Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States.

Serodiagnosis of human immunodeficiency virus (HIV) infection in the United States has traditionally relied on a sequential two-test algorithm: an initial screen with an enzyme immunoassay (EIA) and reflex testing of EIA-reactive specimens with a more specific supplemental test such as Western blotting or immunofluorescence. The supplemental tests are tedious, subjective, and expensive. In addition, there have been major improvements in the performance and accuracy of the EIA tests as well as the introduction of rapid serologic tests (RT) and HIV nucleic acid amplification tests (NAAT). Related to these improvements is the possibility that alternative algorithms using combinations of currently approved HIV tests may function as well as if not better than the current algorithm, with more flexibility, improved accuracy, and lower cost. To this end, we evaluated the performance of 12 currently licensed tests and 1 in-house HIV test (6 EIA, 4 RT, and 3 NAAT) on panels of plasma samples from HIV-infected (n = 621 HIV type 1 [HIV-1] and 34 HIV-2) and uninfected (n = 513) people and of sequential specimens from people early in seroconversion (183 specimens from 15 patients). Test combinations were analyzed in two dual-test (sensitivity-optimized and specificity-optimized) algorithms and in a three-test (tie-breaking) algorithm, and performance was compared to the conventional algorithm. The results indicate that alternative algorithm strategies with currently licensed tests compare favorably with the conventional algorithm in detecting and confirming established HIV infection. Furthermore, there was a lower frequency of discordant or indeterminate results that require follow-up testing, and there was improved detection of early infection.

Cd4+ T cells programmed to traffic to lymph nodes account for increases in numbers of cd4+ T cells up to 1 year after the initiation of highly active antiretroviral therapy for human immunodeficiency virus type 1 infection.

Cells programmed to traffic through lymph nodes dominate initial increases in total CD4(+) T cell numbers after highly active antiretroviral therapy (HAART) is begun for human immunodeficiency virus type 1 (HIV-1) infection. However, it is unknown whether this dominance continues throughout the first year of treatment. To examine this question, 10 subjects who had a positive response to HAART for 1 year were selected from a cohort of 20 who were receiving this treatment. Flow cytometry, which was used to characterize CD4(+) T cell subsets by immunophenotype, demonstrated that cells programmed to traffic through lymph nodes, irrespective of their memory or naive phenotype, continued to best account for increases in CD4(+) T cells, even 1 year after starting HAART. This suggests that, although this pool is preferentially depleted during HIV-1 infection, HAART allows for reaccumulation of these cells for at least 1 year. Furthermore, it suggests that phenotypic differences based on markers of lymphocyte trafficking may be more relevant for understanding HIV-1 pathogenesis than are naive and memory markers alone.

CCR5 and CXCR4 expression on memory and naive T cells in HIV-1 infection and response to highly active antiretroviral therapy.

OBJECTIVE: To measure CCR5 and CXCR4 chemokine receptor expression on CD4 and CD8 T cells in HIV-1 infection and to relate levels to the distribution of CD45RO memory and CD45RA-naive subsets, measures of disease activity, and response to highly active antiretroviral therapy (HAART). DESIGN: Fourteen untreated HIV-1-infected patients, 18 patients at 3-to 4-weeks after beginning HAART, and 35 uninfected control subjects were studied. METHODS: Four-color cytofluorometry with appropriate conjugated monoclonal antibodies (mAbs) was performed to define CD45RA and CD45RO subsets of CD4 and CD8 T cells and measure their expression of CCR5, CXCR4, and CD38. RESULTS: HIV-1-infected patients had higher CCR5 levels and lower CXCR4 levels on CD4 and CD8 T cells and their CD45RO/CD45RA subsets than control subjects did. However, CCR5 elevation was statistically significant only for CD4 T cells and their subsets, and CXCR4 depression was significant for CD8 T cells and their subsets (and for CD4:CD45RO cells). The elevation of CCR5 and depression of CXCR4 were not due to shifts in CD45RO/CD45RA subset proportions but to upregulation or downregulation within the subsets. CCR5 elevation on CD4 T cells was significantly restored toward normal by HAART, but the CXCR4 depression was not. CCR5 expression but not CXCR4 expression correlated with other measures of immunodeficiency (CD4 T-cell levels), active infection (viral load), and cellular activation (CD38). CONCLUSIONS: CCR5 elevation is a concomitant of immune activation and viral replication that occurs in HIV-1 infection, but the relation of CXCR4 depression to severity of infection, disease progression, and response to therapy remains undefined.

Assessment of antibody assays for identifying and distinguishing recent from long-term HIV type 1 infection.

