Development of vaccines against

Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage-display technology. We designed a new lambda vector to express protein fragments as N-terminal fusions to the D capsid protein and generated high-complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage-displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients. © 2003 Wiley-Liss, Inc. Key words: breast cancer; immune response; phage displayTumor-associated antigens recognized by humoral effectors of the immune system are an attractive target for diagnostic and therapeutic approaches to human cancer. A considerable number of this type of antigen have been identified over recent years through screening expression cDNA libraries from human solid tumors with sera of the autologous patients (SEREX, serologic identification of antigens by recombinant expression cloning). [1][2][3][4] This type of screening of a cDNA expression library by conventional methods requires the preparation of a large number of membrane filters blotted with bacteriophage plaques that are then searched with a specific probe. In the case of the SEREX experiments, the screening is performed using sera from cancer patients, which are usually available in very limited quantity. The second limitation is that such immunoscreening procedure does not allow selection of antigens that are recognized by sera from different patients.We tried to overcome these limitations and to simplify the screening procedure by performing affinity selection of cDNA libraries in very small volumes by combining the SEREX approach with that of phage-display technology. Phage-display technology is based on the insertion of foreign nucleotide sequences into genes encoding for various coat proteins of filamentous phage, resulting in a heterogeneous mixture of phages, each displaying the different peptide sequence encoded by a corresponding insert. A physical link between a displayed fusion protein and DNA encoded for it make this phage target selectable.Phage-display technology was introduced in 1985 by Smith 5 and has been widely used since for generating and screening peptide libraries to identify ligands for various kinds of receptor molecules. 6 -8 However, significantly less striking progress has been reported on the use of filamentous phage as display vector for constructing cDNA libraries. In most cases, foreign peptides are displayed on the filamentous phage capsid as N-terminal fusions to the major (pVIII) or the minor (pIII) coat protein. Large protein domains fused to pVIII disturb (with some rare...

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The objective of this work was to develop an antibody-specific immunoglobulin G (IgG) avidity assay to discriminate between acute and latent phases of Toxoplasma gondii infection by using recombinant antigens. One hundred twenty-one serum samples from women who developed IgG antibodies against Toxoplasma during pregnancy were used. The IgG avidities of antibodies directed against epitopes carried by fragments of GRA3, GRA7, MIC3, and SAG1 antigens were measured by performing parallel enzyme immunoassays. The avidity index for Toxoplasma-specific antibodies against a homogeneous mixture of recombinant GRA3, GRA7, MIC3, and SAG1 antigens correlated closely with the IgG avidity of antibodies against lysed whole-cell T. gondii antigen. The avidity assay performed with the recombinant MIC3 antigen highlighted the presence of avidity low-antibodies IgG exclusively in sera collected within 2 months after primary infection. The presence of T. gondii-specific, low-avidity IgG antibodies against recombinant MIC3 antigen can be used to determine the point of infection with T. gondii within a 2-month time frame after infection.

Infection with Toxoplasma gondii causes morbidity and mortality in congenitally infected and immunocompromised individuals. Both humoral and cell-mediated immunity are involved in host resistance to invasion of the parasite. Among putative vaccine candidates, the T. gondii microneme proteins appear to be promising, because they are responsible for the invasion process. The present work focused on studying the immunogenicity of microneme proteins in infected individuals and in a mouse model of chronic toxoplasmosis. We identified 5 distinct antigenic regions within MIC2, MIC4, MIC2-associated protein, and apical membrane antigen 1 gene products, which were recognized by (1) T cells from both adults with acquired infection and children with congenital infection and (2) antibodies from all patients. Finally, we demonstrated that DNA immunization with microneme fragments elicited effective protection in mice (84% reduction in brain-cyst burden), suggesting that a combination of these antigenic regions should be considered in the design of potential vaccines against toxoplasmosis.

We have evaluated the diagnostic utility of six antigenic regions of the Toxoplasma gondii MIC2, MIC3, M2AP, GRA3, GRA7, and SAG1 gene products, assembled in recombinant chimeric antigens by genetic engineering, in order to replace the soluble, whole-cell tachyzoite extract in serological assays. Serum samples from 100 adults with acquired T. gondii infection and from 30 infants born to mothers with primary toxoplasmosis contracted during pregnancy, of whom 20 were congenitally infected, were included. Immunoglobulin G (IgG) and IgM antibodies against epitopes carried by chimeric antigens were measured by performing parallel enzyme immunoassays (recombinant enzyme-linked immunosorbent assays [Rec-ELISAs]), and the results obtained by standard commercial assays with the whole-cell Toxoplasma antigen and assays with the chimeric antigens were compared. Our results demonstrate that IgG and IgM Rec-ELISAs with individual chimeric antigens have performance characteristics comparable to those of the corresponding commercial assays. Furthermore, we show that IgM-capture assays based on chimeric antigens improve the ability to diagnose congenital toxoplasmosis postnatally compared with the ability to diagnose congenital toxoplasmosis by the use of standard assays. The use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected individuals from T. gondii-uninfected individuals and shows that immunoassays based on recombinant products could provide the basis for standardized commercial tests for the serodiagnosis of toxoplasmosis.Human infection by Toxoplasma gondii is generally asymptomatic and induces a self-limiting disease. In contrast, primary T. gondii infection acquired during gestation can be transmitted to the fetus through the placenta and may cause miscarriage, permanent neurological damage, premature birth, and visual impairment (15,29,32). Toxoplasmosis during gestation represents a formidable task for the clinician due to its subclinical course in the majority of pregnant women and the unpredictable long-term outcome of congenital infection (11,16,32). To implement suitable therapies in good time and to avoid neonatal malformations or reduced eyesight in newborns, it is essential to establish when the primary infection has been acquired in the mother and to determine if vertical transmission to the fetus has occurred.The diagnosis of T. gondii infection can be established by detecting parasite-specific DNA sequences in body fluids and tissues or Toxoplasma-specific immunoglobulins in sera from infected individuals (2,20,30). Serological methods are generally preferred, since the sensitivities and the specificities of other methods can be affected by the appropriateness of sample handling and shipping and storage conditions. Furthermore, parasitemia may be of short duration, further limiting the value of detection of DNA in amniotic fluid and peripheral blood.The diagnosis of toxoplasmosis by serological methods routinely uses immunoenzymatic assays to detect the presence of the var...