IFN-gamma-independent IgG2a production in mice infected with viruses and parasites.

After infection with some viruses and intracellular parasites, antibody production is restricted to IgG2a. We first observed that, whereas live viruses such as lactate dehydrogenase-elevating virus (LDV) or mouse adenovirus induced mostly an IgG2a response, a large proportion of antibodies produced against killed viruses were IgG1. This IgG1 antiviral response was suppressed when live virions were added to inactivated viral particles. These results indicate that the IgG2a preponderance is related to the infectious process itself rather than to the type of antigen involved. Since IFN-gamma is known to stimulate IgG2a production by activated B lymphocytes and to be secreted after infection, we examined the role of this cytokine in the antibody isotypic distribution caused by LDV. Most IgG2a responses were relatively unaffected in mice deficient for the IFN-gamma receptor or treated with anti-IFN-gamma antibody. A similar IFN-gamma-independent IgG2a secretion was observed after infection with the parasites Toxoplasma gondii and Trypanosoma cruzi. However, the IFN-gamma-independent IgG2a production triggered by infection still required the presence of functional T(h) lymphocytes. Therefore, signal(s) other than IFN-gamma secretion may explain the T(h)-dependent isotypic bias in antibody secretion triggered by viruses and parasites.

Antibodies against enteroviruses in intravenous Ig preparations: great variation in titres and poor correlation with the incidence of circulating serotypes.

Antibody titres in immunoglobulin preparations for intravenous use were tested against 24 different enterovirus serotypes and varied between 1:100 and 1:10,000 within a single batch. Differences up to 8-fold were found for homologous titres between two different batches that were prepared 6 years apart. The lowest titre obtained was 1:40. The observed differences within and between the two batches could not be explained by different incidence of serotypes of enteroviruses circulating at the time blood was collected. Differences in titres of up to 18-fold were observed when several strains of the same serotype were tested suggesting that intratypic variation influences antibody titres. It is concluded that immunoglobulin preparations contain antibodies against many enteroviruses, but that titres can be low and cannot be predicted from the incidence of any particular serotype circulating in the community. Because of intratypic variation, selection of a batch for specific treatment should be based on results obtained with the patient's own isolate, and not with a reference strain.

Coxsackie B1 virus-induced murine myositis: a correlative study of muscular lesions and serological changes.

We investigated the role of humoral factors in the pathogenesis of Coxsackie B1 virus-induced murine myositis (CB1-myositis). At 2, 4 and 8 weeks after inoculation, serum was studied for circulating immune complexes (CIC) (Raji-cell assay), haemolytic complement activity (CH50 titre) and anticytoplasmic autoantibodies (Western blotting, immunoprecipitation) in relation to degree of mononuclear cell infiltration and muscle fibre necrosis. At 2, 4 and 8 weeks, cell infiltration correlated positively to muscle fibre necrosis. From 2 weeks on, moderate quantities of CIC were found in nearly all CB1-inoculated mice, but without correlation to histological changes. Except for a positive correlation of CH50 titre to muscle necrosis at 4 weeks (r = 0.60; P = 0.02), CH50 titres did not correlate to muscle lesions. Anticytoplasmic and other known autoantibody specificities were absent. In conclusion, first, in CB1-myositis CIC occurred from 2 weeks on, but no correlative evidence was found for their involvement in pathogenesis, neither for that of complement nor for anticytoplasmic autoantibodies. Secondly, cell infiltration correlated positively to muscle necrosis, underscoring the importance of cellular mechanisms. Thus, our data do not support, or conclusively exclude, a role for humoral processes in CB1-myositis.

Coxsackie B1 virus-induced murine myositis: relationship of disease severity to virus dose and antiviral antibody response.

