Investigation of the exclusive 3He(e,e' pn)1H reaction.

Cross sections for the 3He(e,e' pn)1H reaction were measured for the first time at energy transfers of 220 and 270 MeV for several momentum transfers ranging from 300 to 450 MeV/c. Cross sections are presented as a function of the momentum of the recoil proton and the momentum transfer. Continuum Faddeev calculations using the Argonne V18 and Bonn-B nucleon-nucleon potentials overestimate the measured cross sections by a factor 5 at low recoil proton momentum with the discrepancy becoming smaller at higher recoil proton momentum.

Quasifree pi0 and pi- electroproduction on 4He in the Delta-resonance region.

The reactions 4He(e, e' p3He)pi- and 4He(e, e' p3He)pi0 were studied simultaneously, and for the first time, in a large kinematical domain including the Delta-resonance region. This was achieved by detecting the recoiling 3He and 3H nuclei instead of the emitted pions. The dependences of the cross section on the recoil momentum p(rec), the invariant mass WpiN, and the direction thetapi,q' and phipi,q' of the produced pion, are globally well described by the results of (quasifree) distorted-wave impulse approximation calculations. However, in the Delta-resonance region there are clear discrepancies, which point to medium modifications of the Delta in 4He.

Electron-induced neutron knockout from 4He.

The differential cross section for electron-induced neutron knockout in the reaction 4He(e,e(')n)(3)He has been measured for the first time with a statistical accuracy of 11%. The experiment was performed in quasielastic kinematics at a momentum transfer of 300 MeV/c and in the missing-momentum range of 25-70 MeV/c. The comparison of the data with theoretical calculations shows an impressive increase of the cross section resulting from final state interaction effects. Specifically, the p-n charge-exchange process dominates the cross section in this kinematical regime.

Vaccination of cats against feline immunodeficiency virus (FIV): a matter of challenge.

So far, most apparently successful immunodeficiency virus vaccines have only been tested against challenge with cell culture-adapted virus. However, even live priming of cats with feline herpesvirus (FHV) vectors expressing the FIV gag and env gene followed by inactivated booster vaccination with fixed FIX infected cell vaccine proved non-protective against infection with a primary FIV isolate. An effective FIV vaccine for field applications therefore is still not underway.

Evaluation of subunit vaccines against feline immunodeficiency virus infection.

Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase (K-SU3) or glutathione-S-transferase (G-SU3). Quantitative and qualitative differences in the humoral immune response were observed with three adjuvants of which Quil A was the best in terms of total and virus neutralizing antibody. Notwithstanding the responses induced, 19 of 20 immunized cats did not resist challenge and became infected. To determine whether priming with a live viral vector would confer protection, cats were inoculated oronasally and subcutaneously with a feline herpesvirus (FHV) mutant expressing the FIV env gene; two booster immunizations followed using the K-SU3 product in either Quil A or a mineral oill Al(OH)3 adjuvant. FIV-specific antibody responses were only weak, and the vaccinates did not withstand challenge with a low dose of homologous virus.

Vaccination against feline immunodeficiency virus using fixed infected cells.

Crandell feline kidney cells and feline thymocytes, either feline immunodeficiency virus (FIV) infected or uninfected, were fixed with paraformaldehyde and used to vaccinate cats. The cells were mixed with a 30:70 water/mineral oil emulsion containing 250 micrograms ml-1 N-acetyl-D-glucosaminyl-beta-(1-4)-N-acetyl-muramyl-L-alanyl-D-isoglutam ine. Eighteen specific pathogen-free cats were vaccinated three times with 3-week intervals and challenged 21 days after the final boost with a low dose of the homologous FIV-UT113 strain. Eight out of ten cats that had received FIV-infected cell vaccines developed significant anti-FIV antibody titres to the envelope and core antigens. Neutralizing antibodies were detectable at the moment of challenge in the sera of these animals. Within 5 weeks after challenge 15 out of 18 cats became viraemic. Three animals, two that had been vaccinated with FIV-infected thymocytes and did not develop antibody, and one that had received an uninfected thymocyte preparation, remained uninfected for 6 months. Upon rechallenge of the three animals, two again resisted infection; these cats had been immunized with the infected and the uninfected thymocyte preparations, respectively.

