Sialic acid-dependent binding of baculovirus-expressed recombinant antigens from Plasmodium falciparum EBA-175 to Glycophorin A.

The Plasmodium falciparum Erythrocyte Binding Antigen-175, EBA-175, is a soluble merozoite stage parasite protein which binds to glycophorin A surface receptors on human erythrocytes. We have expressed two conserved cysteine-rich regions, region II and region VI, of this protein as soluble His-tagged polypeptides in insect cell culture, and have tested their function in erythrocyte and glycophorin A binding assays. Recombinant region II polypeptides comprised of the F2 sub-domain or the entire region II (F1 and F2 sub-domains together) bound to erythrocytes and to purified glycophorin A in a manner similar to the binding of native P. falciparum EBA-175 to human red cells. Removal of sialic acid residues from the red cell surface totally abolished recombinant region II binding, while trypsin treatment of the erythrocyte surface reduced but did not eliminate recombinant region II binding. Synthetic peptides from three discontinuous regions of the F2 sub-domain of region II inhibited human erythrocyte cell binding and glycophorin A receptor recognition. Immune sera raised against EBA-175 recombinant proteins recognized native P. falciparum-derived EBA-175, and sera from malaria-immune adults recognized recombinant antigens attesting to both the antigenicity and immunogenicity of proteins. These results suggest that the functionally-active recombinant region II domain of EBA-175 may be an attractive candidate for inclusion in multi-component asexual blood stage vaccines.

Clinical and immune responses in advanced melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside GD2.

PURPOSE: To determine immune responses and toxicity to the anti-idiotype vaccine, as well as clinical responses and survival, we initiated a clinical trial for patients with advanced melanoma treated with an anti-idiotype antibody (TriGem) that mimics the disialoganglioside GD2. PATIENTS AND METHODS: Forty-seven patients with advanced melanoma received either 1-, 2-, 4-, or 8-mg doses of TriGem (Titan Pharmaceuticals Inc, South San Francisco, CA) mixed with QS-21 adjuvant (Aquila Biopharmaceuticals, Inc, Worcester, MA) 100 microg subcutaneously weekly for 4 weeks and then monthly until disease progression. Median age was 57 years, there were 32 men and 15 women, 43% of patients had undergone prior therapy for metastatic disease, 55% had disease confined to soft tissue, and 45% had visceral metastasis. RESULTS: Hyperimmune sera from 40 of 47 patients showed an anti-anti-idiotype (Ab3) response. Patient Ab3 was truly Ab1' because it specifically bound purified disialoganglioside GD2. The isotypic specificity of the Ab3 antibody consisted of predominantly immunoglobulin (Ig)G, and all IgG subclasses were represented. One patient had a complete response that persisted at 24 months, and 12 patients were stable from 14+ to 37+ months (median, 18+ months). Disease progression occurred in 32 patients on study from 1 to 17 months (median, 5.5 months), and 21 have died at 1 to 16 months (median, 6 months). The Kaplan-Meier-derived overall median survival has not been reached. Median survival has not been reached for the 26 patients with soft tissue disease only and was 13 months for 21 patients with visceral metastasis. Toxicity consisted of local reaction at the site of injection and mild fever and chills. CONCLUSION: TriGem has minimal toxicity and generates robust and specific IgG immune responses against GD2. Objective responses were minimal, but there may be a favorable impact on disease progression and survival that will require prospective randomized trials.

Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen.

PURPOSE: We generated an anti-idiotype antibody, designated CeaVac, that is an internal image of the carcinoembryonic antigen (CEA). We previously demonstrated that the majority of patients with advanced colorectal cancer generate specific anti-CEA responses. The purpose of the current study was to treat patients with surgically resected colon cancer with CeaVac to determine the immune response and clinical outcome to treatment with vaccine. We also compared the immune responses between patients treated with fluorouracil (5-FU) chemotherapy regimens plus vaccine versus vaccine alone. PATIENTS AND METHODS: Thirty-two patients with resected Dukes' B, C, and D, and incompletely resected Dukes' D disease were treated with 2 mg of CeaVac every other week for four injections and then monthly until tumor recurrence or progression. Fourteen patients were treated concurrently with 5-FU chemotherapy regimens. RESULTS: All 32 patients entered onto this trial generated high-titer immunoglobulin G and T-cell proliferative immune responses against CEA. The 5-FU regimens did not have a qualitative or quantitative effect on the immune response. Three of 15 patients with Dukes' B and C disease progressed at 19, 24, and 35 months. Seven of eight patients with completely resected Dukes' D disease remained on study from 12 to 33 months; one patient with resected Dukes' D disease relapsed at 9 months. One patient with incompletely resected Dukes' D disease remained on study at 14 months without evidence of progression; eight experienced disease progression at 6 to 31 months. CONCLUSION: CeaVac consistently generated a potent anti-CEA humoral and cellular immune response in all 32 patients entered onto this trial. A number of very high-risk patients continue on study. 5-FU regimens, which are the standard of care for patients with Dukes' C disease, did not affect the immune response. These data warrant a phase III trial for patients with resected colon cancer.

