Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity- purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity- purified, allergen-specific anti-idiotypic antibodies induced anti- allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.

The use of murine polyclonal anti-idiotypic antibodies as surrogate allergens in the diagnosis of Parthenium hypersensitivity

Background: Anti-idiotypic antibodies (Ab-2), which are the mirror images of idiotypic antibodies (Ab-1), may be useful as diagnostic reagents and for use as immunogen to induce antigen-specific immune responses. Methods and Results: To explore the biologic potential of Ab-2 as diagnostic reagents in allergic diseases, murine mouse (m) Ab-2 were raised by immunizing Balb/c mice with affinity purified rabbit (r) Ab-1 specific for the pollen of Parthenium hysterophorus, an allergenic weed that grows wild on the Indian subcontinent and in Australia, Mexico, and the southern United States. Affinity purified Parthenium-specific human (h)AB-1 could successfully inhibit the binding of mAb-2 to immobilized rAb-1. Further, Balb/c mice immunized with mAb-2 induced Parthenium-specific anti-anti-idiotypic IgE and IgG antibodies. Specificity of the Ab-2 was confirmed by the ability of Parthenium pollen extracts to inhibit the binding of allergen-specific IgE and IgG Ab-1 in the sera of patients with rhinitis to immobilized mAb-2. Parthenium-sensitive patients with rhinitis who had positive results on skin prick tests to Parthenium pollen extracts also responded with a positive skin reaction to mAb-2. Conclusion: Our data demonstrate that Parthenium-specific mAb-2 may be of value as surrogate allergens in allergen standardization and for in vitro diagnosis.

Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: Critical roles of IFN gamma

Mycobacterium indicus pranii (MIP) is approved for use as an adjuvant (Immuvac/Cadi-05) in the treatment of leprosy. In addition, its efficacy is being investigated in clinical trials on patients with tuberculosis and different tumors. To evaluate and delineate the mechanisms by which autoclaved MIP enhances anti-tumor responses, the growth of solid tumors consisting of Sp2/0 (myeloma) and EL4 (thymoma) cells was studied in BALB/c and C57BL/6 mice, respectively. Treatment of mice with a single intra-dermal (i.d.) injection of MIP 3 days after Sp2/0 implantation greatly suppresses tumor growth. MIP treatment of tumor bearing mice lowers Interleukin (IL)6 but increases IL12p70 and IFN? amounts in sera. Also, increase in CD8+ T cell mediated lysis of specific tumor targets and production of high amounts of IL2 and IFN? by CD4+ T cells upon stimulation with specific tumor antigens in MIP treated mice is observed. Furthermore, MIP is also effective in reducing the growth of EL4 tumors; however, this efficacy is reduced in Ifn?-/- mice. In fact, several MIP mediated anti-tumor responses are greatly abrogated in Ifn?-/- mice: increase in serum Interleukin (IL)12p70 amounts, induction of IL2 and lysis of EL4 targets by splenocytes upon stimulation with specific tumor antigens. Interestingly, tumor-induced increase in serum IL12p70 and IFN? and reduction in growth of Sp2/0 and EL4 tumors by MIP are not observed in nonobese diabetic severe combined immunodeficiency mice. Overall, our study clearly demonstrates the importance of a functional immune network, in particular endogenous CD4+ and CD8+ T cells and IFN?, in mediating the anti-tumor responses by MIP.

DC-SIGN and alpha 2,6-sialylated IgG Fc interaction is dispensable for the anti-inflammatory activity of IVIg on human dendritic cells

Intravenous immunoglobulin (IVIg) is widely used to treat autoimmune diseases. Several mutually nonexclusive mechanisms are proposed to explain the beneficial effects of IVIg in patients (1, 2). Lately, Ravetch and colleagues (3) demonstrate that anti-inflammatory activity of IVIg is mediated mainly by antibodies that contain terminal _2,6-sialic acid linkages at the Asn297-linked glycan of Fc region.

