Serum Neutralizing Activity Research Articles

Use of protein A-treated sera in unmasking herpes simplex virus type 1 (HSV-1) immunoglobulin A and identifying HSV-1 immunoglobulin G as the predominant neutralizing antibody

Treatment of human sera with protein A reduced the amounts of immunoglobulin G (IgG), IgA, and IgM detected by radial immunodiffusion. This treatment also decreased the amount of herpes-specific IgG and IgM detected by radioimmunoassay, whereas it increased and even unmasked the amount of herpes-specific IgA detected. Comparison of protein A-treated sera with untreated sera indicated that herpes simplex virus type 1 IgG was responsible for more than 92 to 99% of the serum neutralizing activity.

Kinetics of different specific immunological parameters after rabies vaccination in mice.

A significant protection to an intracerebral challenge of 70 mean lethal doses of a standard live rabies virus strain was obtained in BCG-pretreated mice or in normal mice which had been immunized with a single subcutaneous injection of a beta-propiolactone-inactivated rabies vaccine. Concomitantly, levels of delayed-type hypersensitivity (measured in vivo by the footpad test) and serum-neutralizing activity were evaluated at various times after immunization. All immune criteria were significantly augmented in the BCG-pretreated, rabies-immune mice as compared to normal, rabies-immune mice. However, peak levels of protection, delayed-type hypersensitivity, and serum-neutralizing activity did not occur at the same times. For instance, in the BCG-pretreated, rabies-immune mice, delayed-type hypersensitivity peaked on day 7, protection peaked on day 21, and serum-neutralizing activity peaked on day 60. In BCG-pretreated mice, which did not receive the rabies vaccine, positive delayed-type hypersensitivity, some protection, and serum neutralizing activity were observed 4 to 5 weeks after BCG pretreatment. The possible relationships between specific and nonspecific immunity provoked by rabies virus antigens, tissue culture cell-associated antigens (derived from the bovine fetal kidney cells in which the rabies virus was grown, and BCG are discussed.

Congenital Rubella after Maternal Reinfection

This report concerns a boy with congenital rubella virus infection. The diagnosis was confirmed by virus isolation, demonstration of rubella-specific serum IgM and by persistence of serum antibody at the age of 9 months. In 2 sera from the mother sampled 2 weeks apart 20 months before the birth of the boy, low titers of rubella antibody were demonstrated by hemagglutination-inhibition, hemolysis-in-gel and complement fixation tests, but not by neutralization. Significant rises in titer were demonstrable by all serologic reactions--including neutralization--at the time of birth of the infected child. The mother was not aware of any rubella-like illness or exposure to such disease during pregnancy. The case is discussed against findings of neutralizing activity in sera from natural immunes and rubella vaccinees.

Antibody-Independent Neutralization of Vesicular Stomatitis Virus by Human Complement

Abstract Normal human serum neutralized up to 5 logs10/ml of vesicular stomatitis virus (VSV) grown in hamster (BHK), mouse (L), or human (HFF) cell lines when equal volumes of virus (∼105 PFU/ml) and serum were incubated together for 30 min at 37°C. The neutralizing activity of fresh serum was heat labile, thus distinguishing this activity from that of immune human serum which was heat stable. The lack of an antibody requirement for VSV neutralization by normal human serum was demonstrated in three ways: first, isolated human immunoglobulins failed to neutralize VSV or potentiate neutralization by fresh serum; second, absorption of fresh serum with uninfected cells or cells bearing VSV antigens did not reduce neutralizing activity; third, agammaglobulinemic serum mediated C3b deposition on the viral surface. Neutralization by normal human serum was complement (C) dependent and required the early reacting components of the classical C pathway (C1q, C2, C4) but not the late reacting components (C5, C6, C7, C8) or the alternative C pathway (factor B). Incubation of serum with concentrated VSV reduced the CH50, C2, C3, and C4 titers by 23.5, 21, 22, and 31%, respectively, and was accompanied by deposition of C3b on the viral surface. Purified VSV and the isolated external glycoprotein of VSV, G protein, bound 125I-C1q, and purified VSV also bound C1̄. However, VSV did not activate purified reconstituted C1, and VSV was not neutralized by isolated C1, C2, C3, C4, and C5. Therefore, an additional serum factor is apparently required for neutralization. These studies indicate that neutralization of VSV by normal human serum involves the components of the classical C pathway through C3b and a nonimmunoglobulin serum factor. The reaction is initiated by the direct attachment of C1 to the G protein, which leads to C activation and the sequential deposition of the C components through C3b on the viral envelope. The mechanism of action of the nonimmunoglobulin serum factor remains to be established.

