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Administration of a single broadly neutralizing human immunodeficiency virus (HIV)-specific antibody to HIV-infected persons leads to the development of antibody-resistant virus in the absence of antiretroviral therapy (ART). It is possible that monotherapy with UB-421, an antibody that blocks the virus-binding site on human CD4+ T cells, could induce sustained virologic suppression without induction of resistance in HIV-infected persons after analytic treatment interruption.We conducted a nonrandomized, open-label, phase 2 clinical study evaluating the safety, pharmacokinetics, and antiviral activity of UB-421 monotherapy in HIV-infected persons undergoing analytic treatment interruption. All the participants had undetectable plasma viremia (20 copies of HIV RNA per milliliter) at the screening visit. After discontinuation of ART, participants received eight intravenous infusions of UB-421, at a dose of either 10 mg per kilogram of body weight every week (Cohort 1) or 25 mg per kilogram every 2 weeks (Cohort 2). The primary outcome was the time to viral rebound (≥400 copies per milliliter).A total of 29 participants were enrolled, 14 in Cohort 1 and 15 in Cohort 2. Administration of UB-421 maintained virologic suppression (20 copies per milliliter) in all the participants (94.5% of measurements at study visits 2 through 9) during analytic treatment interruption, with intermittent viral blips (range, 21 to 142 copies per milliliter) observed in 8 participants (28%). No study participants had plasma viral rebound to more than 400 copies per milliliter. CD4+ T-cell counts remained stable throughout the duration of the study. Rash, mostly of grade 1, was a common and transient adverse event; one participant discontinued the study drug owing to a rash. A decrease in the population of CD4+ regulatory T cells was observed during UB-421 monotherapy.UB-421 maintained virologic suppression (during the 8 to 16 weeks of study) in participants in the absence of ART. One participant discontinued therapy owing to a rash. (Funded by United Biomedical and others; ClinicalTrials.gov number, NCT02369146.).

Background Alpha-synuclein (α-Syn) aggregation is the primary characteristic of synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Immunotherapy targeting α-Syn has shown promising results in animal models of the disease. This study investigates the target specificity of three different active vaccines for pathological α-Syn aggregates found in human brain tissue from synucleinopathies. Methods Guinea pigs were immunised with 3 vaccines developed by United Neuroscience, and IgG fractions purified from the resulting immune sera (IGG-1, IGG-2 or IGG-3) were used to perform immunohistochemical staining of human cases of PD, DLB and MSA. The resulting immunoreactivity was compared to a commercially available α-Syn antibody from Novacastra (NOV) commonly used for diagnostic purposes. Images were captured from the substantia nigra (SN), temporal lobe, internal capsule, insular cortex and putamen and quantified for the percentage area with α-Syn immunoreactivity. Lewy bodies (LB) and Lewy neurites (LN) were further analysed in PD and DLB cases. Results Vaccine-generated antibodies detected more α-Syn pathology compared to NOV. The levels of α-Syn immunoreactivity varied between brain region and disease type with IGG-3 recognising the highest levels of α-Syn in most cases and in all brain regions that are affected early in disease progression. IGG-3 had a high recognition for glial inclusions found in MSA which are known to have a more compact conformation. Slot blot analysis confirmed the specificity of IGG-3 for native oligomers and fibrillar α-Syn. Higher levels of α-Syn were recognised by IGG-2 in cortical regions, and by IGG-3 in SN of PD and DLB cases. This was due to increased immunolabelling of LNs in these brain regions suggesting that IGG-2 and IGG-3 recognised additional α-Syn pathology compared to IGG-1 and NOV. Whether the unique binding properties of the antibodies produced in guinea pigs will translate in the clinic remains to be addressed, which is the main limitation of this study. Conclusions These vaccines induce antibodies that bind α-Syn oligomers and aggregates in the human brain and specifically support the choice of the vaccine generating IGG-3 (i.e. UB-312) as a candidate for clinical trials for synucleinopathies.

A novel multitope protein-peptide vaccine against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and disease is described in this report. The initial development and characterization experiments are presented along with proof-of-concept studies for the vaccine candidate UB-612. UB-612 consists of eight components rationally designed for induction of potently neutralizing antibodies and broad T cell responses against SARS-CoV-2: the S1-RBD-sFc fusion protein, six synthetic peptides (one universal peptide and five SARS-CoV-2-derived peptides), a proprietary CpG TLR-9 agonist at low concentration as an excipient, and aluminum phosphate adjuvant. Through immunogenicity studies in Guinea pigs and rats, we optimized the design of protein/peptide immunogens and selected an adjuvant system, yielding a vaccine that provides excellent S1-RBD binding and high neutralizing antibody responses, robust cellular responses, and a Th1-oriented response at low doses. In challenge studies, UB- 612 vaccination reduced viral load and prevented development of disease in mouse and non-human primate challenge models. With a Phase 1 trial completed, a Phase 2 trial ongoing in Taiwan, and additional trials planned to support global authorizations, UB-612 is a highly promising and differentiated vaccine candidate for prevention of SARS-CoV-2 infection and COVID-19 disease.Author SummarySARS-CoV-2 virus, the causative agent of Coronavirus Disease 2019 (COVID-19), has spread globally since its origin in 2019, causing an unprecedented public health crisis that has resulted in greater than 4.7 million deaths worldwide. Many vaccines are under development to limit disease spread and reduce the number of cases, but additional candidates that promote a robust immune response are needed. Here, we describe a multitope protein-peptide vaccine platform that is unique among COVID-19 vaccines. The advantages of our approach are induction of both high levels of neutralizing antibodies as well as a Th/CTL response in the vaccinated host, which mimics the immune response that occurs after natural infection with SARS-CoV-2. We demonstrate that our vaccine is immunogenic and effective in preventing disease in several animal models, including AAV- hACE-2 transduced mice, and both rhesus and cynomolgus macaques. Importantly, no immunopathology was observed in the lungs of immunized animals, therefore showing that antibody-dependent enhancement (ADE) does not occur. Our study provides an additional, novel vaccine candidate for advancement in clinical trials to treat and prevent SARS-CoV-2 infection and COVID-19 disease.

