Generation of human monoclonal antibodies to human immunodeficiency virus.

Based on the finding that cells producing antibodies to human immunodeficiency virus (HIV) circulate in the peripheral blood of HIV-infected individuals, attempts were made to immortalize such B cells with Epstein-Barr virus. Mononuclear cells from 58 HIV-seropositive subjects at various stages of HIV infection were transformed, and anti-HIV cell lines were derived from 4 subjects, all of whom were in early stages of infection. Seven of these cell lines have been stable with respect to antibody production for up to 15 months. Three lines are producing IgG antibody to the 41-kDa HIV transmembrane glycoprotein gp41 and 4 produce IgG antibodies to the 24-kDa HIV core protein p24, its precursors and a breakdown product. The antibodies are reactive by ELISA, by radioimmunoprecipitation, and by Western blot, demonstrating the feasibility of producing multiple stable cell lines synthesizing human monoclonal antibodies to HIV by immortalization of peripheral blood cells with Epstein-Barr virus.     

Phase I study of low-dose zidovudine and acyclovir in asymptomatic human immunodeficiency virus seropositive individuals

PURPOSE: The combination of zidovudine and acyclovir has shown in vitro antiretroviral activity and led to short-term improvement in patients with symptomatic human immunodeficiency disease (HIV) disease. We performed a phase I study of zidovudine (500 mg/day) plus acyclovir (2 or 4 g/day) in asymptomatic HIV-seropositive men to investigate pharmacokinetics, safety, tolerance, and immunologic effects of the combination. SUBJECTS AND METHODS: Fifty HIV-seropositive homosexual or bisexual men from the San Francisco City Clinic Cohort Study were recruited for the study; of these, 20 met the eligibility criteria. Treatment with zidovudine and acyclovir was open label. Pharmacokinetic, virologic, immunologic, and clinical data were collected periodically over a 24-week period. RESULTS: Pharmacokinetic analysis showed no drug interaction. The combination was generally well tolerated, and hematologic parameters remained stable through 24 weeks. There were no significant changes in total lymphocytes, T4 lymphocytes, overall skin test reactivity, or ability to culture virus from peripheral blood. CONCLUSION: This combination of agents is safe in this population for at least six months. Conclusions about long-term tolerance and efficacy await the results of larger trials with longer follow-up.     

Isolation of human immunodeficiency virus type 1 (HIV-1) from seropositive hemophiliac Japanese

We reported some biological properties of HIV-1 isolated from 16 hemophiliac Japanese and accidentally infected one mother. Peripheral mononuclear cells (PMCs) were obtained from them, one with AIDS, one with lymphadenopathy and the others were asymptomatic carriers. CD 8 depleted PMCs were obtained by panning methods. They were cocultivated with PHA-stimulated PMCs from seronegative donors. Fifteen HIV-1 isolates were obtained from 17 cases. Recovery rate was 87.5%. The replication rate of HIV-1 from AIDS patient was faster than other isolates from asymptomatic carriers. They did not from plaques on MT 4 cells. The host range study showed that all fifteen isolates infected primary macrophages and only two simultaneously infected human T cell line (MT 2). None of them showed infectivity to other T cell, B cell or monocytic cell lines. Although our study population was rather small, these results suggested that the majority of seropositive hemophiliac Japanese were already infected by HIV-1 and had the risk for the development of AIDS. Moreover, we recognized that HIV-1 from hemophiliac Japanese showed characteristic biological features, i,e, such as 1) weak cytopathic effects, 2) narrow host range and 3) tropism to primary macrophages. It is suggested that they may belong to a unique subtype of HIV-1 and their selective infectivity to primary macrophages have some relation to the clinical status of seropositive hemophiliacs. Further study is necessary to clarify these points.     