Generation of chimeric monoclonal antibodies from mice that carry human immunoglobulin Cgamma1 heavy of Ckappa light chain gene segments.

Gene targeting in mouse embryonic stem (ES) cells was used to replace (i) the mouse immunoglobulin heavy chain (IgH) Cgamma2a gene segment (mCgamma2a) with the human Cgamma1 gene segment (hCgamma1), and (ii) the mouse immunoglobulin light chain (IgL) Ckappa gene segment (mC kappa) with its human counterpart (hC kappa). ES cells carrying these gene conversions were used to generate chimeric mice that transmitted the human alleles through the germ line. Mice homozygous for both gene alterations were generated by breeding. Serum from homozygous mutant mice contained comparable amounts of antibodies with chimeric kappa or mouse lambda light chains but only small fractions of basal serum IgG or antibodies elicited against immunizing agents contained chimeric heavy chains. A relative increase in immunogen-specific hCgamma1 antibodies was seen following immunization in combination with the saponin adjuvant QS-21. The effect of this was to shift the IgG1-dominated response to an IgG subclass profile that included significant amounts of IgG2a, IgG2b and IgG3 and chimeric IgG. The amounts of antibody secreted by hybridomas derived from mutant and wild-type mice were similar. Sequencing confirmed correct splicing of hCgamma1 and hCkappa gene segments to mouse J gene segments in hybridoma Ig gene transcripts. In conclusion, IgHhCgamma1/IgLhCkappa double mutant mice provide a useful animal model for deriving humanized antibodies with potential applications in immunotherapy and diagnostics in vivo as well as for investigating hCgamma1 associated functions.

Antibody responses in melanoma patients immunized with an anti-idiotype antibody mimicking disialoganglioside GD2.

We initiated a clinical trial for patients with advanced malignant melanoma treated with an anti-idiotype antibody that mimics the disialoganglioside GD2. We report the clinical and immune responses of the first 12 patients entered into this trial. Patients received 1-, 2-, 4-, or 8-mg doses of the anti-idiotype antibody mixed with 100 microg of QS-21 adjuvant every other week, four times, and then monthly. Twelve patients have been on trial for 2-23 months, and all of them have generated immune responses. Patients were removed from the study if they demonstrated disease progression. Hyperimmune sera from all 12 patients revealed an anti-anti-idiotypic Ab3 response, as demonstrated by the inhibition of Ab2 binding to Ab1 by patients' immune sera. To further test the anti-anti-idiotypic response, patients' Ab3 antibodies were affinity purified on Sepharose 4B columns containing adsorbed immunizing anti-idiotype immunoglobulin. Purified Ab3 of all patients studied inhibited binding of Ab1 to a GD2-positive cell line. Purified Ab3 also inhibited binding of Ab1 to purified GD2, in a manner comparable to equal quantities of purified Ab1. The patient Ab3 was truly an Ab1' because it specifically bound to purified disialoganglioside GD2. The isotypic specificity of the Ab3 antibody was predominantly IgG, with only minimal IgM. The predominant IgG subclass was IgG1, with approximately equal quantities of IgG2, IgG3, and IgG4. These Ab3 antibodies reacted specifically with tumor cells expressing GD2 by immune flow cytometry and immunoperoxidase assays. Five patients' Ab3 antibodies studied for antibody-dependent cellular cytotoxicity were positive. One patient had a complete clinical response, with resolution of soft tissue disease, and six patients had stable disease, ranging from 9 to 23 months, and are being continued on vaccine therapy. Toxicity consisted of local reaction at the site of the injection, including induration and pain that generally resolved within a few days. Mild fever and chills were observed in 75% of the patients but rarely required acetaminophen. There was no additional toxicity, including abdominal pain that was previously seen with infusion of murine monoclonal anti-GD2 antibody. Current trials include patients with stage III melanoma and small cell lung cancer. Future trials will attempt to enhance the antitumor response by the addition of interleukin 2, granulocyte macrophage colony-stimulating factor, and other cytokines, together with the 1A7 vaccine.

Hepatitis B virus activation among central Africans infected with human immunodeficiency virus (HIV) type 1: pre-s2 antigen is predominantly expressed in HIV infection.