United Biomedical, Inc. (UBI) has developed a set of core technologies for the discovery and production of synthetic peptide-based immunotherapeutics and vaccines. These core technologies have led to products that stimulate functional site-directed antibody responses for therapeutic effects. UBI active immunotherapies can be used to modulate physiological processes effective for the control of cell entry by HIV virions, for control of prostate cancer and allergy, and for immunocastration in livestock leading to boar taint elimination and growth promotion in swine. The UBI technologies are also useful to stimulate site-directed antibodies against pathogenic agents such as foot-and-mouth disease virus. UBITh® Immunotherapeutic peptides were developed as antigens to direct antibody responses against targeted epitopes on self-proteins and viral pathogens that are responsible for biological functions and pathogenicity. A collection of promiscuous UBITh® T helper cell epitopes was used to impart these functionally antigenic peptides with immunogenicity. The T cell helper epitopes were covalently linked to the functional antigenic target sites by peptide synthesis, creating well-defined synthetic immunogens. Finally, vaccine formulations were selected appropriate for the delivery of peptide immunogens. Controlled production processes and the means to characterize the final product provide a framework for the GMP-compliant manufacture of UBITh® immunotherapeutics and vaccines.

A standardized and rapid peptide-based SARS assay is characterized for sensitivity and specificity., A peptide-based enzyme-linked immunosorbent assay (ELISA) can be used for retrospective serosurveillance of severe acute respiratory syndrome (SARS) by helping identify undetected chains of disease transmission. The assay was developed by epitope mapping, using synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of SARS-associated coronavirus. The new peptide ELISA consistently detected seroconversion by week 2 of onset of fever, and seropositivity remained through day 100. Specificity was 100% on normal blood donor samples, on serum samples associated with infection by other pathogens, and on an interference panel. The peptide-based test has advantages of safety, standardization, and automation over previous immunoassays for SARS. The assay was used for a retrospective survey of healthy healthcare workers in Taiwan who treated SARS patients. Asymptomatic seroconversions were detected in two hospitals that had nosocomial disease.

mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu–peripheral blood leukocyte (PBL)–severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL–severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.

A microencapsulated vaccine was prepared, containing a branched peptide immunogen (200M), representing a portion of the principal neutralizing determinant of HIV-1, entrapped in poly (lactide-co-glycolide) microparticles. Following extensive in vitro characterization of the microparticles, which included assessments of particle size and size distributions, microparticle surface structure, antigen loading level and efficiency of entrapment, moisture content, the levels of residual solvent, the in vitro release rate, an assessment of antigen integrity, the product bioburden and stability during storage, the microparticles were assessed in vivo. The initial assessments undertaken, involved studies in different animal species to determine the safety and pyrogenicity of the vaccine and also the toxicity following oral administration. Once the microparticles had been shown to be safe, pyrogen free and non-toxic, they were assessed for their ability to induce serum IgG and neutralizing antibody responses in guinea pigs. Following oral immunization alone, and combined oral and subcutaneous immunization, the microparticles were shown to induce high levels of both serum IgG and neutralizing antibodies against HIV. Pending review by the U.S. Food and Drugs Administration, the microparticle based oral vaccine against HIV-1 will be assessed in clinical trials in seronegative human volunteers.

Improved serologic and polymerase chain reaction (PCR)-based tests for hepatitis C virus (HCV) infection provided an opportunity to reexamine a posttransfusion follow-up study done from 1969 to 1972. A total of 213 cardiac surgery patients was prospectively followed after receiving an average of 18 units of blood, 24% of which was from paid donors. Serial sera were tested for antibody to recombinant DNA-derived C100-3 and capsid polypeptides; selected cases were also tested against synthetic peptides derived from different regions of the HCV sequence. PCR and RIBA II immunoblot assays were done on selected sera. Each of 55 probable and 5 of 11 possible hepatitis cases who were seronegative before transfusion seroconverted. Anti-HCV seroconversion also occurred in 6 (4%) of 148 subjects without hepatitis. Among subjects followed > 1 year, PCR positivity persisted in 14 (82%) of 17. If the results of this study can be generalized, all bloodborne non-A, non-B hepatitis may be due to HCV.