Monoclonal antibodies to immunodominant and neutralizing domains of the envelope surface protein of feline immunodeficiency virus.

Hybridomas secreting monoclonal antibodies (MAbs) specific for the surface protein (SU) of feline immunodeficiency virus were generated. Four MAbs were obtained which could be assigned to two groups based on their neutralization and competition behaviour. Using SU protein fragments expressed in Escherichia coli the antigenic site recognized by one of the MAbs (2H11) could be mapped to the c terminus. The neutralizing MAb 1E1 did not bind to any of the SU protein fragments and was directed to a conformational epitope. Binding of the MAb 1E1 to native SU protein could be blocked with a rabbit serum raised against the SU3 fragment (amino acids 361 to 445). These data indicate that at least part of the epitope is located on this SU3 domain. In competition experiments most sera of naturally infected cats were able to inhibit binding of the MAbs. This shows the conserved and immunodominant nature of the epitopes involved.

Antibody response in cats to the envelope proteins of feline immunodeficiency virus: identification of an immunodominant neutralization domain.

Overlapping fragments of the envelope protein of feline immunodeficiency virus (FIV) have been expressed in Escherichia coli. Screening of cat sera for antibodies to these fragments revealed that the immunodominant domain of the FIV envelope is localized within the transmembrane protein (amino acids 687-741) and that both the variable region 3 (SU3, aa 385-417) and the COOH-terminus (aa 599-611) of the surface protein (SU) are highly immunogenic. Of all rabbit sera raised to the envelope protein fragments only the serum directed to SU3 was neutralizing. Both FIV-infected and SU3-immunized cats elicited neutralizing antibodies to SU3. Neutralizing antibodies in sera of infected cats could be absorbed by SU3, showing that SU3 is a major neutralization domain of FIV.

Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis.

The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence cleavage occurred in Sf9 cells expressing the envelope precursor, but further processing was not observed. Crude lysates of insect cells infected with the wild-type baculovirus or with the recombinant viruses were used to develop an enzyme-linked immunosorbent assay for the detection of feline immunodeficiency virus-specific antibodies in cat sera. The assay showed a higher sensitivity and specificity than immunofluorescence and Western blotting (immunoblotting).

Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein.

Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.

Both Fc receptors and lymphocyte-function-associated antigen 1 on human T gamma lymphocytes are required for antibody-dependent cellular cytotoxicity (killer cell activity).

A monoclonal antibody, designated CLB-LFA-1/1, directed to the human lymphocyte-function-associated antigen 1 (LFA-1) was raised by immunization of mice with the peripheral blood lymphocytes of a T gamma lymphocytosis patient. The monoclonal antibody was selected by inhibition of the natural killer cell and the antibody-dependent killer cell activity of the patient's T gamma lymphocytes. In addition, the monoclonal antibody was shown to inhibit the cytotoxic activity of T cell clones specific for either class I or class II HLA molecules. The antigen recognized by CLB-LFA-1/1 consisted of three polypeptide chains with molecular weights of 180 000 (alpha), 155 000 and 94 000 (beta). The antibody reacted with T cells, B cells, monocytes and granulocytes, and stained normal T gamma cells and T gamma cells of patients with T gamma lymphocytosis two- to threefold stronger than normal T cells. It was shown that LFA-1 and the Fc receptor on T gamma cells did not comodulate and it is therefore concluded that Fc receptors and LFA-1 are independent membrane structures, both required for the killer cell activity of T gamma cells.

H-2 control of the cytotoxic antibody response against a newly defined MuLV-related cell-surface antigen: G(B10.A).