Inoculation of Coxsackie B1 virus (CB1) in newborn CD1 Swiss mice induces a chronic myositis of proximal hindlimb muscles (CB1 myositis). To study the possible role of the virus dose, and of antiviral antibodies in the development of CB1 myositis, we infected groups of newborn mice with six CB1 doses, ranging from 30 to 10,000 plaque forming units (pfu); after 4 and 8 weeks we determined morbidity and antiviral antibody titer, and quantified histopathological changes. At 4 weeks, morbidity and mononuclear cell infiltration differed significantly for the various groups, with the most prominent changes in 300 pfu animals. At 4 and 8 weeks diseased animals had significantly higher antibody titers than clinically unaffected animals, and at 4 weeks myopathic and infiltrative changes correlated positively with the serum antibody titer. Our data indicate that the virus dose plays a pathogenic role in CB1 myositis, and they suggest further study on the role of humoral immune mechanisms in the early phase of CB1 myositis.

Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus.

IgG subclass distribution of primary and secondary immune responses concomitant with viral infection.

Infection with mouse hepatitis virus was found to selectively increase the proportion of IgG2a in antibodies elicited by a concomitant administration of unrelated T cell-dependent protein Ag. In contrast, T cell-independent responses were only marginally affected. This isotypic bias, which occurred when the virus was inoculated shortly before or after a primary immunization, persisted in subsequent secondary responses. However, infection concomitant to secondary antibody responses did not affect their isotypic distribution. These observations suggest that the virus can durably modify unrelated T cell responses that are initiated at the time of infection, which could have implications in the pathogenesis of autoimmune reactions.

Rheumatoid factor production and polyclonal non-antiviral antibody responses elicited by infection with common mouse viruses.

Rheumatoid factor (RF) production and antibody responses directed against antigens unrelated to infectious agents were measured in the serum of 129/Sv mice infected with various viruses. Some of these viruses induced a polyclonal B lymphocyte activation reflected by the presence of serum antibodies reacting in ELISA with dinitrophenylated albumin and with transferrin, but failed significantly to trigger the production of RF capable of agglutinating IgG2a-coated latex particles. In contrast, high RF serum levels were detected only in mice infected with an intestinal agent which was not able to induce other non-antiviral antibody responses. This observation indicates that the strong RF production previously reported in 129/Sv mice is a phenomenon specifically elicited by infection with this particular intestinal agent that cannot be explained by mere polyclonal B lymphocyte activation similar to that induced by common viruses.

Diagnosis of herpes simplex virus encephalitis by detection of virus-specific immunoglobulins A and G in serum and cerebrospinal fluid by using an antibody-capture enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was evaluated for detection of intrathecal synthesis of immunoglobulin G (IgG) and IgA antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). Since the antibody-capture principle was used and the assay was carried out at the saturation level of the anti-IgG- or anti-IgA-coated solid phase, correction for blood-brain barrier leakage was not needed. A total of 34 pairs of serum and cerebrospinal fluid specimens obtained from 20 patients with HSVE were examined. Intrathecal synthesis of HSV IgG and IgA was detected from day 7 after the onset of illness in patients with HSVE. Specimens from all 19 patients from whom paired serum and cerebrospinal fluid specimens were obtained at more than 10 days after the onset of illness were positive. Intrathecal synthesis of HSV IgG and IgA was not detected in patients with HSVE before day 7 of illness or in any of the 16 control patients with other causes of (meningo)encephalitis. Use of the antibody-capture enzyme-linked immunosorbent assay for HSV IgG and IgA allows the rapid diagnosis of HSVE during the second week of illness.

Elimination of murine viral pathogens from the caecal contents of mice by anaerobic preparation.

The aim of this study was to investigate methods to eliminate pathogenic viral agents while preserving the 'normalizing' properties of the gut microflora of mice. Mouse hepatitis virus A59 (MHV), Reo 3 virus and Theiler GD VII virus were added to the caecal contents of 'normal' mice and following dilution, with or without subsequent culturing, given to germ-free mice. Four weeks later antibody titres against these and other viruses were determined. MHV and Theiler CD VII virus survived dilution but were eliminated during culturing. Reo-virus survived the 10(-1) dilution-culture step. All dilutions and dilution-cultures of caecal contents resulted in 'normalization' in germ-free recipients of the relative caecal weight, percentage faecal fusiform-shaped bacteria, faecal bile acids and colonization of small intestine by segmented filamentous bacteria.

Virally induced modulation of murine IgG antibody subclasses.