The prevalence of antibodies to hepatitis C virus (anti-HCV) in a hepatitis B virus hyperendemic region was evaluated with a second-generation enzyme immunoassay. A general population of 1500 from Northern Taiwan was tested with this immunoassay which includes synthetic peptides from the capsid and non-structural protein region as the solid-phase antigen. A total of 37 subjects out of 1500 (2.5%) were positive, with no significant difference in distribution between hepatitis B antigenemic and non-antigenemic samples. The prevalence of anti-HCV was higher in subjects with abnormal than those with normal liver function tests (8.2% vs. 1.5%). Prevalence increased with age. By polymerase chain reaction, 43% of anti-HCV-positive samples were HCV-RNA-positive. However, only 1 (0.6%) of 150 anti-HCV-negative subjects had HCV-RNA. Comparison of the results with those from a conventional recombinant C100-3-based assay showed that the capsid/non-structural region synthetic antigen system provided more accurate sensitivity and specificity. The more sensitive assay revealed a high prevalence for HCV infection of 2.5% among this general population in Taiwan.

A titer for homologous viral neutralization activity (greater than 1:19,683) was observed after a 3.5-year immunization period with an octameric, branching peptide representing the principal neutralizing determinant (PND) of the human immunodeficiency virus-1IIIB envelope protein. Booster immunizations elicited persistent and potent antibodies in guinea pigs, exceeding responses produced by a conventional bovine serum albumin conjugate by 100-fold. Peptide length, central presentation of a conserved sequence, and inclusion of an upstream sequence contributed to immunogenicity. Titers (greater than 1:1,000) of heterotypic neutralizing antibodies also developed. Octameric PND peptides are a promising approach for an acquired immunodeficiency syndrome (AIDS) vaccine.

The UBITh ® AD immunotherapeutic vaccine for Alzheimer’s disease uses an amyloid- (A) immunogen having two designer peptides that have been engineered to elicit anti-N terminal A1–14 antibodies while minimizing potential for the generation of adverse anti-A immune responses. The vaccine has been further designed for minimization of inflammatory reactivities through the use of a proprietary vaccine delivery system that biases Th2 type regulatory T cell responses in preference to Th1 pro-inflammatory T cell responses. In vitro studies and in vivo studies in small animals, baboons and macaques show that anti-A antibodies are generated with the expected N-terminus site-specificity, and that these antibodies have functional immunogenicities to neutralize the toxic activity of A and promote clearance of plaque deposition. The antibodies appear to draw A from the CNS into peripheral circulation. Results indicate that the UBITh ® AD vaccine did not evoke anti-A cellular responses in a transgenic mouse model for AD. The vaccine was safe and well tolerated in adult Cynomolgus macaques during a repeat dose acute and chronic toxicity study. © 2007 Elsevier Ltd. All rights reserved.

An immunotherapeutic vaccine for allergy was produced by designing IgE-based synthetic peptide immunogens and selecting them for functional immunogenicity. The vaccine targets the binding site on IgE for the high affinity receptor Fc eRI, by active immunization. The peptide target site on IgE heavy chain was selected from among the amino acid sequences for the Ce2, Ce3, and Ce4 domains. These were characterised by epitope mapping studies for cross-reactivity to IgE and functional antigenicity. A peptide, modified from positions 413–435 of a loop region of Ce3 and subjected to conformational constraint, elicited anti-IgE antibodies that blocked IgE-mediated histamine release. It was immunopotentiated by linkage to a promiscuous T helper site to produce a wholly synthetic chimaeric immunogen. This immunogen was shown to induce polyclonal site-specific anti-IgE antibodies that obstruct binding to Fc eRI, inhibit histamine release by IgE-sensitised basophils, inhibit passive cutaneous anaphylaxis, and do not signal degranulation. Immunized dogs experienced significant reductions in total serum IgE. © 2002 Elsevier Science Ltd. All rights reserved.

We have identified continuous antigenic determinants within the amino acid sequences of the conserved nonstructural region containing proteins 2C and 3ABC of foot-and-mouth disease virus which can distinguish between the sera from vaccinated and infected animals. An ELISA based on a 3B peptide gave a positive reaction with sera from cattle, pigs, sheep and guinea pigs infected with all seven serotypes of the virus, but not with sera from vaccinated animals. In experiments with cattle and pigs to determine the duration of the antibody response, positive reactions were obtained as late as one year after infection. The advantages of using peptides from the nonstructural viral proteins instead of recombinant proteins for differentiating vaccinees from infected animals include their exquisite specificity, nonreactivity with antibodies against host cell-derived proteins (e.g. E. coli and insect cell proteins), and their ease of preparation.

Peptides from Plasmodium berghei circumsporozoite protein (CS) and influenza A virus nucleoprotein (NP) were entrapped in microparticles prepared from poly (lactide-co-glycolide) polymers, and the microparticles were administered parenterally to mice. After immunization with single or multiple doses, splenocytes were tested for a cytotoxic T cell (CTL) response and high levels of CTL activity were detected. The CTL induced were CD8+, MHC class I restricted, and could recognize virus infected cells. Peptide entrapped in microparticles of mean size500 nm were better inducers of CTL than larger microparticles (mean2 microns and above). Microparticles could also be used to deliver lipid modified peptides (lipopeptides) and elicited higher levels of cytolytic activity than either free peptide in microparticles or lipopeptide alone. Microparticles provide a novel way of inducing a CTL response using synthetic peptides.