H-2-congeneic C57BL mice with milk transmission of B-tropic murine leukemia virus (V+ mice) have a much higher lymphoma incidence than the same strains without milk-transmitted virus (V- mice). Gene(s) within the major histocompatibility complex (H-2) influence virus titers, lymphoma incidence, lymphoma type and the anti-MuLV envelope antibody response. In this paper, we report that the prevalence of cytotoxic antibodies to virus-induced lymphomas is also regulated by the H-2 complex. Milk transmission of MuLV resulted in the formation of cytotoxic antibodies against primary virus-induced C57BL lymphomas. These antibodies detect an antigen that is also present on the RADAI tumor-cell line, and on normal spleen cells of young adult B10.A (H-2a) mice of both V+ and V- sublines, but not on spleen cells of young adult B10 (H-2a) mice of either subline. These cytotoxic antibodies were detected in the sera of B10V+ and B10.A(5R)V+ animals, but not in the sera of B10.AV+ mice. This indicates that the prevalence of these antibodies is controlled by a gene in the K- and/or I-A region of the H-2 complex. The presence of these cytotoxic antibodies in serum is recessively inherited. The specificity of the cytotoxic antibodies was investigated with a standard panel of transplantable tumor-cell lines. Of these, only the RADAI cells expressed the target antigen in direct cytotoxicity tests and by absorption. The ability of B10V+ sera to lyse the B10.AV+ and RADAI tumor cells is ascribed to antibody activity against a new MuLV-related cell-surface protein: G(B10.A). Immunochemical analysis and absorption experiments with different types of purified MuLV and MuLV-infected cell lines indicate that the cytotoxic antibodies belong to low-avidity IgM antibodies that are directed to MuLV.

Naturally occurring leukemia viruses in H-2 congenic C57BL mice. III. Characterization of C-type viruses isolated from lymphomas induced by milk transmission of B-ecotropic virus.

The prevalence of different host-range classes of murine leukemia virus (MuLV) was studied in C57BL mice with (V+) and without (V-) milk transmission of a naturally occurring B-tropic ecotropic MuLV. Virus isolates were studied with respect to growth properties, XC-plaque formation, antigen profiles of their envelope proteins (gp70 and P15(E)), gp70 tryptic-peptide maps, and their potential to induce lymphomas after inoculation into newborn mice. B-tropic ecotropic MuLV with the capacity to cause plaques in XC cells was isolated from almost all lymphomas of both V+ and V- sublines. The reaction patterns of these ecotropic isolates with monoclonal antibodies reactive with MuLV-env proteins and the tryptic-peptide maps of the gp70 molecule indicate that they are similar to each other and differ only slightly from the ecotropic MuLV in the spleens of young V+ animals, which is identical to the milk-transmitted virus. XC-, B-tropic dualtropic mink cell focus-inducing (MCF) viruses were isolated from the majority of different types of lymphoma (B cell, T cell, or neither B nor T cell derived), but not from the spleens or milk of young V+ or V- animals. The env proteins of the MCF isolates are highly heterogeneous, but most isolates originating from B10.AV + T-cell lymphomas share MCF-related epitopes in their gp85 envelope precursor with AKR MCF-247 virus. Most MCF viruses isolated from non-T lymphomas do not possess these epitopes. The results indicate that also in this model the generation of dualtropic MCF viruses might be important in lymphoma induction, although only some of the cloned MCF viruses show enhanced oncogenic properties in comparison with ecotropic isolates. A cloned oncogenic MCF virus induced different lymphoma types in C57BL/10 (= B10, H-2b) and B10.A (H-2a) mice, similar to what was found earlier with the milk-transmitted virus. Hence, the lymphoma-type differences are not due to differences in the B-tropic ecotropic viruses transmitted through the milk in these strains, but reflect an influence of the H-2 complex on the phenotype of the virus-induced lymphomas.

Identification of Rauscher murine leukemia virus-specific mRNAs for the synthesis of gag- and env-gene products.

Polyadenylylated mRNA isolated from cells infected with Rauscher murine leukemia virus was fractionated by centrifugation in in a denaturing sucrose gradient into different sizes. Each RNA fraction was injected into oocytes of Xenopus laevis and the virus-specific products were analyzed by immunoprecipitation with polyvalent and monospecific antisera against polypeptides of Rauscher murine leukemia virus, and then by gel electrophoresis and scintillation autoradiography. It was shown that a 35S mRNA species directs the synthesis of a precursor of the internal or group-specific antigens of the virion (the gag-gene products). A 22S mRNA species directs the synthesis of two viral envelope polypeptides and their precursor polypeptide (env-gene products). The results indicate that the gag- and env-related polypeptides of Rauscher murine leukemia virus are synthesized uncoordinately and provide evidence for open and closed cistrons on the virus-specific mRNAs.