Defining human immunodeficiency virus (HIV) type 1 immunotypes with six human monoclonal antibodies

Studies of HIV-1 immunological relatedness have revealed that genetic diversity does not parallel antigenic diversity and have recently shown that HIV-1 strains from different geographic regions from around the world can be grouped into a small number of immunologically defined groups (immunotypes). Previously, the binding patterns of 28 monoclonal antibodies (mAbs) (specific for V3 and C5 of gp120 and cluster I of gp41) with 26 HIV-1 virions obtained globally were determined in a virus binding assay. Analysis of the binding patterns of these 728 mAb/virus combinations now reveals that a particular subset containing six of the 28 mAbs can correctly immunotype 24 of the 26 isolates (92%) into three immunotypes. Like the original panel of mAbs, the subset of six mAbs identified was directed against epitopes in the V3 and C5 regions of gp 120 as well as cluster I of gp41. The binding patterns ("profiles") of these six mAbs with 24 additional HIV-1 virions from Cameroon confirmed that epitopes in V3 and C5 of gp120 and cluster I of gp41 are well exposed on these viruses. Multivariate analysis of the binding patterns of these six mAbs with all 50 viruses (26 obtained globally and 24 obtained from Cameroon) indicates that the viruses from Cameroon have binding profiles similar to viruses from the rest of the world and can be classified into the same three immunotypes that were previously described. This study suggests that a vaccine against HIV-1 need not be based on geographic origin of the virus or on clade, but may better be based on antigenic properties that classify the plethora of different HIV-1 viruses into immunologically defined groups.     

Antibodies to CD4 in individuals infected with human immunodeficiency virus type 1.

The attachment of human immunodeficiency virus type 1 (HIV-1) to target cells is mediated by a specific interaction between the viral envelope glycoprotein (gp120) and the CD4 receptor. Here we report that approximately 10% of HIV-1-infected individuals produce antibodies that recognize the extracellular portion of the CD4 molecule. Carboxyl-terminal deletions of CD4 that do not affect HIV-1 gp120 binding eliminate recognition of CD4 by patient antisera. In contrast, mutations in the amino-terminal domain of CD4 that attenuate HIV-1 gp120 binding do not diminish CD4 recognition by patient antisera. These results suggest that HIV-1 infection can generate antibodies directed against a region of the viral receptor distinct from the virus-binding domain.     

Antigenic implications of human immunodeficiency virus type 1 envelope quaternary structure: oligomer-specific and -sensitive monoclonal antibodies.

A majority of monoclonal antibodies (mAbs) raised against soluble oligomeric human immunodeficiency virus type 1 isolate IIIB (HIV-1IIIB) envelope (env) glycoprotein reacted with conformational epitopes within the gp120 or gp41 subunits. Of 35 mAbs directed against gp41, 21 preferentially reacted with oligomeric env. A subset of these mAbs reacted only with env oligomers (oligomer-specific mAbs). In contrast, only 1 of 27 mAbs directed against the gp120 subunit reacted more strongly with env oligomers than with monomers, and none were oligomer-specific. However, 50% of anti-gp120 mAbs preferentially recognized monomeric env, suggesting that some epitopes in gp120 are partially masked or altered by intersubunit contacts in the native env oligomer. Two mAbs to oligomer-dependent epitopes in gp41 neutralized HIV-1IIIB and HIV-1SF2, and binding of these mAbs to env was blocked by preincubation with HIV-1-positive human serum. Thus, immunization with soluble, oligomeric env elicits antibodies to conserved, conformational epitopes including a newly defined class of neutralizing antibodies that bind to oligomer-specific epitopes in gp41, and may also minimize the production of antibodies that preferentially react with monomeric env protein.     

Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries.

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.     

Cerebrospinal fluid and human immunodeficiency virus. Findings in healthy, asymptomatic, seropositive men

Involvement of the central nervous system by human immunodeficiency virus is an important cause of morbidity and mortality. We have undertaken a longitudinal study of asymptomatic individuals found to be human immunodeficiency virus seropositive to identify and characterize cerebrospinal fluid abnormalities early in the disease process. Our findings in 25 individuals have been notable for a frequent incidence of cerebrospinal fluid abnormalities. Pleocytosis or elevated cerebrospinal fluid protein was found in 12 (48%) of 15 patients studied. Oligoclonal banding was present in 6 (26%) of 23 patients. Human immunodeficiency virus was isolated by culture in 4 asymptomatic patients. The cerebrospinal fluid abnormalities we observed indicate an active process occurring in the central nervous system, even in early human immunodeficiency virus infection in asymptomatic patients. Serial observation of these patients for development of neuropsychiatric findings may provide answers to the significance of cerebrospinal fluid abnormalities identified in these patients.     