Immunoglobulin M (IgM) antibody to hepatitis C core antigen (anti-HCV-core) was tested by enzyme immunoassay against a synthetic peptide representing amino acids 1 to 62 of the core protein. Of 214 patients with different categories of histological activity, 193 (90%) showed positive results for IgM anti-HCV-core, and 207 (97%) had HCV RNA; most cases (186, 87%) had both markers detectable simultaneously. No differences in the frequency of IgM anti-HCV-core were observed with respect to epidemiological, biochemical, or histological parameters. In 175 interferon alfa (IFN-alpha) recipients, and in 39 untreated controls, pretreatment IgM anti-HCV-core frequencies were similar: 28 of 32 (88%) in sustained responders; 55 of 61 (90%) in responders with relapse; 72 of 82 (88%) in nonresponders; and 38 of 39 (97%) in untreated controls. After IFN-alpha therapy, IgM anti-HCV-core levels became undetectable with significantly greater frequency in sustained responders (P = .014); a similar trend was observed for HCV RNA (P < .0001). IgM anti-HCV-core levels decreased after therapy in responders (P < .001) but increased in nonresponders. Fifty-one cases were longitudinally tested in relation to long-term disease outcome. Both markers remained detectable in most nonresponders with persistent liver disease, in most responders before relapse, and in all but one case at the time of biochemical relapse. IgM anti-HCV-core and HCV RNA became undetectable in most sustained responders, but reappeared despite a long-lasting transaminase normalization, behaving as asymptomatic HCV carriers; the possibility that disease reactivation may take place years afterwards cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemic cells from eight patients with chronic lymphocytic leukemia were isolated and cultured in the continuous presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) at a concentration of 1.6 × 10−9 M for 4–10 days. Aliquots of cells were then analyzed at intervals of 24–72 hr for changes in morphology, acid phosphatase staining (AP), and expression of two hairy cell-associated surface antigens, HCL1 (CD22, Leu 14) and HCL3 (CD11c, Leu M5). All cases studied showed typical B-CLL phenotype, and only a small proportion of cells expressed CD22 and CD11c (mean 7% and 4.9%, respectively). TPA treatment induced the coexpression of CD22 (mean 49%) and CD11c (mean 48%) and tartrate-resistant acid phosphatase in seven of eight cases. Morphologically, cells in TPA cultures expressed hairy cell features that were evident in light and electron microscopic studies. Collectively these changes indicate that TPA can induce hairy cell features on CLL cells in vitro, suggesting the later maturational stage of HCL compared with CLL. © 1992 Wiley-Liss, Inc.

Cloning and expression of hepatitis C virus have allowed the development of immunoassays to detect hepatitis C virus infection. However, currently available recombinant fusion protein C100-3 assays, based on a nonstructural protein of the virus, are limited in sensitivity, particularly for detecting acute infection. In this report seroconversion panels showed that an assay based on synthetic peptides, derived from immunodominant regions of both capsid and nonstructural proteins, accelerated hepatitis C virus antibody detection by 4-10 weeks. In screening, this enzyme immunoassay increased detection from 47% to 64% in plasmapheresis donors with elevated alanine aminotransferase levels (greater than 100 international units per liter), from 15% to 24% in anti-hepatitis B core antigen-positive blood donors, and from 28% to 42% in renal dialysis patients when compared with nonstructural peptide-based assays. The screening assay was repeatedly reactive for 27 of 2902 volunteer blood donor samples (0.93%); four sera reacted only with the capsid antigen. The peptide test distinguished true from false positive results in agreement with recombinant immunoblot assay in 96% of blood donor samples repeatably reactive on a recombinant hepatitis C virus enzyme immunoassay.

Two monoclonal antibodies were obtained that showed unique specificities for the leukemic T cells used for immunization. One antibody, S160, was totally specific for the antigen. The other antibody, S511, also reacted with a small population of normal T cells. This was made especially evident by concentrating these normal T cells with the antibody. Considerable evidence was obtained that both antibodies reacted with the same membrane molecules. In the unreduced state a major component of approximately 80 kdaltons was observed; after reduction this split into two components of approximately 43 and approximately 38 kdaltons. The reaction of the two antibodies with different antigenic sites on the same molecule, one representing a private site and the other a more cross-reactive site, strongly suggests an antibodylike molecule, but composed of polypeptide chains differing from immunoglobulins.

