                Affiliations
                [1 ]Laboratory for Molecular and Cell Immunology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
                [2 ]The School of Medical Technology, Jiangsu University, Zhenjiang, Jiangsu 212001, China
                [3 ]Kunming Chinawave Biotechnology Company Ltd., Kunming, Yunnan 650106, China
                Article
                Publisher ID: 1471-2334-3-30
                DOI: 10.1186/1471-2334-3-30
                PMC ID: 320486
                PubMed ID: 14693038
                SO-VID: e511ffad-3098-4803-a85d-50cf5fb7b832
                Copyright © Copyright © 2003 Zhang et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.
                History
                Date received : 5 October 2003
                Date accepted : 23 December 2003
                Categories
                Subject: Technical Advance

                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: mtt,microtiter neutralization assay,hiv-1,serum or plasma,neutralizing antibody
                Data availability:
                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: mtt, microtiter neutralization assay, hiv-1, serum or plasma, neutralizing antibody

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