To evaluate the impact of human immunodeficiency virus (HIV) type 1 on the natural history of hepatitis B virus (HBV) infection, sera from Central Africans with and without antibodies to HIV were examined for HBV markers of ongoing replication, recovery from infection, and reactivation or reinfection. The prevalence of HBV infection and HBV surface antigen (HBsAg) was similar for HIV-positive and -negative persons. AIDS patients were more likely to be HBsAg positive and have markers of viral replication. Unlike pre-surface (pre-s) 1 antigen (Ag), which did not differ significantly with respect to HIV infection, pre-s2Ag was more common among HIV-positive persons than among HIV-negative ones and was more common among AIDS patients than among HIV-positive asymptomatic carriers. HIV-positive persons had more markers consistent with HBV reactivation and lower levels of antibody to HBsAg.

Infection with human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic viruses among leprosy patients and contacts: correlation between HIV-1 cross-reactivity and antibodies to lipoarabinomannan.

To determine the association between leprosy and human retroviral infections, 57 leprosy patients, 39 leprosy contacts, and 500 pregnant women were investigated serologically for antibodies to human immunodeficiency virus type 1 (HIV) and human T cell lymphotropic virus (HTLV) types I and II. Antibodies to Mycobacterium leprae phenolic glycolipid I (PGL-I), and lipoarabinomannan (LAM) were also analyzed. A low prevalence of HIV-1 infection was observed among leprosy patients (3.5%), leprosy contacts (0), and pregnant women (3.6%). Antibodies to HTLV-I but not -II were found more often in leprosy patients (8.7%) and contacts (12.8%) than in pregnant women (0). Sera from leprosy patients and leprosy contacts were often false-positive for HIV-1 by ELISA and were indeterminate by Western blot. LAM IgM and PGL-I IgM antibodies in sera from leprosy patients yielded significant cross-reactivities with HIV-1 pol and gag proteins. These data suggest that mycobacterial cell wall antigens may share common epitopes with HIV. Caution should be exercised when interpreting HIV-1 ELISA and Western blot data from regions where leprosy or other mycobacterial diseases are endemic.

Humoral aspects of anti-HIV immune responses in Zairians with AIDS: lower antigenemia does not correlate with immune complex levels.

Serological patterns of anti-HIV immune responses of 150 HIV-infected (65 asymptomatic, 19 ARC, 66 AIDS) and 150 HIV-negative healthy Zairians were studied to determine the clinical significance of p24 antigen, and anti-p24 antibody, particularly in relation to p24 relative binding capacity (RBC) and circulating immune complexes (CICs). Levels of p24 antigen, anti-p24 antibody titers, and p24 RBC were evaluated by means of enzyme-linked immunosorbent assay (ELISA). Circulating immune complexes were measured by C1q-binding assay. Human immunodeficiency virus CICs were detected by polyethylene glycol (PEG) precipitation followed by 6 M guanidinium lysis, ELISA, Western blot, or radioimmunoprecipitation of the lysed precipitates. Immunoglobulin levels for IgG, IgM, IgA, and beta 2-microglobulin (beta 2-M) were quantified in all study participants by laser nephelometry and ELISA. All immunoglobulin levels were significantly elevated among HIV-positive vs. HIV-negative individuals. Immunoglobulin levels correlated well with disease progression among infected patients. Similarly, beta 2-M levels were significantly higher in HIV-positive vs. HIV-negative individuals and correlated well with progression to AIDS. Free p24 antigen in serum was detected in 1.33% of all patients. However, p24 reactivity increased to 6% (9 of 150 cases) after PEG precipitation and CIC dissociation. There was a good correlation between p24 reactivity and disease development. High titers of anti-p24 antibody (> 44,100) occurred in at least 80% of all patients, and did not correlate with disease stage. Similarly, more than 60% of patients had high levels of p24 RBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotypic distribution of HIV-1-specific antibodies in individuals from central Africa.

Individuals infected with human immunodeficiency virus type 1 (HIV-1) develop a humoral immune response to the virus's major structural gene products env, gag, and pol. The distribution of antibodies to env, gag, and pol proteins in Central African populations is of interest as they have a high level of immune system activation compared to non-African populations. Using the Western blot technique, we analyzed the isotypic distribution of anti-HIV antibodies in 45 HIV-1-infected individuals from Central Africa that were either symptomatic or asymptomatic. We observed two basic differences between the isotypic profile of individuals from Central Africa and non-African populations. Central African individuals had a strong polyisotypic response to gag and pol, which has only been observed for gag in American and European populations. In addition, individuals from Central Africa had a high frequency of IgG4 to gag and pol, 75 and 51%, respectively, as compared to 29 and 6% in a non-African population. The elevated IgG4 response may result from the high basal level of immune stimulation seen in Africans due to multiple and frequent exposures to viral, bacterial, and parasitic antigens.