Peripheral blood and/or bone marrow leukemic cell suspensions from 49 patients with 'non-T, non-B' acute lymphoblastic leukemia (ALL) were analysed by flow cytometry using a new panel of four monoclonal antibodies. Anti-BL1 and anti-BL2 originating from NALM-6 and B35M lymphoblastoid cell lines, respectively. These antibodies recognize B-cell differentiation antigens: a heat stable non-immunoprecipitable antigenic determinant, and a 68 000 daltons glycoprotein molecule, respectively. BL5 and BL6 were derived by immunization with the promyelocytic cell line HL-60, recognizing antigens present on early hematopoietic cells: an 85 000 daltons MW glycoprotein (Pro-Im 1) and a heat stable antigen (Pro-Im2), respectively. All ALL patients studied had L1 or L2 morphology by the FAB classification and a blast count exceeding 50 per cent. There were 25 males and 24 females. Median age was 8 years (range 1-67 years). Thirty-nine cases were studied at initial presentation and 10 at relapse. Cells from 46/49 cases expressed BL2 and/or BL1, but were not reactive with BL5 or BL6. Three of 49 cases did not express BL1 or BL2. However, a small percentage of blasts from one case was positive for BL5 (13 per cent) and the other 2 cases were reactive with BL6 (20 per cent and 36 per cent, respectively). These were one adult and 2 pediatric patients that had other ALL markers and achieved a complete remission with appropriate ALL therapy. One of the BL6+ cases relapsed after 19 months with a change in phenotype to BL1+ BL2+ BL5- BL6-. This analysis shows that the majority of 'non-T, non-B' ALL's do express B-cell associated antigens (BL1/BL2) argumentative of their B-cell origin. A small subgroup does not express such antigens and may arise from a more immature cell, since they expressed antigens on early hematopoietic stem cells.

In the course of generating monoclonal antibodies to human thymus-dependent differentiation antigens, we were able to define specificities shared by T cells and by cells from patients with chronic lymphatic leukemia that were not detectable on normal B cells. In particular, one of these antibodies was reactive by indirect immunofluorescence with greater than 95% of the thymocytes and 80--95% of nonadherent sheep erythrocyte-rosetting peripheral blood lymphocytes (PBL), but was unreactive with normal B cells or cell lines derived from PBL by Epstein-Barr virus transformation. However, the leukemic cells from 11 of 14 patients with B-type chronic lymphatic leukemia were found to express detectable concentrations of this surface determinant. The target antigen recognized by this monoclonal antibody was shown by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a p69,71 complex. Our findings suggest a possible relationship between this antigen and the previously described GIX system in the mouse.

To define cell surface antigens associated with hairy cell leukemia (HCL), and to gain better insight into the origin of this disease, we developed monoclonal antibodies against spleen cells of a patient with this disease. Although none of these antibodies alone proved specific for the leukemic cells, two of them, designated alpha S-HCL 1 (alpha Leu-14) and alpha S-HCL 3 (alpha Leu-M5) were found to be valuable in the diagnosis of HCL when used in combination. alpha S-HCL 1 recognizes an antigen associated with greater than 95% of B cells in the peripheral blood. Biochemical analysis identified this antigen as a single polypeptide chain with a molecular weight of 150,000 daltons (150 kilodaltons). alpha S-HCL 1 expression on hairy cells is markedly increased when compared with normal B lymphocytes isolated from peripheral blood, tonsils, and spleens. alpha S-HCL 3 reacts with an antigen present on hairy cells but also on monocytes, macrophages, in a lower density on neutrophils, and a small percentage (less than 2%) of lymphocytes. The antigen recognized by alpha S-HCL 3 is composed of a non-covalently linked biomolecular complex of 90 and 150 kilodaltons. Since the HCL 3 antigen was not detectable on other lymphomas of either T or B cell type, the co-expression of S-HCL 1 and S-HCL 3 on hairy cells is a unique marker for this disease.

Human peripheral blood B lymphocytes differentiate into immunoglobulin (Ig)-secreting cells in the absence of pokeweed mitogen (PWM) when cultured in medium containing fetal calf serum. As increasing numbers of T cells were added to a fixed number of B cells, polyclonal plaque-forming cells (PFC) reached a similar level to that produced by PWM. The PFC response was not due to a certain effect rendered by the cell-separation procedures, nor was it due to a nonspecific killer cell effect provided by a high concentration of T cells. When T cells were cultured with irradiated non-T cells, T cells proliferated as shown by DNA synthesis. Inhibition of T-cell proliferation by irradiation resulted in decreased helper activity for the B-cell differentiation. An anti-Ia reagent inhibited proliferation of autologous reactive T cells as well as decreased the PFC response. These results suggest that the PFC response in the culture condition used was due to the autologous mixed lymphocyte reaction which requires a higher number of T cells to be effective in helping B cells differentiate.