The effect of altered lymphocyte function on the immunologic disorders of NZB/NZW mice: Response to anti-thymocyte globulin

Defective cellular-immune responses are characteristic of NZB/NZW mice and are important in the pathogenesis of their lupus-like immune-complex nephritis. T-cell function was further suppressed by the administration of antithymocyte globulin (ATG) to young female mice; controls received rabbit IgG or no treatment. Administration of ATG resulted in decreased circulating lymphocytes and diminished ability of spleen cells to induce graft-versus-host reactions and to function as helper or suppressor cells in antibody formation. ATG treatment accelerated proteinuria and deaths from nephritis and increased circulating antibodies to sDNA and nDNA. Renal eluates from ATG-treated mice contained increased quantities of mouse γ-globulin and anti-DNA antibodies, but antibodies to ATG were not demonstrable. Levels of serum neutralizing activity and antibodies against xenotropic and ecotropic murine C type viruses were not altered. The administration of ATG probably accelerated anti-DNA antibody formation and immune nephritis by reducing T-cell suppression of autoantibody formation.

Antibodies against human lymphokines: I. Methods for induction of antibodies capable of neutralizing stable (α) and unstable (β) lymphotoxins released in vitro by activated human lymphocytes

Various methods were employed to induce antibodies in rabbits that were capable of neutralizing different families of lymphotoxins (LT). Both stable (α-LT) and unstable (β-LT) molecules, released by activated human lymphocytes in vitro, were neutralized. The different LT families were first separated into their respective groups by physical-chemical methods. Immunization with small quantities of antigen yielded a high percentage of responder animals. Techniques were developed for eliciting α-LT antibodies using as little as 2–3 ml of a cell-free supernatant. The situation was more difficult, however, when the unstable β-LT molecules were employed as antigens. We found that because of the low concentration and lability of β-LT in supernatants, the immunizing dose had to be: a) handled rapidly, b) larger than that used with the α-LT, and c) injected at closer intervals and over a longer immunization protocol. Physical-chemical studies supperted the concept that the LT neutralizing activity in the immune serum was immunoglobulin.

Humoral Immunity in Experimental Syphilis

Abstract Evidence for a humoral mechanism in immunity to experimental syphilis was provided by the demonstration of immune rabbit serum factor(s) capable of inactivating virulent Treponema pallidum, Nichols strain, in an in vitro-in vivo neutralization test. After intratesticular infection, rabbits were bled periodically and their resistance to reinfection was determined by challenge with T. pallidum. The results of challenge showed that resistance to reinfection begins to develop by 11 days after infection, becomes complete by 3 months, and persists for at least 2 years. In the neutralization test, a mixture of treponemal suspension and serum from the infected animals was incubated anaerobically at 34°C and the virulence of the treponemes was determined by intradermal inoculation into normal rabbits. Complete inactivation of treponemes by immune serum required heat-stable and heat-labile (56°C, 30 min) serum components and 16 hr of incubation, and was accelerated by pre-incubation of the treponemes for 4 hr with nonimmune serum but not by 100 µg/ml of added lysozyme. Serum-neutralizing activity, first demonstrable 1 month postinfection, was quantitated by a neutralizing endpoint (NEP). A relatively close quantitative correlation was shown between the development of resistance to symptomatic reinfection and the appearance and persistence of both TPI antibody and neutralizing serum factor(s). The nature of the serum factor(s), the mechanism of treponemal inactivation, and the application of the test in assessing the immune status are discussed.

The immune response of zebu cattle to infection withTrypanosoma congolenseandT. vivax

Zebu cattle were infected with Trypanosoma congolense and T. vivax and changes in the levels of serum immunoglobulins were measured during infection and after treatment. Increases in the levels of IgM occurred within 14 days of infection coincidentally with the first appearance of trypanosomes in the peripheral blood. IgM levels rose to 2-10 times preinfection levels and remained high until the animals were treated with diminazene aceturate. Levels then declined to approximately pre-infection levels within 25 days. IgG levels showed little change. In one animal infected with T. congolense which died of trypanosomiasis, there was an initial increase in IgM levels but subsequently both IgM and IgG levels declined until the animal died. The neutralizing activity of whole serum, and serum IgM and IgG fractions from an ox infected with T. congolense were examined. High levels of neutralizing antibodies against trypanosomes isolated on the eighth day after infection were found in sera collected 12-29 days after infection. Most of the antibody activity was located in IgG fractions which showed a peak in neutralizing activity between day 17 and 22. IgG fractions of the sera showed only slight antibody activity.

Virus-specific neutralization by a soluble non-immunoglobulin factor found naturally in normal mouse sera.

A low-molecular-weight substance found naturally in mouse serum neutralizes mouse xenotropic C-type virus. It has no effect on endogenous ecotropic viruses. This neutralizing factor does not belong to the known immunogloblin classes, and its activity is not associated with the antivirus immunoglobulins that can be detected by radioimmunoprecipitation. Preparations of xenotropic virus absorb out this neutralizing activity in mouse sera. The specificity of this factor for X-tropic virus suggests that it represents a newly recognized type of response of the host to an endogenous virus. Its possible role in the regulation of endogenous C-type viruses is considered.