We evaluated 16 antibody assays for their performance in discriminating recent from established HIV-1 infection. These approaches were based on antigen specificity, quantity, conformation dependence, and avidity/affinity of HIV-specific antibodies. A panel of 41 sera from subjects who had seroconverted in the previous 2-6 months (n = 20) and from subjects with established infection (>1 year, n = 21) were run in each assay. Compared with anti-Gag and anti-Pol responses, quantitative anti-Env antibody levels were initially lower and ultimately higher, resulting in the greatest spread and least overlap between incident and established infection. Quantitative measurement included end-point titer in Western blot, end-point titer or response at a given dilution in solid-phase enzyme immunoassays (EIAs) with recombinant proteins or synthetic peptides, and IgG capture assays that reflect the relative proportion of IgG that is anti-HIV antibody. Focusing on the anti-env response, we measured specific responses to the V3 region of gp120, to the CD4-binding site of gp120, to a peptide corresponding to the immunodominant region of gp41, and to conformation-dependent epitopes of gp120. We also measured antibody affinity for gp41 peptide and the relative avidity for gp120 or gp41 peptide by thermal or urea-elution assays. These assays also discriminated recent from established infection but were not necessarily superior to the quantitative anti-Env assays. Appropriate approaches, based on distinct principles or combination of principles, can be used to develop simple assays for identifying individuals recently infected with HIV-1.

Markers of lymphocyte homing distinguish CD4 T cell subsets that turn over in response to HIV-1 infection in humans.

In HIV-1 infection, the abrupt rise in CD4 T cells after effective antiretroviral therapy has been viewed as a measure of HIV-1-related CD4 T cell turnover in the steady state. The early (2-4 wk) response is reportedly dominated by CD4 T cells with a memory (CD45RO) phenotype. It is controversial whether the measurement of steady-state kinetics identifies cells that otherwise would have been recruited into a short-lived, virus-producing pool or reflects lymphoid redistribution/sequestration. We performed detailed phenotypic and kinetic analysis of CD4 T cell subsets in 14 patients. Turnover occurs in memory (CD45RO) as well as naive (CD45RA) cells, if the latter are present at baseline. Most of the turnover occurs in those memory (CD45RO) and naive (CD45RA) cells that are programmed for recirculation through lymphoid organs (CD62L+ and CD44low), whereas very little turnover occurs in memory cells (CD45RO) destined for recirculation from blood to tissue (CD62L- and CD44high). Turnover occurs in both activated (CD25+ and HLA-DR+) and nonactivated populations, although it is restricted to CD38-positive cells, indicating that turnover does not measure cells that are already infected. More likely, turnover occurs in cells that replace infected cells or are on their way to becoming infected. Taken together, markers of lymphocyte trafficking better describe cell turnover related to virus replication than do naive and memory markers per se, and lymph organs, not tissue-destined cells or peripheral blood cells, appear to be the important site of virus replication and CD4 T cell turnover, destruction, and redistribution.

Lymphocyte kinetics and precursor frequency-dependent recovery of CD4(+)CD45RA(+)CD62L(+) naive T cells following triple-drug therapy for HIV type 1 infection.

New therapeutic regimens have dramatically altered morbidity and mortality attributed to HIV-1 infection. Changes in lymphocyte subsets after treatment may mirror salutary clinical changes. Over 4 months we analyzed lymphocyte subsets in 20 patients starting new HIV-1 therapy. Absolute numbers of lymphocytes, CD4+ T cells, CD8+ T cells, and B cells increased significantly by 4 months, but CD8+ T cell and B cell increases were restricted to late-stage patients. Subset analysis revealed that the magnitude of recovering naive-phenotype CD4+ T cells (slope) correlated with the number of these cells present at baseline, equaling or exceeding the memory-phenotype slope within days if these naive cells were abundant at baseline. Five of 10 patients in whom naive-phenotype CD4+ T cells were absent at baseline partially repopulated these cells by 4 months. These findings have important implications for the origin and mechanisms of renewal of naive-phenotype CD4+ T cells following effective treatment for HIV-1 infection.

A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity.

OBJECTIVE: To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. METHODS: Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti-double-stranded DNA (anti-dsDNA), anti-single-stranded DNA, antihistone, anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 (DNA topoisomerase I), anticentromere, and anti-Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. RESULTS: Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anticentromere, and anti-Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. CONCLUSION: No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases.

Neutralizing antibody and perinatal transmission of human immunodeficiency virus type 1. New York City Perinatal HIV Transmission Collaborative Study Group.

