Induction of gp70-specific suppressor T-cells in mice inoculated with virus-producing tumor cells.

Inoculation of BALB/c mice with syngeneic, murine leukemia virus (MuLV)-producing (murine sarcoma virus-transformed) Balb/3T3 tumor cells resulted in diminution of alloreactivity as measured in the mixed leukocyte culture (MLC) reaction. Cells that suppressed this response were identified in the spleens of tumor-bearing mice 10--14 days after inoculation. The suppressor cells were adherent, radiosensitive T-lymphocytes of the Lyt 1-, 2, 3+ phenotype. Mice inoculated with Gross MuLV (G-MuLV)-producing tumor cells, but not those inoculated with a nonproducing subclone of the same tumor cells, developed suppressor T-cells. The T-cell-mediated suppression of the MLC could be augmented by the admixture of G-MuLV antigen, similar to that replicated by the tumor cell, but not by the admixture of a Rauscher-type MuLV antigen which lacked the cross-reactive, type-specific antigens of the G-MuLV. Furthermore, this augmented suppression could be abrogated by the addition of monoclonal anti-gp70 antibody. These findings indicated that antigen-specific suppressor T-cells were induced in response to leukemia virus antigen shed from and/or expressed on tumor cells and that the suppressive activity involved the specific recognition of the gp70 portion of the virus.

Comparison of the allospecific and viral-specific immune responses to irradiated versus formaldehyde-fixed allogeneic Moloney lymphoma cells in CBA mice.

Two groups of adult CBA mice were immunized with 10-7 allogeneic Moloney lymphoma (YAC) cells. These YAC (H-2a) cells, which were either irradiated with 6000 R (Group i) or were formaldehyde fixed (Group II), were injected i.p. at weekly intervals for 3 weeks. Four days following the last injection, sera and lymphocytes were collected and tested in vitro for activity against either allospecific antigens (H-2d target cells) or viral-specific antigens, namely, Moloney leukemia virus (MLV). Both groups of animals developed measurable cellular and humoral immunity to the virally determined antigens. However, only the animals in Group i, immunized with irradiated cells, developed detectable immunity to H-2d. Immune and control lymphocytes were tested in microcytotoxicity tests and by 51Cr release. Antibody was assessed by complement-dependent cytotoxicity, indirect membrane immunofluorescence, virus neutralization, and antibody-dependent lymphocyte cytotoxicity. Group I serum, which had both anti-MLV and anti-H-2 antibodies, was absorbed with either living or formaldehyde-fixed YAC cells. The living cells were able to remove both H-2 and MLV antibodies. On the other hand, the formaldehyde-fixed cells removed no H-2 antibody but were able to remove MLV antibody, although less efficiently than living cells. These data indicate that formaldehyde fixation selectively impaired the H-2 antigens, leaving the viral antigenicity relatively intact. Differences between the immune responses to MLV-determined antigens and to H-2 antigens were demonstrated in many of the parallel in vitro tests.

Inactivation of tumor cell-associated feline oncornavirus for preparation of an infectious virus-free tumor cell immunogen.

Ultraviolet (UV) and thermal methods of inactivating the oncogenic potential of C-type particle-producing feline oncornavirus-induced tumor cells were developed. The techniques were evaluated by several parameters for their use in preparation of cellular immunogens. The UV inactivation dose required to reduce the number of focus-forming units per ml by 1 log10 for FL-74 lymphoblastoid cell-associated feline leukemia virus was 44,000 ergs/sq mm, and the thermal inactivation dose required to reduce the number of focus-forming units per ml by 1 log10 at 45 degrees was 16 min. Inactivation of greater than 6 log10 of virus per ml associated with 4 x 10(8) cells required a UV dose of 270,000 ergs/sq mm, 100 min at 45 degrees or 3 min at 56 degrees. All three treatments concomitantly destroyed the replicating potential of FL-74 cells as shown by their inability to propagate under normal growth conditions and to incorporate [3H]thymidine into nuclear DNA. UV inactivation and thermal inactivation at 45 degrees allowed the best retention of feline oncornavirus-associated cell membrane antigen. A 50% loss in antigenic activity was observed as a result of 56 degrees treatment, but this method was the only one that did not destroy the surface structural integrity of FL-74 cells.

Accelerated regeneration of trypsin-treated surface antigens of simian virus 40-transformed BALB/3T3 cells induced by X-irradiation.

The antigens of SV40-transformed BALB/3T3 cells measured by a radioisotopic footpad assay after removal by trypsin treatment regenerated in vitro in 3 to 6 hr. After X-irradiation with 3000 R, however, the antigens were regenerated to normal levels within 1 h. X-ray doses of between 1000 and 5000 R accelerated the regeneration of cell surface antigens, while X-irradiation with the larger dose of 8000 R did not. X-irradiation of nontrypsinized tumor cells was without effect. Possible mechanisms of this phenomenon are discussed.

Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses.

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or ß-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.

Endogenous murine leukemia viruses: frequency of radiation-activation and novel pathogenic effects of viral isolates.

Female C57BL/6 and BALB/c mice were injected i.p. with 0.06 microCi/kg or 0.5 microCi/kg of the short-lived alpha-emitting radionuclide 224radium at 3-day intervals. Infectious N-ecotropic XC+, and xenotropic C-type retroviruses were activated in several tissues in both strains. In C57BL/6 mice the activation of ecotropic and xenotropic virus was dose-dependent as observed 4 weeks after the start of irradiation. In BALB/c mice a few animals showed activation of ecotropic virus after four weeks of irradiation. The expression of xenotropic virus was similar in irradiated mice and controls. Viral antigen, indicative for viraemia, was not detected in irradiated or control animals. Antiviral antibodies were found in both control and irradiated mice but higher titers were found in the irradiated mice. Bone tissue-derived N-tropic XC+ virus isolates were found to be non-oncogenic in newborn mice of the parental strain. In contrast, the same virus isolates induced a novel pattern of disease, such as osteopetrosis and osteomas together with malignant lymphomas in NMRI mice. The data indicate that the pattern of endogenous murine leukemia virus activation by internal alpha-irradiation is dependent on the dose rate, and on the genetics of the mouse strain.

A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.

A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described.

The effect of some common inactivation procedures on the antigens of bovine herpesvirus 1.

Bovine embryonic kidney cells were infected with bovine herpesvirus 1 (BHV1) or were sham-inoculated. When cytopathic effect was apparent, the cells were treated with beta-propiolactone, formalin, heat (56 degrees C), or ultraviolet irradiation until the virus was inactivated. Infected-treated, infected-untreated (IU) and sham-inoculated cultures were solubilized using Triton X-100 detergent. Resulting preparations were tested by 2-dimensional- and fused rocket-immunoelectrophoresis and were evaluated for their ability to inhibit virus neutralization by BHV1 antiserum. Eleven viral antigens were detected consistently in IU preparations, which strongly inhibited virus neutralization. Eight or more IU antigens were detected in beta-propiolactone-treated, formalin-treated and heat-treated preparations; these inhibited virus neutralization less strongly than the IU preparations. No IU antigens were detected in ultraviolet-treated preparations, nor did this material inhibit virus neutralization. One of the IU antigens was reduced preferentially by all treatments. The selective destruction of antigens by the various treatments might allow antigen-specific serological testing to distinguish vaccinated from naturally-exposed cattle.

Gamma irradiation alters bluetongue virus protein antigen.

Bluetongue virus (BTV) antigen, prepared for a monoclonal antibody (MAb)-based competitive enzyme-linked immunosorbent assay (C-ELISA), was exposed to 1, 2, 3, 4, 5 and 6 Mrad of gamma irradiation. The major group-specific BTV protein (VP7) reactive with the Mab was altered at higher doses of radiation, as revealed by immunoblotting studies. As well, a reduction in immunoreactivity was noted when irradiated antigen was used in the ELISA.

UV-inactivation of Epstein-Barr virus: differences in early antigen expression in two different non-productive cell lines and influence of caffeine.

Two non-productive Epstein-Barr (EB) virus genome-carrying lymphoblastoid cell lines, namely Raji and NC37, were used for studying the effect of UV irradiation on the ability of P3HR-1 EB virus to induce early antigen (EA) formation. In NC37 cells infected with UV-irradiated virus the formation of EA was delayed; thus the slope of inactivation curve based on the early (24 hr) reading was steeper than that based on the late (72 hr) reading. This was not observed in Raji cells. Caffeine did not influence the percentage of EA positive cells in cultures infected with untreated virus; however, the drug exhibited a marked inhibitory effect on EA production after infection with UV-irradiated virus. The sensitivity to caffeine effect decreased more rapidly with time after infection of Raji than of NC37 cells, suggesting a higher degree of readiness of the host cell repair system in the former than in the latter cells. The caffeine effect was merely directed against the synthesis of R (restricted) component of EA; its influence on the D (diffuse) component formation was negligible.

[Principles of selective inactivation of the virus genome. IV. The effect of UV-irradiation of phage MS2 on its binding with anti-MS2-immunoglobulins]. / Printsipy selektivnoi inaktivatsii virusnogo genoma. IV. Vliianie UF-oblucheniia faga MS2 na ego sviazyvanie s anti-MS2-immunoglobulinami.