Directed selection of recombinant human monoclonal antibodies to herpes simplex virus glycoproteins from phage display libraries.

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.     

CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1-infected individuals.

PCR was used to clone isolates of the human immunodeficiency virus type 1 (HIV-1) nef gene directly from peripheral blood leukocytes of HIV-1-infected individuals. A transient expression system with human CEM T cells was used to assess the effect of nef on CD4 antigen expression on the cell surface. We show that CD4 down-regulation is a frequent property of primary HIV-1 nef alleles. Mutations in conserved amino acid motifs of Nef disrupted CD4 down-regulation. Our observations strongly suggest that CD4 down-regulation reflects a conserved function of nef, which is selected in vivo in human HIV-1 infection. Methodology described here provides quantitative assays to establish whether alterations in nef correlate with the dynamics of disease progression in human AIDS.     

Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals.     

The chest roentgenogram in pulmonary tuberculosis patients seropositive for human immunodeficiency virus type 1

To determine the impact that co-infection with HIV has on the radiographic presentation of pulmonary tuberculosis, we examined the chest roentgenograms obtained before treatment in 225 HIV-tested adult Haitians with bacillary (smear or culture or both) positive pulmonary tuberculosis. There were 67 HIV-seropositive and 158 HIV-seronegative patients. Intrathoracic adenopathy alone was more common and parenchymal infiltrates less common in HIV-seropositive patients (p less than 0.05). Although a parenchymal infiltrate was less likely to be cavitating in the HIV-seropositive group (p less than 0.05) when cavitary parenchymal disease was present, HIV seropositivity did not affect the number of cavities (single or multiple) or the size of the largest cavity. Patients with AIDS were significantly more likely to have a chest radiographic pattern consistent with primary tuberculosis (80 percent) than HIV-seropositive patients without AIDS (30 percent), and the latter were significantly more likely to have such a pattern than HIV-seronegative patients (11 percent) (p less than 0.05). The HIV-seropositive patients were equally infectious, regardless of the pattern of disease (primary vs postprimary). Even though pulmonary tuberculosis in an HIV-seropositive adult probably results from reactivation of dormant foci or reinfection, the pattern on the chest roentgenogram often suggests primary disease, especially if the patient has AIDS.     

Prediction of optimal peptide mixtures to induce broadly neutralizing antibodies to human immunodeficiency virus type 1.

Sequences of the principal neutralizing determinant (PND) of the external envelope protein, gp120, from 245 isolates of human immunodeficiency virus type 1 are analyzed. The minimal set of peptides that would elicit antibodies to neutralize a majority of U.S. and European isolates of human immunodeficiency virus type 1 is determined with the assumption that peptides of a given length including the central Gly-Pro-Gly triad are required. In spite of the hypervariability of the PND, 90% of these 245 sequences include peptides from a set of 7 pentapeptides, 13 hexapeptides, or 17 heptapeptides. Tests of these peptide sets on 78 additional PND sequences show that 95% are covered by the 7 pentapeptides, 94% by the 13 hexapeptides, and 86% by the 17 heptapeptides. To anticipate variants not yet observed, single amino acid mutation frequencies from the 245 isolates are used to calculate an expanded set of the 10,000 most probable PND sequences. These sequences cover 86% of the total distribution expected for the central portion of the PND. Peptide lists derived from this expanded set when tested on the 78 additional sequences show that 7 pentapeptides cover 95%, 13 hexapeptides cover 94%, and 17 heptapeptides cover 94%. These results suggest that peptide cocktails of limited size with the potential to cover a large fraction of PND sequence variation may be feasible vaccine candidates.     

Production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the human immunodeficiency virus type 1 envelope glycoprotein.

