Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1.

This study investigates whether cell-derived glycosylphosphatidylinositol-linked complement control proteins CD55 and CD59 can be incorporated into HIV-1 virions and contribute to complement resistance. Virus was prepared by transfection of cell lines with pNL4-3, and primary isolates of HIV-1 were derived from patients' PBMCs. Virus was tested for sensitivity to complement-mediated virolysis in the presence of anti-gp160 antibody. Viral preparations from JY33 cells, which lack CD55 and CD59, were highly sensitive to complement. HIV-1 preparations from H9 and U937 cells, which express low levels of CD55 and CD59, had intermediate to high sensitivity while other cell line-derived viruses and primary isolates of HIV-1 were resistant to complement-mediated virolysis. Although the primary isolates were not lysed, they activated complement as measured by binding to a complement receptor positive cell line. While the primary isolates were resistant to lysis in the presence of HIV-specific antibody, antibody to CD59 induced lysis. Likewise, antibody to CD55 and CD59 induced lysis of cell line-derived virus. Western blot analysis of purified virus showed bands corresponding to CD55 and CD59. Phosphatidylinositol-specific phospholipase C treatment of either cell line-derived or primary isolates of HIV-1 increased sensitivity to complement while incubation of sensitive virus with purified CD55 and CD59 increased resistance to complement. These results show that CD55 and CD59 are incorporated into HIV-1 particles and function to protect virions from complement-mediated destruction, and they are the first report of host cell proteins functioning in protection of HIV-1 from immune effector mechanisms.

Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies.

Characterization of the sites recognized by antibody on the V3 loop of the envelope glycoprotein gp120 of HIV-1 was done by competition ELISAs on a series of four mouse mAbs, a human mAb and a human Fab. The solid-phase antigen consisted of biotin-YNKRK-RIHIGPGRAFYTTKN, a sequence from the center of the V3 loop of gp120MN, applied to streptavidin-coated wells. Competing antigens were two series of peptides with the HIV-1MN sequence each serially deleted at either the N or C terminus but kept constant at the other terminus. For each series, the amino acid at the deleting end needed to give a minimum KD was identified. The epitope was defined as the sequence including both of the identified amino acids as terminal amino acids. For the six antibodies reported, the epitope length ranged from seven to 14 amino acids. Use of a cyclic peptide as competing fluid-phase antigen suggested the influence of conformational constraints on presumed "linear" epitopes. The operationally-defined epitope was longer than the contact residues in one of two instances in which the X-ray crystallographic structure had been determined. The longer estimates of epitope length in the current study based on competition ELISAs with serial deletions suggest that non-contact residues are significant both in epitope definition and in functional applications including immunogen design.

Presentation of HIV V3 loop epitopes for enhanced antigenicity, immunogenicity and diagnostic potential.

OBJECTIVE: To evaluate the immunological properties of a panel of human mucin MUC1/HIV V3 loop chimeras. DESIGN: The immunodominant epitope of MUC1 (APDTR) was found to be structurally isomorphous with the tip of the principle neutralizing determinant (PND) of HIV-1 (MN) (GPGRA). A panel of 120 residue, six tandem repeat (TR) and 60 residue, three TR chimeric antigens were constructed in which the repeating MUC1 epitope is replaced by HIV-1 PND. Each 20 residue TR contains one PND epitope. The PND of HIV-1 is presented in the native beta-turn conformation at the crest of each repeating knob structure of the mucin-like protein. METHODS: The antigenicity of the chimeric antigens were compared using enzyme-linked immunosorbent assay (ELISA) and HIV-infected patient sera. Structural effects of antibody-antigen interactions were determined using surface plasmon resonance, with human monoclonal antibodies, chimeric antigens and the cyclic and linear V3 loops. Immunogenicity of three versus six TR was measured in mice. RESULTS: Nine residues of the HIV PND substituted into the mucin backbone were equivalent to the 36 residue cyclic V3 loop in ELISA. The 120 residue antigens induced high titer, immunoglobulin (Ig) M and IgG, and HIV-specific antibodies in mice. CONCLUSIONS: MUC1/V3 chimeras efficiently detect HIV-specific antibodies in patient sera. Multivalent presentation of the PND is advantageous for higher affinity antibody-antigen interactions and for inducing HIV-specific IgM and IgG antibodies.