Two antigenically distinct species of human interferon.

Rabbit antisera prepared against interferon produced in human fibroblast cell cultures stimulated with poly(1).poly(C) neutralized the activity of interferon preparations produced in various human fibroblast cultures timulated either with poly(1)poly)C) or with viruses. However, these antisera showed no detectable neutralizing activity against interferon produced in cultures of human leukocytes. On the other hand, most rabbit antisera against the human leukocyte interferon were active in neutralizing both homologous interferon and fibroblast interferons. A preparation of antiserum against leukocyte interferon, active against both leukocyte and fibroblast interferons, was shown by affinity chromatography to have two distinct antibody populations, one of which was specific for the fibroblast interferon. We conclude that the heterologous neutralizing activity of sera from rabbits immunized with leukocyte interferon is liekly to be due to the presence of two antigenic species of interferon. The major antigenic species of leukocyte interferon preparations (designated "Le") is distinct from huamn fibroblast interferon. The minor species of leukocyte interferon ("F") is either identical with, or closely related to, interferon produced in human fibroblast cultures.

Neutralizing antibody response of guinea pigs to lymphocytic choriomeningitis virus.

A 24-h neutralization test that is based on fluorescent cell counting was used for the detection and quantitative determination of serum-neutralizing antibody against lymphocytic choriomeningitis virus. The earliest manifestation of serum-neutralizing antibody in guinea pigs was shown to occur within 1 week after inoculation with lymphocytic choriomeningitis virus. Within 11 weeks, serum-neutralizing antibody increased from 20- to 300-fold. Neutralizing end points of serum samples, obtained from 3 through 11 weeks, were enhanced as much as sevenfold by complement. Anti-guinea pig immunoglobulin G or anti-whole guinea pig serum potentiated the neutralizing activity of serum as much as 20- and 40-fold, respectively.

Neutralization of channel catfish virus by serum of channel catfish.

Sera from 71 adult channel catfish (Ictalurus punctatus), with a history of channel catfish virus (CCV) association, were assayed for CCV neutralization activity. Sixty-seven serum samples had positive CCV neutralization indices. Sera from 10 fish with no known history of CCV exposure, showed no evidence of virus neutralization activity. Viable CCV was not isolated from 232 organs or excretory products of studied fish. Serum from channel catfish experimentally immunized with viable CCV reached peak neutralization indices 60 days after virus injection. Detection of CCV serum neutralization activity can afford a possible method of identifying channel catfish populations with a previous exposure to CCV.

Heat-labile accessory factor involved in vaccinia virus plaque neutralization.

A heat-labile accessory factor present in native (unheated) normal serum was found to replace the vaccinia virus neutralizing activity of immune serum destroyed by heating at 56° C for 30 minutes. Treatment of serum samples with ammonium hydroxide or cobra snake venom did not significantly reduce the activity of immune serum. Also, inactivation studies with the properdin deficient (PR) fraction of immune serum after zymosam treatment did not alter the neutralizing activity of immune serum. The neutralization kinetics of vaccinia virus were altered in the presence of the accessory factor. However, the optimum conditions for neutralization were not changed. In the hemagglutination-inhibition (HI) test procedure, heating decreased the HI activity of vaccinia immune serum, but the addition of native normal rabbit serum did not restore the lost activity. Although individual components of complement might affect vaccinia neutralization, it appeared that a heat-labile accessory factor, other than properdin or complement, was required for the complete neutralizing activity of vaccinia immune serum.

Comparison of neutralizing activity in nasal secretion and serum of ferrets in response to infection with influenza A viruses.

Comparison of neutralizing activity in nasal secretion and serum of ferrets in response to infection with influenza A viruses.

Serum enterotoxin-neutralizing antibody in human shigellosis.

THE neurotoxin of Shiga's bacillus (Shigella dysenteriae 1), known for 70 years1 and nearly as potent as botulinus toxin2, has not been considered to play a role in human shigellosis3, partly because no strain of shigella other than Shiga's has been found to produce the exotoxin2. Isolation of an exo-entero-toxin in broth culture filtrates of S. dysenteriae 1 (ref. 4), closely related if not identical to this neurotoxin5, has warranted thorough reassessment of its pathogenetic significance. Our report is the first to document neutralizing activity of sera from patients with disease due to S. dysenteriae 1, as well as other shigellae, including dysenteriae 2 and flexneri and sonnei strains.

Foot-and-mouth disease: changes in serum-neutralizing activity of immunized cattle shortly after virus exposure.