The isotypic distribution of murine IgG was examined after infection with several viruses. The results indicate that when a hypergammaglobulinemia was induced by the infection, it was restricted to the IgG2a and, to a lesser extent, to the IgG2b subclasses. In addition, when mice were infected with some viruses concomitantly with the immunization with a soluble protein antigen, a modification in the isotypic distribution of antiprotein antibodies was observed, with a preferential production of IgG2a. These observations indicate that viral infections can actively influence the switch of Igs and selectively stimulate the production of the IgG2a subclass.

Antibody isotype response after human cytomegalovirus infection.

The antibody isotype response to human cytomegalovirus (CMV) was studied in paired sera from patients with primary and recurrent CMV infection. The majority of sera was obtained from immunocompromised patients. In the 48 patients with primary CMV infection, CMV-specific IgM (CMV-IgM) and IgG (CMV-IgG) antibodies were found in all patients, and CMV-specific IgA (CMV-IgA) and IgE (CMV-IgE) antibodies in 46 patients. CMV-IgM, -IgA, and -IgE antibodies were found in, respectively, 21, 31 and 4 of the 53 patients with recurrent CMV infection, and in, respectively, 3, 3 and 1 of the healthy controls. The results indicate that CMV-IgE is a better marker of primary CMV infection than CMV-IgM, and confirm that detection of CMV-IgM and -IgA may also be useful for diagnosis of recurrent CMV infection.

IgG2a restriction of murine antibodies elicited by viral infections.

The isotypic distribution of IgG antibodies was determined in the serum of mice after infection with a panel of RNA and DNA viruses representative of 11 different genera. The antiviral response induced by all these viruses showed a striking preponderance of the IgG2a subclass whatever the strain of mice tested or the time elapsed after infection. Together with the predominance of IgG1 in antiprotein and of IgG3 in anticarbohydrate response, this IgG2a restriction of antiviral antibodies strongly suggests the existence of highly specific mechanisms for the regulation of individual subclasses in the mouse.

Rheumatoid factor production in 129/Sv mice: involvement of an intestinal infectious agent.

High titers of IgG2a-specific rheumatoid factors (RF) are frequently observed in colonies of 129/Sv mice. The involvement of a transmissible agent in this phenomenon was shown by the following findings: (i) cesarean-derived and isolator-reared offsprings of RF-positive dams were free of RF, ii) a single intragastric inoculation with intestinal fluid from RF-positive donors elicited chronic RF production in RF-negative recipients, and iii) intestinal fluid collected from these primary recipients induced a comparable RF response in a second set of animals. The nature of this RF-inducing agent, however, remained elusive. Although its ability to pass through filters that efficiently retained bacteria could be unequivocally established, systematic serological analysis failed to detect any significant correlation between RF production and antibody responses to common mouse viruses or Mycoplasma. Moreover, all attempts to identify the RF-inducing agent by electron microscopy or to grow it in nude or newborn mice, as well as in cell cultures, remained unsuccessful.

Diagnosis of parainfluenza virus infection in children and older patients by detection of specific IgM antibody.

The significance of detecting specific antibody of the IgM class for the diagnosis of parainfluenza infections was examined. Paired sera from 763 children and adults admitted to the hospital for acute respiratory disease were tested for significant antibody titer rises in the hemagglutination inhibition (HI) test and for specific IgM antibody with the hemadsorption immunosorbent techniques (HIT). Sera were collected during two 6-month periods, January through June, 1982 and 1983. Evidence of parainfluenza infections was found in 122 patients (16%): 83 (25%) in 1982 and 39 (9.1%) in 1983. The HIT was superior to the HI test for detection of parainfluenza infection, in particular in infants and aged patients, 94 patients were positive only in the HIT test, 12 in the HI test, and 16 in both tests. In a control group of 120 persons (time- and age-matched to the patients of 1982) admitted for nonrespiratory illness, six (5%) showed parainfluenza IgM in their serum. Blocking experiments and retrospective clinical information indicated that the IgM antibody detected in these individuals is specific IgM acquired after a mild parainfluenza infection. Most (66%) patients showed IgM antibody titer rises or high titers (greater than 1,280) in both sera, and in 23%, a fall in IgM antibody titer was found. Detection of specific IgM antibody by HIT permitted early presumptive diagnosis in 71% of the patients with parainfluenza infection. IgM antibody persisted for 2-11 weeks. The HIT appears to be an important supplement for the diagnosis of parainfluenza infections.