The expression of HLA-DC/DS antigen detected by the monoclonal antibody Leu 10 was studied in three human precursor and pre-B cell lines (Josh 7, Reh, and Nalm 12). Flow cytometric analysis showed that none of these cell lines stained for the HLA-DC/DS antigen. In the presence of 1.6 X 10(-9) M of 12-O-tetradecanoylporbol-13-acetate (TPA), expression of this antigen was detected. The expression was completed after 168 h of incubation. Iodination of cell surface, immunoprecipitation by Leu 10 antibody, and two-dimensional gel analysis revealed that TPA-treated Josh 7 cells synthesized and expressed a 29,34 kD bimolecular complex with both alpha and beta chains different from those of HLA-DR antigen. Quantitative absorption experiments with cell lysates indicated a greater than 25-fold increase in HLA-DC/DS antigen in TPA-treated cells. With the induction of HLA-DC/DS antigen expression, there are concomitant decreases in the expression of the common acute lymphoblastic leukemia antigen (CALLA) and the enzymatic activity of terminal deoxynucleotidyl transferase. No appreciable changes in HLA-DR and Ig expression were observed. There was also no change in HLA-SB expression as detected by antibody ILR-1. However, DNA synthesis was markedly inhibited by TPA treatment. These results indicate that precursor and pre-B cell lines can be induced to mature in vitro. They also suggest that the expression of HLA-DC/DS antigen which precedes the expression of membrane Ig and follows the HLA-DR expression is relevant to human B cell development and cell interaction.

We studied the ability of phytohemagglutinin (PHA) and two anti-T-cell monoclonal antibodies, OKT3 and Pan T2, to induce interleukin-2 (IL2) production and proliferation in peripheral blood lymphocytes (PBL) from 14 patients with combined varied immunodeficiency (CVI). The median values of endogenous IL2 produced by mitogen-stimulated PBL was significantly lower in patients than controls irrespective of the mitogen used. The patients, taken as a group, had a significantly decreased in vitro PBL response to mitogen stimulation when compared to controls. With the addition of a highly purified human IL2 preparation, the proliferative response in the majority of patients was significantly improved with all mitogens. Three patient groups could be distinguished: Group A (3/14) had full restoration of proliferative response with the addition of IL2, Group B (5/14) had partial restoration, and Group C (6/14) had no significant response. The monoclonal antibody, Pan T2, recognized a T-cell proliferative defect in 5 of 14 patients which neither PHA nor OKT3 recognized. This was not significantly corrected by the addition of IL2. This T-cell proliferative defect correlated with the lack of B-cell proliferation and immunoglobulin production in response to B-cell mitogens in three-fourths of the patients assayed. These data show that CVI patients are a heterogeneous group but have in common a decreased in vitro production of IL2 resulting in a proliferative defect which is correctable at least in part, in vitro, in the majority by the addition of purified IL2.

The induction of mRNA synthesis and accumulation of TNF/cachectin and lymphotoxin (LT) mRNAs in T leukemic cell lines and freshly isolated T cells were studied by Northern blot analyses. Without stimulation, TNF mRNA was barely detected in four T cell lines (CEM, KE4, MT-1, and SKW-3) and not detectable in Molt-4 and Jurkat cells, while a considerable amount of TNF mRNA was observed in HSB-2 cells. When stimulated by PMA, these T cell lines accumulated varying levels of TNF mRNA. All seven T cell lines expressed LT mRNA when unstimulated and responded well to PMA by increased accumulation of LT mRNA. The calcium ionophore A23187 by itself had no effect on TNF and LT mRNA accumulations in these cell lines. The CD3+ T cell lines did not respond to anti-CD3 mAb T3-II alone. However, A23187 and mAb T3-II further elevated TNF and LT mRNA accumulations in PMA-treated T cell lines. Synergism between PMA and mAb T3-II was modest in the CD3+ cell lines. A slight difference in kinetics of TNF and LT mRNA accumulations was noted. In addition, heterogeneities in TNF and LT expressions by these cell lines in responses to PMA and other stimuli were observed. In monocyte-depleted peripheral blood T cell populations. PMA was able to induce both TNF and LT mRNA syntheses. This effect was potentiated markedly by the addition of anti-CD3 mAb T3-II. This synergistic response to anti-CD3 mAb and PMA provided further evidence that T cells were the source of TNF synthesis in these cultures. There was a difference in the kinetics of TNF mRNA accumulation and that of LT mRNA. Maximal accumulation of TNF mRNA occurred at 4 h while 8-18 h was required for maximal LT mRNA accumulation. IL-2 mRNA accumulated at an intermediate peak time of 4-8 h. Western blot analyses and cytotoxicity assays with L cells as targets indicated that these T cell lines and peripheral blood T cells secreted TNF. These results provide further evidence that human T cells are capable of making TNF as well as LT under appropriate stimulations. Their productions are an integral part of T cell response to activation signals. In addition, it appears that the production of these two closely related molecules is independently regulated.

Human malignant melanoma cell lines were found to bear Ia-like cell surface determinants demonstrable by hetero- or alloantisera and by direct identification of the characteristic bimolecular glycoprotein complex. Immunoprecipitation confirmed the presence of Ia determinants on the bimolecular complex. The quantity of Ia molecules determined by these methods and by absorption experiments was relatively constant for each cell line. Among different lines, however, the amount of Ia antigens ranged from a level equal to that expressed by B-cell lines to a small fraction of this amount. This variation in level of Ia contrasted with the more uniform amount of beta2-microglobulin detected on the cell surface. The Ia alloantigen specificity DRw2 was the most frequently encountered specificity. Ia determinants were also found on the surface of an epidermoid carcinoma line, but not on various other cell lines of normal or neoplastic origin.