Model-based optimization of infectivity parameters: a study of the early epidemic in San Francisco.

By combining dynamic modeling of human immunodeficiency virus (HIV) transmission with mathematical optimization techniques, we calculate values of epidemiological parameters characterizing the early epidemic (1978-1986) among homosexual and bisexual men in San Francisco. The seroconversion fraction data reported by the San Francisco hepatitis B vaccine trials cohort study for this period is accurately simulated by a model assuming varying infectivity among three stages of HIV infection (early antigen stage, HIV antibody-positive stage, and AIDS stage). Using optimization techniques, we generate curves of the annual number of new partners and the annual number of risk contacts as functions of time. We project future case rates using optimized parameter values, and we study the sensitivity of these projections to variations in parameters, including the population size and the incubation period.

Studies with monoclonal antibodies to the V3 region of HIV-1 gp120 reveal limitations to the utility of solid-phase peptide binding assays.

Using human monoclonal antibodies (HuMAbs) r(1)-447 (L-736,523) and 19b to the V3 region of HIV-1 gp120, we have explored epitope presentation on V3-peptides and on the corresponding gp120 proteins. HuMAb r(1)-447 binds strongly to the MN and SF-2 peptides and gp120 proteins. In contrast, while this HuMAb binds equally avidly to both the HxB2 and the BRU/BH10 peptides, it binds but weakly to the HxB2 V3 loop on gp120 and fails to bind at all to BH10 gp120. Thus, the solid-phase peptide binding assay can falsely predict reactivity of an MAb with a gp120 protein. Conversely, HuMAb 19b fails to bind to a peptide from the V3 loop of HIV-1 AD-6 in solid-phase assays, but binds to the same peptide in solution and also to AD-6 gp120. Thus, the solid-phase peptide binding assay can fail to predict reactivity of an MAb with a gp120 protein. Furthermore, serum antibodies from individual AD-6 do not react well with the AD-6 V3-peptide in a solid-phase assay, but react strongly with the corresponding MN V3-peptide. On the basis of peptide binding assays, we had assumed that the AD-6 virus was "MN-like" with a prototypic North American/European subtype B GPGR motif at the crown of the V3 loop. However, direct sequencing demonstrates that the AD-6 V3 loop contains a variant GPGK motif. This highlights a limitation of V3-peptide-based assays for serotyping viruses.

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function.

We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens.

An immuno-dot blot assay for the detection of antibody to HIV.

This paper describes a simple and rapid immuno-dot blot assay for the detection of antibody to HIV. It utilizes nanogram quantities of HIV lysate, immobilized on nitrocellulose paper, and microliter quantities of serum or culture supernatants for the detection of antibody. The test is highly sensitive and specific. It offers the opportunity to perform multiple assays at a minimal cost and provides the additional advantage of not requiring any sophisticated or expensive equipment to perform the test or interpret the results.

Characterization of human monoclonal antibodies selected with a hypervariable loop-deleted recombinant HIV-1(IIIB) gp120.

Recombinant gp120 of the HIV-1(IIIB) isolate (BH10 clone) has been mutated to form the PR12 protein with the first 74 C-terminal amino acids and the V1, V2 and V3 hypervariable loops deleted. A variety of studies have shown that the CD4 binding domain (CD4bd) is very well exposed in PR12 in contrast to rgp120(LAI). Using PR12 for selection of human monoclonal antibodies (MAbs) from HIV-infected individuals, five MAbs were generated with specificities to the epitopes overlapping the CD4bd (1570A,1570C,1570D,1595 and 1599). The three MAbs, 1570A, C and D, generated from one HIV-infected individual, represent one MAb as determined by sequence analysis of the V(H)3 region. Since the epitopes overlapping the CD4bd exhibit variability among HIV-1 clades, the specificity of anti-CD4bd MAbs were distinguished by differing patterns of binding to recombinant envelope proteins derived from clade A, B, C, D and E viruses. The PR12-selected MAbs were also compared with a panel of gp120-selected anti-CD4bd MAbs and showed a different range of specificities. MAb 1599 is clade B specific, MAb 1595 reacts with the A, B and D clades, while MAb 1570 recognises the most conserved epitope, as it binds to all proteins. The results show that the exposure of different epitopes in the CD4bd of the PR12 protein allows this protein to serve as an immunogen and to induce anti-CD4bd antibodies.