The major immunologic determinants for perinatal transmission of human immunodeficiency virus type 1 (HIV-1) remain largely unknown. The presence of maternal neutralizing antibodies has been proposed as an explanation for why the majority of infants born to untreated HIV-1-infected women do not become infected. Using maternal and infant specimens collected as part of a longitudinal cohort study of perinatal transmission in New York City between 1991 and 1995, we successfully obtained primary viral isolates from 10 of 20 perinatally nontransmitting (NTR) women, 14 of 20 perinatally transmitting (TR) women, and 13 of 13 of their HIV-1-infected infants. Neutralizing antibody titers were then determined using a titer reduction assay. TR and NTR women did not differ in their ability to neutralize autologous virus or laboratory strains LAI and MN. Infant viruses were not less sensitive to neutralization by maternal sera than autologous viruses. Similarly, TR and NTR isolates were neutralized equally well using a reference serum with broad neutralizing ability. Finally, a heteroduplex tracking assay (HTA) was used to analyze the degree of viral homology within 13 TR maternal-infant pairs. In eight pairs, maternal and infant isolates were highly homologous. In five pairs, lesser degrees of homology were observed, consistent with perinatal transmission of a minor species. However, these isolates were no more or less resistant to maternal sera than were homologous isolates. Thus we found no association between the presence of neutralizing antibody in maternal sera as measured by a titer reduction neutralization (inactivation) assay and perinatal transmission of HIV-1.

Range of antinuclear antibodies in "healthy" individuals.

OBJECTIVE: To determine the range of antinuclear antibodies (ANA) in "healthy" individuals compared with that in patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc; scleroderma), Sjögren's syndrome (SS), rheumatoid arthritis (RA), or soft tissue rheumatism (STR). METHODS: Fifteen international laboratories experienced in performing tests for ANA by indirect immunofluorescence participated in analyzing coded sera from healthy individuals and from patients in the 5 different disease groups described above. Except for the stipulation that HEp-2 cells should be used as substrate, each laboratory used its own in-house methodology so that the data might be expected to reflect the output of a cross-section of worldwide ANA reference laboratories. The sera were analyzed at 4 dilutions: 1:40, 1:80, 1:160, and 1:320. RESULTS: In healthy individuals, the frequency of ANA did not differ significantly across the 4 age subgroups spanning 20-60 years of age. This putatively normal population was ANA positive in 31.7% of individuals at 1:40 serum dilution, 13.3% at 1:80, 5.0% at 1:160, and 3.3% at 1:320. In comparison with the findings among the disease groups, a low cutoff point at 1:40 serum dilution (high sensitivity, low specificity) could have diagnostic value, since it would classify virtually all patients with SLE, SSc, or SS as ANA positive. Conversely, a high positive cutoff at 1:160 serum dilution (high specificity, low sensitivity) would be useful to confirm the presence of disease in only a portion of cases, but would be likely to exclude 95% of normal individuals. CONCLUSION: It is recommended that laboratories performing immunofluorescent ANA tests should report results at both the 1:40 and 1:160 dilutions, and should supply information on the percentage of normal individuals who are positive at these dilutions. A low-titer ANA is not necessarily insignificant and might depend on at least 4 specific factors. ANA assays can be a useful discriminant in recognizing certain disease conditions, but can create misunderstanding when the limitations are not fully appreciated.

Mechanisms of human immunodeficiency virus Type 1 (HIV-1) neutralization: irreversible inactivation of infectivity by anti-HIV-1 antibody.

An assay for the neutralization of human immunodeficiency virus type 1 (HIV-1) is described in which the reduction in infectious titer of HIV-1 after preincubation at 37 degrees C with antibody-positive serum is the measure of neutralization. The assay format and its controls allow several experimental manipulations that, taken together, indicate an effect of antibody on HIV-1 infectivity that occurs before or independently of HIV-1 attachment. The direct inactivation of HIV-1 infectivity by antibody is irreversible and temperature dependent, requires a bivalent antibody directed against accessible envelope determinants, and does not require a heat-labile or (Ca2+)- or (Mg2+)-dependent cofactor. The mechanism of inactivation cannot be explained by agglutination of virus, nor is it associated with disruption or dissociation of envelope protein from virions. Rather, the antibody is likely to perturb some metastable property of the envelope that is required for entry. Laboratory-adapted HIV-1 isolates were more sensitive to the inactivating effects of sera than were primary patient isolates. The latter were particularly resistant to inactivation by contemporary autologous sera, a feature not explained by blocking antibodies. Additional studies showed a weak relationship between disease course and serum inactivation of the reference LAI laboratory strain of HIV-1. Heteroduplex analysis and autologous inactivation assays of sequential specimens from individual patients indicate that over time, the viral quasispecies that emerge and dominate are resistant to the inactivating effects of earlier sera.

Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates.

CD4-IgG2 is a novel fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. This tetrameric protein is being developed as an immunoprophylactic agent to reduce the probability of infection following HIV-1 exposure, in settings such as occupational or perinatal exposure to the virus. CD4-IgG2 has been expressed in Chinese hamster ovary cells and is secreted as a fully assembled heterotetramer. The protein binds with nanomolar affinity to purified gp120 from both a laboratory-adapted strain and a primary isolate of HIV-1. Pharmacokinetic studies in rabbits demonstrated that CD4-IgG2 has a plasma terminal half-life greater than 1 day, compared with 15 min for soluble CD4 (sCD4). CD4-IgG2 does not bind to Fc receptors on the surface of U937 monocyte/macrophage cells. Compared to molecules that incorporate the Fc portion of IgG1, CD4-IgG2 has less potential to mediate functions such as antibody-dependent enhancement of infection or transplacental transmission of HIV-1. When tested in a virus-free HIV-1 envelope glycoprotein-mediated cell fusion assay, the tetrameric CD4-IgG2 molecule inhibited syncytium formation more effectively than monomeric sCD4 or a dimeric CD4-gamma 2 fusion protein. This suggests the protein will block cell-to-cell transmission of HIV-1. Moreover, CD4-IgG2 effectively neutralized a panel of laboratory-adapted strains and primary isolates of HIV-1, including strains with different tropisms and isolated from different stages of the disease, at concentrations that should be readily achieved in vivo.

Increase in sensitivity to soluble CD4 by primary HIV type 1 isolates after passage through C8166 cells: association with sequence differences in the first constant (C1) region of glycoprotein 120.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) were obtained by coculture of peripheral blood lymphocytes (PBLs) from HIV-1-infected people with PBLs from uninfected donors. These viral stocks tend to be resistant to neutralization/inactivation by soluble CD4 (sCD4). When these stocks were passed through the T cell line C8166, virus stocks emerged that were sensitive to sCD4. Pre- and post-C8166 stocks maintained their sCD4-resistant and -sensitive phenotypes, respectively, with further passage in PBLs. Pre- and post-C8166 stocks were biologically cloned by two cycles of limiting dilution. The majority (14 of 17) of pre-C8166 clones were sCD4 resistant, and, conversely, the majority of post-C8166 clones (11 of 12) were sensitive to sCD4. Nucleotide and predicted amino acid sequence analysis in the env (gp120) region revealed a limited number of differences between the clones. The only differences that sorted with biological phenotype were in the first constant (C1) region of gp120. Adaptation to growth in C8166 cells and conversion from the sCD4-resistant to the sCD4-sensitive phenotype represent the emergence to prominence of viral species in the pre-C8166 stock that have a replication advantage in C8166 coincident with increased sensitivity to sCD4.

The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes.

The C-nitroso compound 3-nitrosobenzamide, which has been shown to remove zinc from the retroviral-type zinc finger of p7NC nucleocapsid proteins, inhibits acute infection of human immunodeficiency virus type 1 in cultured human lymphocytes. The attachment of the virus to lymphocytes and the activities of critical viral enzymes, such as reverse transcriptase, protease, and integrase, are not affected by 3-nitrosobenzamide. However, the process of reverse transcription to form proviral DNA is effectively abolished by the drug, identifying the mode of action of 3-nitrosobenzamide as interrupting the role of p7NC in accurate proviral DNA synthesis during the infectious phase of the virus life cycle.

Cross-reactivity of 75 monoclonal antibodies to human immunoglobulin with sera of non-human primates.

We systematically analyzed a panel of 75 murine monoclonal antibodies (mAbs) reactive with human immunoglobulins IgG, IgA, IgM, IgD, and kappa and lambda light chains for reactivity with serum immunoglobulins of higher primates. In the great apes, and to a lesser extent in other primates, epitopes related to human light chains, IgM, IgA, IgD, and all 4 IgG subclasses were identified with many of the mAbs. Those mAbs identified as reactive with a given species may be useful for immunologic studies of these species. Cladistic analysis of antigenic relatedness generated a phylogenetic tree consistent with current anatomic or molecular taxonomies.

Inactivation of HIV-infected H9 cells in whole blood preparations by lysing/fixing reagents used in flow cytometry.