The presence of Ia-like antigens was demonstrated on a small population (2-6%) of highly purified human circulating T lymphocytes by immunofluorescence with a rabbit anti-Ia serum raised against the isolated bimolecular Ia structure. The Ia+ T lymphocytes have no surface or intracellular immunoglobulins. The expansion of this Ia+ T-cell population was encountered in certain patients. Ia antigens were also found on T blasts grown in long-term cultures with conditioned medium generated by phytohemagglutinin-stimulated lymphocytes. In addition, leukemia blasts which stained for Ia antigens and formed E rosettes were identified in the peripheral blood of two leukemic patients. This evidence further supports the existence of Ia-bearing T cells in man. The Ia+ T-lymphocyte population was shown to contain cells responsible for the generation of allogeneic helper activity. Elimination of Ia+ lymphocytes from a purified T-cell population by the anti-Ia antiserum and complement abolished its ability to help an allogeneic B-cell preparation to generate plaque-forming cells against sheep erythrocytes in vitro in the presence of the antigen.

The efficacy of immunomagnetic beads to purge human myeloma cells from bone marrow ex vivo was evaluated. The optimal conditions for purging were studied first by using three myeloma cell lines: RPMI-8226, SKO- 007, and SKMM-2. Myeloma cells labeled with the vital fluorescent dye Hoechst 33342 were admixed with normal bone marrow cells, and two monoclonal antibodies reactive with the myeloma cells (PCA-1 and BL-3) were added alone or in combination with the cells. Magnetic beads coated with goat antimouse immunoglobulin G were then added, and the tumor cells to which beads were attached were separated from the mixture with a magnet. The efficacy of tumor cell removal was dependent on the bead-to-tumor ratio; a ratio of more than 500 was optimal in the presence of excess normal marrow cells. The combination of monoclonal antibodies PCA-1 and BL-3 increased the tumor cell removal as compared with either antibody alone. Two cycles of treatment were more effective than one cycle was. Under optimal conditions, 2.3 to 4 logs of tumor cells could be removed from the mixture containing 10% myeloma cells without a significant loss of normal hematopoietic progenitors as measured by CFU-GM, CFU-GEM, and BFU-E. When the efficacy of this procedure was tested on fresh bone marrow from patients with multiple myeloma (MM) by using the combination of PCA-1, BL-3, and J-5, 1.6 to 2.5 logs of tumor cells could be removed by one cycle of treatment, even from marrows containing less than 10% myeloma cells. These observations support the use of monoclonal antibody combinations and immunobeads as a reliable and nontoxic method to eliminate contaminating myeloma cells ex vivo in preparation for autologous bone marrow transplantation in patients with MM.

A monoclonal antibody designated as C21 reacting with a p43,12 complex was developed against human thymocytes. It stained predominantly the early hematopoietic cells of the lymphoid lineage and also thymocytes, peripheral B-cells and activated T- and B-cells similarly to OKT10. The heavy chain of this antigen was a glycoprotein of M, 43,000 (p43). Sequential immunoprecipitation with C21 and OKT10 antibodies indicated that they both reacted with an identical heavy-chain molecule. This observation was further documented by two-dimensional analysis. Monoclonal antibody C21 was used to probe a p43,12 complex further. Structural polymorphism of the p43 heavy chain isolated from T- and B-cells of different individuals was not detected by chymotryptic peptide mappIng, although molecules from these cell types possessed a different charge on two-dimensional gels. An unusual observation was made regarding this complex on MOLT4 cells. The light chain co-precipitated from these cells was 12,000 daltons and had a pI distinct from that of β2-microglobulin but similar to the pI of the βt molecule. Comparison between chymotryptic peptide maps of the p43 heavy chain and those of the human and murine class I molecules such as HLA, T6, H-2K, Qa-2 and TL revealed no apparent homology. We have shown, however, that the peptide backbone of p43, as studied by both tunicamycin treatment of cells and endoglycosidase F digestion of immunoprecipitates, was identical in size to that of murine Qa-1. These results suggest that the p43 antigen may be homologous to murine Qa-1 or another class I antigen encoded in the murine TL:Qa region.

Five monoclonal antibodies (A7, B24, I14, L12, and M2) recognizing different epitopes of the human natural IFN-gamma were prepared by immunizing BALB/c mice with a highly purified human natural IFN-gamma preparation (10(7) U/mg). All five antibodies had high IFN-gamma-binding activity but exhibited differential IFN-gamma-neutralizing activities. Furthermore, none of them neutralized the antiviral activity exhibited by either IFN-alpha or IFN-beta preparations, indicating thus their specificity for IFN-gamma. The A7, L12, M2, and I14 monoclonal antibodies, but not the B24, blocked the augmentation of natural killer cytotoxicity, mediated by peripheral blood monocyte-depleted lymphocytes, by Escherichia coli-derived IFN-gamma or natural IFN-gamma but not by IFN-alpha 2. All five monoclonal antibodies precipitated an identical molecular complex containing two major protein components with molecular weights of 20,000 (20 kD) and 25,000 (25 kD) and two minor components with molecular weights of 17,000 (17 kD) and 45,000 (45 kD). Treatment of the immunoprecipitated IFN-gamma molecule with endoglycosylase F led to a stepwise removal of the carbohydrate portions on both the 25 and 20 kD chains, which resulted in the appearance of both 16 kD and 18 kD chains. The hereby reported monoclonal anti-IFN-gamma antibodies will prove useful as probes for purification and for rapid assay of human IFN-gamma molecule.