After infection of the upper respiratory tract with foot-and-mouth disease (FMD) virus in immunized cattle, a drop in specific serum-neutralizing antibody activity was observed. Vaccinated cattle with high preexposure serum antibody titers had a precipitous drop for the first 4 days after virus exposure, followed by a rise in titer. This drop was also seen in passively immunized cattle with high preexposure titers; however, their antibody titers decreased for 5 to 7 days after exposure to virus. Some vaccinated and passively immunized cattle with low preexposure antibody titers showed fluctuations in their titers shortly after exposure. These data suggest that the course of infection with FMD virus in immunized cattle depends on the rapidity of new antibody formation as well as on the level of neutralizing antibody titer at the time of exposure.

Isolation of a herpes simplex virus-specific antigenic fraction which stimulates the production of neutralizing antibody.

Infection of mammalian cells with herpes simplex virus (HSV) results in the production of a number of virus-induced soluble antigens. Immunodiffusion analyses of the soluble antigen mixture (SAM) obtained from HSV-infected KB or BHK cells revealed at least six well-defined immunoprecipitin bands. Calcium phosphate chromatography (Brushite) was employed to separate one immunoprecipitin (designated CP-1) from the remaining viral and host antigens. We conclude that CP-1 is a viral-specific antigen because (i) specific antiserum, which had been repeatedly absorbed with uninfected cell extracts or serum components, still retained the capacity to react in gel diffusion with CP-1 antigen; (ii) anti-CP-1 serum reacted in gel diffusion with SAM, yielding one precipitin band in identity with the band formed against human gamma globulin; (iii) the CP-1 fraction stimulated the production of HSV-neutralizing antibody of high capacity. The last observation suggests that fraction CP-1 contains a biologically active structural component of the virus which is associated with the envelope. The CP-1 immunoprecipitin was separated from SAM by an alternative method by using a cyanogen bromide-linked immunosorbent prepared from anti-CP-1 gamma globulin. The observation that the CP-1 antigen isolated from the immunosorbent effectively blocked serum-neutralizing activity provided further evidence that neutralizing antibody was directed against CP-1. Acrylamide gel electrophoresis and immunological experiments suggest that the CP-1 antigen is in part a glycoprotein. The finding that CP-1 contains only one antigenic component of the virus will permit future biological studies to be made with a monoprecipitin antiserum. In addition, the techniques described in this paper represent initial steps in the purification of HSV antigens.

Infectious mononucleosis: appearance of neutralizing antibody to Epstein-Barr virus measured by inhibition of formation of lymphoblastoid cell lines.

Sera taken before illness and during convalescence from patients with infectious mononucleosis (IM) were examined for their ability to neutralize the capacity of Epstein-Barr virus (EBV) to stimulate formation of lymphoblastoid cell lines from human leukocytes in vitro. Neutralizing activity was not detected in 13 of 15 sera taken before illness and was present at a dilution of 1:4 or higher in all of 24 sera taken after illness. EBV-neutralizing activity was found in sera collected during the first week of illness and was also demonstrated in a serum obtained many years after illness. The titer of neutralizing activity in sera of two patients studied sequentially did not change significantly during the first year after IM. The results demonstrate appearance of EBV-neutralizing antibody in IM and support previous seroepidemiologic studies which indicate that EBV is the etiologic agent of IM.

Neutralizing Activity of Sera from Guinea-Pigs Inoculated with Foot-and-Mouth Disease Virus Variants

Summary The neutralizing activity of sera from guinea-pigs inoculated with small-plaque (SP) and large-plaque (LP) variants of foot-and-mouth disease virus (FMDV) was studied. Virus neutralization tests were performed in suckling mice using the SP and LP variants as well as a low passage virus of the same type and strain. Higher titres were obtained with homologous virus than when a vims of low passage in tissue culture was used. Neutralizing activity was not demonstrable in some guinea-pig sera with a low passage virus, whereas a significant level of neutralizing activity was detected when homologous virus-serum mixtures were tested.

Herpesvirus Neutralization: the Role of Complement

Abstract Complement functions in the neutralization of herpesvirus by causing immunoaggregation. Complement-requiring antibody (CRA) in early immune serum appears to behave like a monovalent antibody since it sensitizes virus to the action of complement, but immunoaggregates fail to develop until complement is added. It appears that each aggregate contains many virions and antibody molecules, but it functions only as a single infectious unit. The titer is reduced in proportion to the degree of immunoaggregation. Late immune serum is polyvalent since it is able to aggregate and neutralize virus in the absence of complement. However, complement may also enhance the neutralizing activity of such late immune serum. This enhancement can be detected only if monodispersed virus is used in the test system. Aggregates present in usual virus harvests resist neutralization (persistent fraction). Antibody can decrease virus titers only to the level of the number of virus aggregates present in the harvest.