Analysis of a Genomic DNA Expression Library of Mycobacterium tuberculosis Using Tuberculosis Patient Sera: Evidence for Modulation of Host Immune Response

DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments, The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins, Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, others whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients, This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab2) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab2 was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab2 resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab2, which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab2. It provides evidence that Ab2 is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab(2)) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab(2) was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab(2) resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab(2), which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab(2). It provides evidence that Ab(2) is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

Timing the Emergence of Resistance to Anti-HIV Drugs with Large Genetic Barriers

New antiretroviral drugs that offer large genetic barriers to resistance, such as the recently approved inhibitors of HIV-1 protease, tipranavir and darunavir, present promising weapons to avert the failure of current therapies for HIV infection. Optimal treatment strategies with the new drugs, however, are yet to be established. A key limitation is the poor understanding of the process by which HIV surmounts large genetic barriers to resistance. Extant models of HIV dynamics are predicated on the predominance of deterministic forces underlying the emergence of resistant genomes. In contrast, stochastic forces may dominate, especially when the genetic barrier is large, and delay the emergence of resistant genomes. We develop a mathematical model of HIV dynamics under the influence of an antiretroviral drug to predict the waiting time for the emergence of genomes that carry the requisite mutations to overcome the genetic barrier of the drug. We apply our model to describe the development of resistance to tipranavir in in vitro serial passage experiments. Model predictions of the times of emergence of different mutant genomes with increasing resistance to tipranavir are in quantitative agreement with experiments, indicating that our model captures the dynamics of the development of resistance to antiretroviral drugs accurately. Further, model predictions provide insights into the influence of underlying evolutionary processes such as recombination on the development of resistance, and suggest guidelines for drug design: drugs that offer large genetic barriers to resistance with resistance sites tightly localized on the viral genome and exhibiting positive epistatic interactions maximally inhibit the emergence of resistant genomes.

An avidin-biotin microELISA for rapid measurement of total and allergen-specific human IgE

his paper describes an improved microtiter solid-phase enzyme immunoassay for the determination of total and allergen-specific human IgE. This assay technique is unique in its use of the avidin-biotin interaction to increase sensitivity. The avidin-biotin microtiter enzyme-linked immunosorbant assay (AB-microELISA) was performed in polyvinyl chloride microtiter plates using biotinylated anti-IgE and horseradish peroxidase (HRP)-avidin conjugate. This AB-microELISA technique enabled the quantitation of human serum IgE in the range of 0.1–5 ng/ml (10–500 pg/test) in less than 3 h. Total serum IgE, whether measured by the AB-microELISA or the paper radioimmunosorbant test (PRIST) was similar (correlation coefficient, r = 0.92). Further, the presence or absence of positive skin tests to 7 specific allergens determined in serum donors generally agreed with the presence or absence of allergen-specific IgE in their sera as measured by the AB-microELISA. The quantity of short ragweed allergen-specific IgE as determined by the AB-microELISA agreed with values obtained by the radioimmunosorbant test (RAST) (correlation coefficient, r = 0.89). No significant interference by ragweed-specific IgG (blocking antibody) was observed in the quantitation of allergen-specific IgE. The AB-microELISA is not only rapid and inexpensive, but also more sensitive than other published ELISA procedures and comparable to solid-phase radioimmunoassays in the quantitation of total and allergen-specific IgE.

Protein A: nature's universal anti-antibody

Staphylococcal protein A specifically interacts with immunogobulins. This fact is being used in various disciplines of biology and some of the unique properties of protein A and their applications are summarized in this review.

Crystallization and preliminary X-ray studies of the anti-T lectin from peanut (Arachis hypogaea)

The anti-T lectin from peanut (Arachis hypogaea) crystallizes in the orthorhombic space group P21212 with one tetrameric molecule (Mr 110,000) in the asymmetric unit in a cell of dimensions a = 129.3 Å, B = 126.9 Å and C = 76.9 Å. The crystals are suitable for high resolution work.

Structure and conformation of an anti-depressant drug, nitroxazepine hydrochloride monohydrate

CIsH20N3Oa+.C1-.H2 O, M r = 395, orthorhombic, Pn21a, a = 7.710 (4), b = 11.455 (3), c -- 21.199 (3)/k, Z = 4, V = 1872.4/k 3, D m = 1.38, D C = 1.403 g cm -3, F(000) = 832, g(Cu Kct) = 20.94 cm -l. Intensities for 1641 reflections were measured on a Nonius CAD-4 diffractometer; of these, 1470 were significant. The structure was solved by direct methods and refined to an R index of 0.045 using a blockdiagonal least-squares procedure. The angle between the least-squares planes through the benzene rings is 125.0 (5) ° and the side chain is folded similarly to one of the independent molecules of imipramine hydrochloride.