The human T6 antigen was studied by two monoclonal antibodies: OKT6 and Leu-6. A third monoclonal antibody, C56 (developed in our laboratory), was found to have similar properties to those of OKT6. On SDS-PAGE, all three antibodies precipitated a 48,000-12,000-dalton heterodimer. Two-dimensional gel electrophoresis and chymotryptic peptide map analysis revealed that these antibodies precipitated in identical 48,000-dalton heavy chain which was distinguishable from the HLA-A,B,C heavy chains. The single 12,000-dalton light chain precipitated with OKT6 antibody was shown to be distinct from beta 2-microglobulin by its pI. The two light chains precipitated with Leu-6 antibody were resolved by charge into beta 2-microglobulin and the more basic 12,000-dalton peptide identical to that precipitated with OKT6. In addition to beta 2-microglobulin, the latter component (presumably beta t) was also found in the light-chain fraction precipitated from the thymocytes with a monoclonal antibody recognizing the framework of HLA-A,B,C heavy chains. Using chymotryptic peptide mapping, no polymorphism was detected among the heavy chains of the T6 antigen isolated from thymocytes of four individuals. All three monoclonal antibodies failed to precipitate murine TL from ASL1 leukemia cell lysates. Similarly, none of the six monoclonal and two conventional anti-TL antibodies reacted with T6. Although a high degree of homology was found by peptide map analysis among the TL molecules encoded by the Tlaa, Tlad and Tlae alleles, a comparison between their peptide maps and that of T6 revealed no similarity. Despite previous suggestions that T6 is homologous to murine TL, the present biochemical studies do not support this hypothesis.

This work was aimed at understanding the mechanisms of T-lymphocyte function by studying the cellular distribution and traffic of molecules of the T-cell receptor complex. The accumulation of specific molecules in intracytoplasmic vesicles is related to the activation of T lymphocytes. Some of these molecules include acid hydrolases, the transferrin receptor, and class I antigens of the major histocompatibility complex. Molecules of the T-cell receptor complex have now also been found in intracytoplasmic vesicles in a human T-cell line derived from a lymphoblastic leukemia. Such vesicles were tightly associated with the cytoplasmic microtubule network. One functional aspect of this association is a cellular pathway by which vesicles traveling to and from the cell surface converge in an area of the cells that is rich in processing enzymes.

We studied the ability of phytohemagglutinin (PHA) and two anti-T-cell monoclonal antibodies OKT3 and Pan T2, to induce proliferation and interleukin 2 (IL2) production in peripheral blood lymphocytes (PBL) from 21 homosexual patients: 12 with Kaposi's sarcoma (KS), 4 with reactive lymphadenopathy, and 5 with opportunistic infections. All patients with KS and opportunistic infections had significantly lower mitogen-stimulated DNA synthesis, as compared to the controls, irrespective of the mitogen used (P less than 0.01). The patients with lymphadenopathy exhibited significantly lower responses only in the OKT3 assay as compared to normals (P = 0.009). The production of endogenous IL2 was significantly lower in PBL cultures from patients with KS and with opportunistic infections, irrespective of the mitogen used, as compared to healthy male controls, and also significantly lower in the Pan T2-stimulated cultures from patients with lymphadenopathy. The addition of highly purified IL2 was able to restore partially lymphocyte proliferation in vitro in the presence of these mitogens in all patients. Our studies demonstrate (1) that male homosexuals even without clinical manifestations of immunodeficiency frequently exhibit a proliferative T-cell defect when anti-T-cell monoclonal antibodies rather than PHA are used as mitogens, (2) that this proliferative defect is associated with defective IL2 production, and (3) that this defect is at least in part correctable in vitro by highly purified IL2.

Two different human T cell leukemias were compared, using antiidiotype-like murine monoclonal antibodies. In each case these antibodies immunoprecipitated disulfide-linked heterodimer molecules from their respective leukemic cells. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the two idiotype-bearing molecules a major difference in molecular weight was observed, which could be attributed to a similar difference in size of the heavily iodinated chain of either heterodimer. The lightly iodinated chains of both molecules co-migrated at 43 Kd, but appeared to have different isoelectric points on two-dimensional gel analysis. The possibility that these two different heterodimers correspond to different classes of the putative T cell receptor for antigen is discussed. Assays of proliferation of the leukemic cells using Sepharose-bound antiidiotype-like monoclonal antibody showed that one of the leukemic cell types proliferated readily in response to its antiidiotypic antibody. This proliferation was not associated with measurable production of IL-2 and appeared to be a direct effect of the antiidiotypic antibody, which may mimic antigen in its interaction with the T cell receptor for antigen. The other leukemic cell type did not respond to Sepharose-bound antiidiotypic antibody and was generally unresponsive to lymphokines and mitogens. It is possible that the two leukemic cell types represent different stages of T cell differentiation.