Reagents that lyse red blood cells and fix white blood cells were tested for their ability to inactivate cell-associated human immunodeficiency virus (HIV). Whole blood was spiked with cells from an HIV-positive cell line (H9), lysed, and fixed. The cell preparations were then cocultured with T cell blasts in serial ten-fold dilutions to rescue infectious virus and measure viral titer. All commercial lysing and fixing reagents tested inactivated cell-associated HIV by 3-5 logs, while ammonium chloride had little effect. Although an additional incubation with 1% formaldehyde for 30 min did not increase the effectiveness of the commercial lysing/fixing reagents, it did inactivate cell-associated HIV in blood treated with ammonium chloride.

Inactivation of human immunodeficiency virus type I in human milk: effects of intrinsic factors in human milk and of pasteurization.

Human milk was inoculated with human immunodeficiency virus type I (HIV-1) or with HIV-1-infected cells in volumes and containers typically used in human milk banks. The inoculated milk was pasteurized at 62.5 degrees C for 30 minutes in a water bath, i.e., conditions currently in use or proposed for human milk pasteurization. The process of HIV-1 inoculation and pasteurization effectively inactivated the infectivity of both cell-free HIV-1 and HIV-1-infected cells. No virus was recovered after the process, even after repeated subculturing in attempts to rescue the virus. Pasteurization reduced the infectious titer of cell-free HIV-1 and HIV-1-infected cells by more than 5 logs and 6 logs respectively. Human milk contains one or more components that inactive HIV-1 but that are not toxic for the cells used to replicate virus. These components have not been identified, but physical and solubility properties are consistent with characteristics of lipids.

Two mechanisms of soluble CD4 (sCD4)-mediated inhibition of human immunodeficiency virus type 1 (HIV-1) infectivity and their relation to primary HIV-1 isolates with reduced sensitivity to sCD4.

Two assays for measuring inhibition of human immunodeficiency virus type 1 (HIV-1) infection by soluble CD4 (sCD4) are described. Experiments in which sCD4, HIV-1, and cell concentrations and sequence of combination, noninfectious/infectious particle ratio, and temperature were varied produced results that support the conclusion that sCD4 inhibits HIV-1 infection by two mechanisms: reversible blockage of receptor binding and irreversible inactivation of infectivity. Fresh isolates obtained from HIV-1-infected persons were tested in both assays and found to be more resistant to both mechanisms of sCD4-mediated inhibition than multiply passaged laboratory strains. Binding studies revealed similar affinities for sCD4 in detergent lysates of sensitive and resistant strains at both 4 and 37 degrees C. The avidity of intact virions for sCD4 was lower at 4 than at 37 degrees C, and in the presence of excess sCD4, less sCD4 was bound at 4 than at 37 degrees C. The avidity differences were similar for fresh isolates and laboratory strains. However, fresh isolates were more resistant to sCD4-induced shedding of envelope glycoprotein gp120 from intact virions than was the laboratory strain. Relative resistance to sCD4 by certain isolates does not represent a lower intrinsic affinity of their envelope for sCD4 or a lower capacity for sCD4 binding. Rather, an event that occurs after binding may account for the differences. This postbinding event or feature may be determined by regions of the envelope outside the CD4 binding site.

Inhibition of HIV-1 infectivity by zinc-ejecting aromatic C-nitroso compounds.

Retroviral nucleocapsid and gag-precursor proteins from all known strains of retroviruses contain one or two copies of an invariant sequence, Cys-X2-Cys-X4-His-X4-Cys, that is populated with zinc in mature particles. Modification of cysteine or histidine residues results in defective packaging of genomic viral RNA and formation of non-infectious particles, making these structures potentially attractive targets for antiviral therapy. We recently reported that aromatic C-nitroso ligands of poly(ADP-ribose) polymerase preferentially destabilize one of the two (Cys-X2-Cys-X28-His-X2-Cys) zinc-fingers with concomitant loss of enzymatic activity, coincidental with selective cytocidal action of the C-nitroso substituted ligands on cancer cells. Based on the occurrence of (3Cys, 1His) zinc-binding sites in both retroviral nucleocapsid and gag proteins and in poly(ADP-ribose) polymerase, we reasoned that the C-nitroso compounds may also have antiretroviral effects. We show here that two such compounds, 3-nitrosobenzamide and 6-nitroso-1,2-benzopyrone, inhibit infection of human immunodeficiency virus HIV-1 in human lymphocytes and also eject zinc from isoalted HIV-1 nucleocapsid zinc fingers and from intact HIV-1 virions. Thus the design of zinc-ejecting agents that target retroviral zinc fingers represents a new approach to the chemotherapy of AIDS.