Production of human monoclonal antibodies specific for conformational and linear non-V3 epitopes of gp120.

Three IgG1 human monoclonal antibodies (MAbs) directed against conformational epitopes of the gp120 envelope protein of HIV-1 were produced, as was a single human MAb to a linear epitope spanning amino acids 487-509 in the C-terminal portion of gp120. All three conformation-dependent MAbs reacted optimally with recombinant gp120 (rgp120) captured on plastic via its carbohydrate moieties with Concanavalin A. These MAbs were able to block the interaction between recombinant CD4 (rCD4) and rgp120; they were also able to achieve 50% neutralization of HTLV-IIIB and MN strains of HIV-1 in a concentration range of 0.5-12.8 micrograms/mL. The MAb to the linear determinant is the first reported human MAb specific for the immunodominant portion of gp120; this MAb was most reactive with rgp120 when it was coated directly on plastic. It could neither inhibit rCD4-rgp120 binding nor neutralize either HTLV-IIIB or MN. The binding affinities of the four human MAbs for rgp120 in solution, reflected by their dissociation constants (Kd), ranged from 0.5 x 10(-8) to 7.5 x 10(-8) M.

Functional activities of 20 human immunodeficiency virus type 1 (HIV-1)-specific human monoclonal antibodies.

Antibodies that are useful in the treatment of HIV infection should result in virus neutralization or lysis of infected cells but should not enhance infection. In this study, the potential clinical use of 20 HIV-1-specific human monoclonal antibodies (HuMAbs) was determined by measuring their enhancing (C-ADE) activities using HIVLAI as the target virus. Two HuMAbs mediated both C-ADE and ADCC, two exclusively neutralized, and five exclusively mediated ADCC. Ten HuMAbs demonstrated no activity in any of the three assays. Three antibodies that neutralized HIVLAI were tested against HIVSF2; all three also neutralized HIVSF2. Four of five HuMAbs mediating ADCC against HIVLAI that were also tested against HIVSF2 had ADCC activity against HIVSF2. These results demonstrate that many HuMAbs have unique functions, allowing the separation of potentially beneficial and harmful activities. Combinations of HuMAbs with ADCC and neutralizing functions may have therapeutic utility.

Anti-CD4-binding domain antibodies complexed with HIV type 1 glycoprotein 120 inhibit CD4+ T cell-proliferative responses to glycoprotein 120.

HIV-specific CD4+ helper T cell responses, particularly to the envelope glycoproteins, are usually weak or absent in the majority of HIV-seropositive individuals. Since antibodies, by their capacity to alter antigen uptake and processing, are known to have modulatory effects on CD4+ T cell responses, we investigated the effect of antibodies produced by HIV-infected individuals on the CD4+ T cell response to HIV-1 gp120. Proliferative responses of gp120-specific CD4+ T cells were inhibited in the presence of either serum immunoglobulin from HIV-infected individuals or human monoclonal antibodies specific for the CD4-binding domain (CD4bd) of gp120. Human monoclonal antibodies to other gp120 epitopes did not have the same effect. The anti-CD4bd antibodies complexed with gp120 suppressed T cell lines specific for varying gp120 epitopes but did not affect T cell proliferation to non-HIV antigens. Moreover, inhibition by the anti-CD4bd/gp120 complexes was observed regardless of the types of antigen-presenting cells used to stimulate the T cells. These results indicate that the presence of anti-CD4bd antibodies complexed with gp120 can strongly suppress CD4+ helper T responses to gp120.

A human monoclonal antibody specific for the V3 loop of HIV type 1 clade E cross-reacts with other HIV type 1 clades.

To ascertain the antigenic relationship between HIV-1 viruses belonging to various genetically defined subgroups (clades), shared epitopes need to be defined. Human monoclonal antibodies (MAbs) are particularly useful for this purpose because they can detect complex regions of viral proteins that may be missed by sequence analysis and because, by definition, they react with epitopes that stimulate the human immune system. Monoclonal antibodies derived from the cells of HIV-1 clade B-infected subjects have been used extensively for this purpose. Here we describe the first human MAb derived from a clade E-infected individual; the MAb is specific for the V3 loop, recognizing a core epitope represented by the amino acids TRTSVR on the N-terminal side of the crown of the V3 loop. The IgG1(kappa) MAb, designated 1324E, binds to the clade E consensus V3 loop, to rgp120 proteins from clade E and to peripheral blood mononuclear cells infected in vitro with the virus that infected the subject from whose cells the MAb-producing heterohybridoma was derived. Strong cross-reactivity of the MAb to the V3 peptides, rgp120 proteins, and native monomeric gp120s representing clades A and C, as well as to cells infected with a clade C primary isolate, revealed a shared V3 epitope between these clades. When tested for its neutralizing ability, MAb 1324E neutralized a clade E isolate that had been adapted for growth in H9 cells but failed to neutralize five clade E primary isolates.