Cell lines secreting IgG1 human monoclonal antibodies (mAb) to the envelope glycoprotein, gp120, of human immunodeficiency virus (HIV) have been produced by transformation of peripheral blood cells from HIV-infected individuals and by fusion of transformed cells to a human-mouse heteromyeloma cell line (SHM-D33). Two human mAbs were site-selected by means of a 23-mer synthetic peptide spanning a portion of the third variable domain of gp120 from the MN strain of HIV. The two heterohybridomas produce three times more IgG than do their parent lymphoblastoid cell lines. The specificities of these mAbs have been mapped to sequences near the tip of the disulfide loop of the gp120 third variable domain, Lys-Arg-Ile-His-Ile and His-Ile-Gly-Pro-Gly-Arg, respectively. The mAbs have dissociation constants of 3.7 x 10(-6) M and 8.3 x 10(-7) M, neutralize HIVMN in vitro at nanogram levels, and bear the characteristics of antibodies associated with protective immunity in vivo.     

Antibodies to human immunodeficiency virus type 1 in the urine specimens of HIV-1-seropositive individuals

Specific antibodies to human immunodeficiency virus type 1 (HIV-1) were detected in 200-fold concentrated urine samples, but none were detected in unconcentrated urine specimens, from 100 randomly selected HIV-1--seropositive individuals by enzyme-linked immunosorbent assay (ELISA) and Western blot techniques using the manufacturer's recommended procedures. Using modified methods for both the ELISA and Western blot tests, antibodies to HIV-1 have also been detected in the unconcentrated urine specimens from the same HIV-1--seropositive individuals. No difference in the frequency of antibodies to HIV-1 were found between unconcentrated and 200-fold concentrated urine samples when tested by the modified methods. HIV-1 core antigen (p24) was not detected in either the concentrated or the unconcentrated HIV-1--seropositive adult urine samples; none of these individuals showed overt clinical or laboratory evidence of renal dysfunction. The titer of the antibodies to HIV-1 found in the urine specimens was found to be parallel with the titer of antibodies to HIV-1 in the corresponding individual's serum. Further elucidation of the pathophysiology and the nature of the specific antibodies to HIV-1 observed in the urine of HIV-1--seropositive individuals is under investigation in our laboratories.     

Herpes simplex virus type 1 and human immunodeficiency virus type 1 antigens in platelets from a hemophilia B patient with human immunodeficiency virus type 1-related thrombocytopenia.

We analyzed platelet-associated antigens from a hemophilia B patient with human immunodeficiency virus type 1 (HIV-1)-related thrombocytopenia. Two bands appeared at 31,000 and 37,000 daltons in the platelet lysate after reaction with autologous serum in SDS-PAGE and Western blots. The band at 37,000 daltons was obtained using anti-herpes simplex type 1 (HSV-1) rabbit antiserum. Doublet bands at 36,000 and 37,000 daltons also appeared after reaction with HSV-1 seropositive human serum. The band at 31,000 daltons appeared after reaction with anti-HIV-1 rabbit serum. These results suggest that the platelet-associated antigens in this patient are components of both HSV-1 and HIV-1 antigens. In addition, acyclovir decreased his PAIgG level and increased his platelet count, and zidovudine increased his platelet count. Thus, we concluded that each of the platelet-associated antigens is partially responsible for the thrombocytopenia by causing deposition of immune complexes in this patient.     

Quantitative analysis of human immunodeficiency virus type 1 antibody reactivity by western immunoblots: evaluation of relative antibody levels in seropositive individuals and mothers.

A quantitative analysis of antibody responses to human immunodeficiency virus type 1 (HIV-1) proteins using Western immunoblots and 125I-labeled protein A is reproducible and can be validated. The antibody levels obtained by Western immunoblots were compared with stoichiometric p24 radioimmunoassay over a wide range of antibody (correlation coefficient, .94; P less than .001). Antibody levels to gp160 and gp120 were validated using purified antigens. Analysis of antibody levels from 31 seropositive individuals revealed a statistically significant correlation between antibody levels to p24 and the other viral proteins except gp120. Anti-gag p24 antibody was strongly correlated with antibodies to other env products, specifically gp41 and gp160. Using the validated assay, HIV-1-infected mothers of infants were found to have highly variable levels of antibody to all viral proteins. Mothers of infected infants did not differ significantly from mothers of uninfected infants in antibody pattern or levels to any viral protein including gp120.