Ultraviolet (254 nm) irradiation of the bacteriophage MS2 results in the decrease of the number of antigenic determinants exposed on the virion surface. The cross-section of the decrease, as measured by the number of anti-MS2 IgG molecules bound per virion, is 10(-16) mm2 per photon. The decrease of the phage-antibody binding proceeds after irradiation with a rate constant of about 5 x 10(-3) min-1. Since the antigenic determinants of the phage MS2 coat protein does not contain photoreactive amino acid residues, the irradiation-induced decrease of the phage antibody binding is determined, most probably, by the shielding of the antigenic determinants. Such shielding could be caused by rearrangement of coat protein molecules and/or of the capsid induced by photomodification of non-antigenic fragments of coat protein and/or of intraphage RNA.

[Immunogenic activity of cultural antirabies vaccine inactivated by gamma-rays]. / Immunogennaia aktivnost' kul'tural'noi antirabicheskoi vaktsiny, inaktivirovannoi gamma-luchami.

Experimental lyophilized tissue culture rabies vaccine inactivated with gamma-rays was prepared from the Vnukovo-32 strain and showed a sufficiently high immunogenic activity. Complete inactivation of the virus and the immunogenic potency of the vaccine depended upon the dose of irradiation and temperature during irradiation. The virus lost infectivity at 18 degrees C when irradiated with 2.6 X 10(6) r and at --70 degrees C with 4.55 X 10(6) r. However, the immunogenic potency of the vaccine inactivated at minus temperature without thawing was significantly higher.

[The immunity indices of BALB/c mice immunized with an inactivated antigen of the Machupo virus]. / Pokazateli immuniteta u myshei linii BALB/c, immunizirovannykh inaktivirovannym antigenom virusa Machupo.

The paper presents the data characterizing parameters of specific and nonspecific immunity in BALB/c mice immunized with gamma-ray-inactivated Machupo virus antigen or its formalinized antigen. The gamma-ray inactivated preparation was shown to be more immunogenic for BALB/c mice. A certain relationship between the time course of activity of nonspecific immunity factors in the immunized animals and the protective activity of the preparation under study was also noted. The decisive role of the T-cell part of the immune system was demonstrated in the resistance of this model animal to Machupo virus infection.

[Antigenic activity of a gamma-ray inactivated, concentrated, purified cultured antirabies vaccine]. / Antigennaia aktivnost' inaktivirovannoi gamma-luchami kontsentrirovannoi ochishchennoi kul'tural'noi antirabicheskoi vaktsiny.

Concentrated purified cultural rabies vaccine inactivated with gamma-rays caused in intramuscular injection (2 ml twice at an interval of 23 and 21 days) production of virus-neutralizing antibodies both in experiments on animals and in the vaccinated volunteers in titres not below those obtained in persons given a complete course of cultural rabies vaccine inoculations. No untoward reactions occurred.

[Biological consequences of the decay of 3H and 14C incorporated into the influenza virus]. / Nekotorye biologicheskie posledstviia raspada 3H i 14C, inkorporirovannykh v virus grippa.

An influenza virus labeled with 3H-uridine loses its infectiousness when stored for a long time. It is suggested that disintegration of tritium incorporated into virus RNA causes lethal intramolecular modifications therein. At the same time, the antigenic activity of virus nucleoprotein decreases perhaps due to the direct effect of tritium. The comparison of the degree of inactivation of various antigenic sites of the nucleoprotein within a virus labeled with 3H-uridine, suggests that they are located at different distances from RNA. A long-term action of 3H disintegration on RNA of a maturing virus decreases the yield probably due to the injury of the intracellular virus RNA during the infections process. Upon storage of the influenza virus labelled with 14C-amino acids the antigenic properties are reduced by the nucleoprotein while the infectiousness remains unaffected. The long-term effect of 14C disintegration on proteins of the maturing virus does not lead to fatal outcome.

Ultraviolet inactivation of Epstein-Barr virus: effect on synthesis of virus-associated antigens.

The relative sensitivity to ultraviolet (UV) light of genome functions of the P3HR-1 strain of Epstein-Barr virus (EBV) was studied. The formation of viral capsid antigen (VCA) appeared to be more sensitive than that of early antigen (EA), while the synthesis of membrane antigen (MA) was most resistant, as seen on examination in the presence of cytosine arabinoside (Ara-C). However, the appearance of both VCA and EA, but not that of MA, was delayed with UV-irradiated virus, in either the presence or absence of Ara-C. The synthesis of EA and VCA induced by UV-irradiated virus was suppressed in the presence of Ara-C, while that of MA was not.

Effect of gamma irradiation on reactivity of rinderpest virus antigen with bovine immune serum in enzyme-linked immunosorbent assays and virus neutralization and indirect fluorescent-antibody tests.

Gamma irradiation effectively inactivated gradient-purified rinderpest virus. Irradiated antigen and sera remained functional in enzyme-linked immunosorbent assays, virus neutralization tests, and indirect fluorescent-antibody tests. Irradiation, however, led to a dose-dependent decrease in reactivity, particularly significant (P < 0.05) when both reagents were irradiated. To avoid false-positive reactions, only one reagent (serum or antigen) may be irradiated.