Anti-human immunodeficiency virus type 1 human monoclonal antibodies that bind discontinuous epitopes in the viral glycoproteins can identify mimotopes from recombinant phage peptide display libraries.

A phage display library screening approach was used to identify peptide sequences that could bind to anti-HIV-1 MAbs whose binding specificities are complex. Most of the antibodies used recognize discontinuous epitopes in gp120 and one recognizes gp41. Both a 15-mer and a 21-mer display library (each with a complexity of greater than 60 x 10[6]) and two constrained, V3 region-biased libraries, all expressed as recombinant pIII protein of filamentous phage, were used. The unmapped anti-gp120 human MAb A32 recognized a set of related linear sequences and repeatedly identified a single phage sequence that could form a cyclic disulfide structure. Selection methods were also developed so that phage could be obtained by competition selection in the presence of antibody bound to native, monomeric gp120 antigen (used with MAb IgG1b12 and the anti-gp120 V3 region MAb 447-52D) or gp120 variable region 3 synthetic peptides (used with anti-gp120 V3 region MAb 19b). The potent, virus-neutralizing MAb IgG1b12 recognized numerous sequences and, when used in competition with gp120, recognized only one sequence. These studies extend the range of antibody determinant studies that can be performed with display phage libraries, demonstrate a workable experimental strategy for use of competition ligands to discriminate among phage mimotopes, and provide a large number of mimotopes that bind potent virus-neutralizing MAbs for HIV-1 vaccine studies.

Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins.

A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo.

Production of immunoglobulins and alloantibodies by lymphocytes from lung cancer patients.

Spontaneous and PWM stimulated production of immunoglobulins (Igs) were measured in culture of lymphocytes isolated from peripheral blood (PBL) of lung cancer patients, blood donors and patients with benign lung diseases. Culture supernatants were tested for antibody activity against lung cancer cells and normal cells and some of them were absorbed with normal lung cells and fetal liver cells. Igs levels and antibody activity in culture supernatants were measured by radioimmunometric assay. Significantly increased level of Igs in unstimulated culture of PBL and low index of PWM-stimulated production of Igs was found in lung cancer patients in contrast to the controls. Supernatants of PBL cultures from lung cancer patients had significant low antibody activity to lung cancer cells and were effectively absorbed by normal lung cells and fetal liver cells. The results suggest in vivo polyclonal activation of B cells in patients with lung cancer and simultaneously diminished production of alloantibodies with some features of natural antibodies following polyclonal activation in vitro.

Immunoregulatory effect of T cell subsets on PWM-induced IgG synthesis by blood lymphocytes in lung cancer.

The mechanism of pokeweed mitogen (PWM) dependent decreased IgG production by blood lymphocytes from lung cancer patients was studied in comparison to control patients and blood donors. It has been shown that the depletion of monocytes has some influence on IgG synthesis but is not a decisive factor. Also, quantitative alterations in the CD4 and CD8 lymphocyte subsets do not significantly influence the PWM stimulation index for IgG synthesis. The assessment of T lymphocyte suppressor activity in lung cancer patients was performed by means of a co-culture with blood mononuclear cells, while helper activity was evaluated through co-culture with donor B lymphocytes. It has been found that lung cancer patient T lymphocytes have no increased suppressor activity, however, especially in the CD4 subset, display the decrease of helper function for B lymphocytes in PWM-induced IgG synthesis. The weakened helper function of CD4 lymphocytes may explain the suppression of specific antibody synthesis do novo which is evident in patients with lung cancer.

Identification of a tumor-related protein antigen in immune complexes derived from pleural effusions of patients with bronchial carcinoma.