Quantitation of immunoglobulin E antibody to cytomegalovirus by antibody capture enzyme-linked immunosorbent assay.

An antibody capture enzyme-linked immunosorbent assay was developed for detection of immunoglobulin E antibody to cytomegalovirus (CMV-IgE). Affinity-purified anti-human IgE-coated microtiter plates were used to separate IgE from other classes of antibody in serum. Virus-specific IgE was detected by subsequent incubation with horseradish peroxidase-labeled CMV antigen and substrate. The assay was shown to be very sensitive, since in most positive sera CMV-IgE was still detected at a dilution of 1:5,000. Of 45 patients with primary CMV infection, 43 (96%) were found to produce CMV-IgE. In contrast, CMV-IgE was detected in only 4 (9%) of 44 patients with recurrent CMV infection and in 1 of 144 healthy controls. Furthermore, the level of CMV-IgE in patients with recurrent CMV infection appeared to be lower than that in patients with primary infection. Preliminary examination of successive sera suggested that CMV-IgE is produced somewhat slower than CMV-IgM and -IgA but persists for a shorter period. These results suggest that CMV-IgE may be used as an indicator of primary CMV infection.

Use of enzyme-labeled antigen for the detection of immunoglobulin M and A antibody to herpes simplex virus in serum and cerebrospinal fluid.

A direct enzyme-linked immunosorbent assay (ELISA that used peroxidase-labeled antigen) was developed for detection of IgM and IgA antibody to herpes simplex virus (HSV). The assay uses immuno-affinity-purified antihuman IgM or IgA antibody-coated wells of microtiter plates to separate IgM or IgA from other classes of antibody in serum or cerebrospinal fluid (CSF). The presence of specific IgM or IgA is detected by subsequent, consecutive incubation with peroxidase-labeled antigen and substrate. HSV antigen was purified by sucrose gradient centrifugation and coupled with peroxidase by the periodate method. By examining sucrose-gradient-fractionated sera the assays were shown to be specific for IgM and IgA classes of antibody. None of the sera from patients with Epstein-Barr virus (n = 20), cytomegalovirus (n = 20), or varicella-zoster virus (n = 8) infection or with both rheumatoid factor and IgG antibody to HSV (n = 13) reacted positively. Only one out of 78 sera from healthy persons was positive for IgA antibody to HSV, and none for IgM antibody. All 33 patients with HSV infection developed HSV-IgA, 22 developed HSV-IgM. Of the 11 patients with primary infection, all had IgM antibody in their first sera and six had IgA antibody. The corresponding figures for the 22 patients with recurrent infection were five and nine. Furthermore, HSV-IgA antibody was found in serum and CSF of all five patients with HSV encephalitis in the second week after onset of symptoms, indicating the usefulness of the assay as a noninvasive technique for diagnosing HSV encephalitis.

Use of the enzyme-linked immunosorbent assay for the early diagnosis of Mycoplasma pneumoniae infection.

IgM and IgG antibodies to Mycoplasma pneumoniae were measured in 147 sera from four groups of patients by means of an indirect enzyme-linked immunosorbent assay (ELISA) and the results compared with those obtained by other methods. A good correlation was demonstrated between the complement fixation test and ELISA-IgM and to a lesser extent ELISA-IgG; for the metabolic inhibition test the reverse was the case. The indirect haemagglutination test appeared to detect mainly IgM antibodies. Low levels of IgM antibodies were detected by ELISA in 60 sera of children not suffering from pneumonia. However, if only high titres (greater than 800) were regarded as indicative of Mycoplasma pneumoniae infection, a presumptive diagnosis could have been made in 42 of 73 single acute phase sera from patients. Comparable results were obtained with IHA. The diagnostic level of IgM antibodies. was reached during the second week of the disease. It is concluded that examination of a single serum sample by either ELISA-IgM or IHA may assist in early diagnosis of Mycoplasma pneumoniae infection.