Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Modification of an existing chromosomal inversion to engineer a balancer for mouse chromosome 15

Chromosomal inversions are valuable genetic tools for mutagenesis screens, where appropriately marked inversions can be used as balancer chromosomes to recover and maintain mutations in the corresponding chromosomal region. For any inversion to be effective as a balancer, it should exhibit both dominant and recessive visible traits; ideally the recessive trait should be a fully penetrant lethality in which inversion homozygotes die before birth. Unfortunately, most inversions recovered by classical radiation or chemical mutagenesis techniques do not have an overt phenotype in either the heterozygous or the homozygous state. However, they can be modified by relatively simple procedures to make them suitable as an appropriately marked balancer. We have used homologous recombination to modify, in embryonic stem cells, the recessive-lethal In(15)21Rk inversion to endow it with a dominant-visible phenotype. Several ES cell lines were derived from inversion heterozygotes, and a keratin-14 (K14) promoter-driven agouti minigene was introduced onto the inverted chromosome 15 in the ES cells by gene targeting. Mice derived from the targeted ES cells carry the inverted chromosome 15 and, at the same time, exhibit lighter coat color on their ears and tails, making this modified In(15)21Rk useful as a balancer for proximal mouse chromosome 15.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.     

Gene mapping and somatic cell hybrid analysis of the role of human lymphocyte function-associated antigen-3 (LFA-3) in CTL-target cell interactions

LFA-3 is expressed on a wide variety of human cell lines, including those which have been used as recipients for gene transfer of human class I gene products, whereas a murine counterpart is either absent or significantly different such that the anti-LFA-3 monoclonal antibody (MAb) does not bind. By using a somatic cell genetic approach, we demonstrate that LFA-3 is not a major histocompatibility complex-encoded molecule, and that its gene locus maps to human chromosome 1. When LFA-3 and HLA-A2 are coexpressed on the mouse cell surface, anti-LFA-3 MAb interfered with specific recognition and lysis of these target cells by human CTL capable of lysing HLA-A2-expressing mouse transfectants. A significant contribution of the LFA-3 molecule to CTL reactivity was not observed, however, because the presence of LFA-3 did not restore recognition by CTL clones previously found incapable of lysing HLA-A2-expressing mouse transfectants, nor was it required by those human CTL that could lyse mouse cell transfectants. Thus, we have used genetic techniques to demonstrate that LFA-3 may serve a role in CTL-target cell interactions at the target cell level, but is not a molecule absolutely required for human allospecific CTL recognition of HLA antigens expressed on mouse cells. We suggest that LFA-3 may not participate directly in CTL function under normal circumstances, but delivers a more general inhibitory signal only when provoked by bound MAb.     

Intercellular adhesion molecule-1 (ICAM-1) is involved in the cytolytic T lymphocyte interaction with a human synovial cell line

The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.     

Acquisition of type IX collagen by the developing avian primary corneal stroma and vitreous

Previous investigations from our laboratory and others have demonstrated that type II collagen, once thought to be a cartilage-specific molecule, is also a component of both the primary corneal stroma and the vitreous of embryonic chickens. In the present immunohistochemical study we have examined the expression in these embryonic matrices of another "cartilage-specific" collagen, type IX, along with type II. In the cornea, type IX collagen is in the primary stroma, but is not detectable in the mature, secondary stroma. Even within the primary stroma this collagen has a brief, transitory existence. It first appears in the peripheral stroma at the time the endothelial cells begin to migrate along its posterior surface, and spreads throughout the stroma during the following 24-36 hr. The epitopes on type IX collagen then suddenly become undetectable just before this matrix swells and becomes populated by the periocular mesenchymal cells (future keratocytes). In comparison, collagen type II (along with type I) is present in the stroma before and long after these events. Deposition of immunodetectable type IX collagen in the developing corneal stroma thus seems to be independent of type II. In the vitreous, we observed type IX collagen along with type II as soon as authentic vitreous could be identified and at all subsequent stages of development. In this tissue, therefore, the expression of collagen types IX and II appears to be coordinate.     

Human T-cell leukemia virus type I infection of CD4+ or CD8+ cytotoxic T-cell clones results in immortalization with retention of antigen specificity.

The human T-cell leukemia virus type I (HTLV-I) is capable of chronically infecting various types of T cells and nonlymphoid cells. The effects of chronic infection on the specific functional activities and growth requirements of mature cytotoxic T lymphocytes (CTL) have remained poorly defined. We have, therefore, investigated the results of HTLV-I infection of both CD4+ and CD8+ human CTL clones. HTLV-I infection resulted in the establishment of functional CTL lines which propagated indefinitely in culture many months longer than the uninfected parental clone. The infected cells became independent of the need for antigen (target cell) stimulation as a requirement for proliferation and growth. Like their uninfected counterparts, however, these HTLV-I-infected clones remained strictly dependent on conditioned medium from mitogen-stimulated T lymphocytes for their growth. This growth factor requirement was not fulfilled by recombinant interleukin-2 alone. Furthermore, the infected lines remained functionally identical to their uninfected parental CTL clones in their ability to specifically recognize and lyse the appropriate target cells. Our findings indicate that the major effects of HTLV-I infection on mature CTL consist of (i) the capacity for proliferation in the absence of antigen stimulation and (ii) a prolonged or immortal survival in vitro, but they also indicate that the fine specificity and cytolytic capacity of these cells remain unaffected.