Pleural effusions from 15 patients with advanced primary bronchial carcinoma, from 2 patients with metastatic lung cancer and from 6 patients with nonmalignant disease were studied. Immune complexes were found in examined fluids in amounts corresponding to 2.5-210 mg/100 ml of aggregated IgG by means of ELISA solid phase anti C3 and 125ICIq binding radioimmunoassay. Following determination of protein content and salting out by ammonium sulfate of examined fluids, the sediments were subjected to subsequent chromatographic procedure including molecular sieving (Sephadex G-200, Sepharose 4B) and affinity chromatography on Protein A-Sepharose CL-4B. The yield--apparently pure immune complexes--was then split by means of chaotropic agent 2.5 M KSCN. It permitted to obtain 2 fractions: one contained IgG while the other was a non-Ig protein of m. w. = 150 000. The latter isolated from malignant effusions possessed antigenic activity in the leukocyte migration inhibition (LMI) assay. It resulted in inhibition of migration of allogenic peripheral blood leukocytes from lung cancer patients in 87% of cases. It had no activity against leukocytes from nonmalignant disease patients. LMI activity of the final second fraction derived from malignant effusion was significantly different from that of other fractions obtained both from malignant and nonmalignant fluids.

Anti-tumor antibodies in lung cancer patients. Immunofluorescence study using various indicator cells.

By means of indirect immunofluorescence a number of primary lung cancer patient sera and control sera were tested for anti-tumor antibody activity on living tumor cells as a substrate. Antibodies against surface antigens were the most frequently detected in autologous system (in 65%) on cells derived from fresh surgical material of lung cancer. They were also found in 50% of cases using tumor cells from primary short-term culture. When established cell line of lung cancer was used (E-14) in allogeneic system, the antibodies were detected in only 22% of examined lung cancer sera. Absorption of positive sera with homogenates of normal tissues did not abolish their specific activity. Positive reactions were confined to squamous cell type of bronchogenic carcinoma.

Incidence of alloantibodies and immune complexes in bronchial secretions of lung cancer patients.

Bronchial secretions may accumulate immunoglobulins (Igs) produced by lymphocytes infiltrating tumor and its vicinity, and tumor associated antigens to higher degree than cancer patient serum. All three classes of Igs were increased in bronchial secretions from lung cancer patients as compared to control patients. Similar relationships were found for IgG and IgA/albumin ratios and IgG and IgA indices. The level of alloantibodies to fetal cells (measured by immunoradiometric--IRMA--assay) and immune complexes (IC, measured by IRMA--Clq and ELISA--anti C3) were significantly higher in lung cancer patients versus control patients in bronchial secretions. The differences were similar in sera but they were not significant. On the contrary, alloantibody binding to tumor cells was higher in control group. Absorption experiments with normal cells abolished antitumor and antifetal activity, what indicates natural character of these alloantibodies. Significant correlations between alloantibody to fetal cells and IC levels indicate that fetal antigens may contribute to antigenic component of IC. These findings suggest the link of alloantibodies and IC levels in bronchial secretions with neoplastic growth.

Evaluation of phenotype of mononuclear host cells isolated from primary tumour and peripheral blood of patients with laryngeal carcinoma.

Mononuclear host cells isolated from primary laryngeal carcinoma were assessed by means of indirect immunofluorescence with a panel of monoclonal antibodies against various lymphocyte subsets and macrophages. Tumours of various staging groups were examined in parallel with cells isolated from patient and donor peripheral blood (PBL). It was found that percentage values of cells bearing T3 and T4 phenotype were reduced both in tumour infiltrating cells (TIC) and in PBL population. The fall in T4+ cells in PBL from cancer patients in T3 and T4 staging groups was statistically significant (p less than 0.01) as compared with donor cells. Corresponding values for T8+ cells from TIC were increased in T1 and T2 staging groups of cancer, but showed a gradual fall in advanced stages. The T4+/T8+ cell ratio was decreased in both TIC and PBL cells. The HNK-1+ (NK) cell pattern in TIC was analogous to that for T8+ cells, i.e. the cell percentage decreased with advance in tumour growth. Corresponding values for OKM-1+ were increased in TIC and in patient blood, though in TIC they grew in proportion to tumour growth. Ia+ (HLA-DR+) cells in peripheral blood were significantly increased in patients versus those of donors (p less than 0.01), but only in T3 and T4 staging groups of examined cancer. These results show that subsets of tumour infiltrating cells in laryngeal carcinoma are a complex phenomenon, associated with growth and progression of tumour.