Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.     

The prevalence of antibodies to human T-lymphotropic virus type I in different population groups in Papua New Guinea

Summary Isolation and partial sequencing of human T-lymphotropic virus type I (HTLV-I) variants from inhabitants of Papua New Guinea (PNG) and the Solomon Islands has confirmed the existence of virus infection in Melanesian populations. To determine the geographical distribution of seropositivity to HTLV-I in PNG we have tested 2907 serum and plasma samples collected between 1972 to 1991 from 16 different population groups. Samples were screened using a particle agglutination assay and confirmed by p21e-enhanced Western immunoblot (WB). From a total of 94 screen positive samples run on WB, 56 (60%) were confirmed positive (positive for bothenv andgag products) and 38 (40%) were WB-indeterminate (gag products only). The prevalence of WB-confirmed antibodies to HTLV-I in lowland and island populations ranged from 0 to 5.4%. There were no confirmed antibody positives in the highland populations surveyed. Geographically isolated populations living on the fringes of the highlands ranged in seropositivity from 0.2 to 5.8%. Two of the subjects surveyed gave WB antibody patterns characteristic of HTLV-II rather than HTLV-I infection.     

Detection of anti-gag antibodies of feline immunodeficiency virus in cat sera by enzyme-linked immunosorbent assay

Summary Usinggag protein of feline immunodeficiency virus (FIV) expressed inEscherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIVgag protein in cat sera. With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIVgag protein were observed in all cases by the ELISA at early stage of infection. When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9%) out of 70 IFA positive sera were judged as negative by the ELISA. However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gp130 but not to p26 by a radioimmunoprecipitation assay. The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.     

Serum antibodies to HIV-1 are produced post-measles virus infection: evidence for cross-reactivity with HLA

Convalescent sera obtained from patients who were recently recovered from an acute measles virus infection were tested for the presence of anti-HIV-1 antibodies by Western blot analysis. While 16% (17/104) of control sera displayed reactive bands to a variety of HIV proteins, 62% (45/73) of convalescent sera demonstrated immunoreactive bands corresponding to HIV-1 Pol and Gag, but not Env antigens. This cross-reactivity appears to be the result of an active measles infection. No HIV-1 immunoblot reactivity (0/10) was observed in sera obtained from young adults several weeks after a combined measles, mumps, and rubella (MMR) vaccination. Interestingly, examination of anti-HLA typing sera specific for either class I and class II molecules revealed that 46% (19/41) of these sera contained cross-reactive antibodies to HIV-1 proteins. Absorption of measles sera with mixed lymphocyte reaction (MLR)-activated lymphocytes and/or HIV-1 recombinant proteins significantly decreased or removed the presence of these HIV-1-immunoreactive antibodies. Together, these findings suggest that the immune response to a natural measles virus infection results in the production of antibodies to HIV-1 and possibly autoantigens.     

GB virus C/hepatitis G virus infection in hemodialysis patients: determination of seroprevalence by a four-antigen recombinant immunoblot assay

GB Virus C/Hepatitis G Virus (GBV-C/HGV) was identified recently and only two assays, consisting of a single recombinant protein, have been described for determination of the seroprevalence of this virus. An immunoblot assay was devised, which contains four recombinant GBV-C/HGV proteins. In this study, serum samples from 154 patients on maintenance hemodialysis were examined to assess the rate of seroreactivity against GBV-C/HGV. All sera were tested for the presence of antibodies by an in-house recombinant immunoblot assay, for GBV-C/HGV viremia by RT-PCR, and for HCV infection by PCR and by serological assays. Antibody reactivity against GBV-C/HGV was detected in 20.8% (n = 32) and viremia was found in 6.5% (n = 10) of the patients. In no case were viremia and GBV-C/HGV antibodies detected in parallel. HCV infection was observed in 15.6% (n = 24) by RT-PCR. In 20 of these patients, HCV antibodies were detected by enzyme immuno assay (EIA) and immunoblot assay. However, four of the HCV PCR-positive patients were negative by both serological tests. Only two patients were viremic for GBV-C/HGV and HCV in parallel. It is concluded that antibody reactivity against GBV-C/HGV is common among patients on maintenance hemodialysis. In contrast to HCV, parallel occurrence of GBV-C/HGV viremia and GBV-C/HGV seroreactivity was not observed. This suggests that GBV-C/HGV infection might be self-limiting.     

Antibodies against varicella-zoster virus and herpes simplex virus deoxythymidine kinase in heterologous infections

Antibody responses to varicella-zoster virus (VZV) deoxythymidine kinase (dTK) and herpes simplex virus (HSV) dTK in homologous and heterologous infections were studied. Antibodies blocking the enzymatic activity of VZV-dTK appeared late after varicella and decreased more or less in parallel with the decreasing complement fixing [CF] titre. In herpes zoster, on the other hand, antibodies to VZV-dTK appeared soon after infection. Antibodies against HSV dTKs appeared long after primary infection, but they were subsequently present in all other HSV-CF positive sera. In recurrent HSV, all acute sera were already HSV-dTK antibody positive, and three of nine persons showed an increase in titer between their acute and convalescent sera. Blocking antibodies to VZV-dTK appeared rapidly in specimens from three of 18 individuals positive by an immunofluorescence VZV-immunity test during HSV infection, whereas all other specimens remained devoid of blocking antibodies against VZV-dTK. A rise in antibody titre against HSV-dTK during VZV infections was observed in serum specimens from three of 13 HSV-CF positive patients, whereas an antibody response against HSV-dTK was not found in HSV-CF negative individuals in connection with VZV infections. The relevance of the sporadic increase in the titres of antibodies against heterologous viral dTKs is discussed.     

The significance of hemolysing-inhibiting antibodies in protection against measles

The occurence of hemagglutinating-inhibiting (HI), hemolysinginhibiting (HLI) and nucleocapsid complement fixing (NC-CF) antibodies in serum samples from individuals immunized with measles virus antigens under various conditions was analyzed. After regular measles infections all three kinds of antibodies were detectable and removal of HI antibodies by absorption with Tween 80 and ether treated virus material only caused an about 2-fold reduction of titers of HLI antibodies. Immunization with further attenuated measles vaccine also induced a production of antibodies detectable in the three different tests. However the response of non-HI HLI antibodies and NC-CF antibodies was relatively less pronounced than after regular measles. Immunization with three or four doses of Tween 80 ether treated inactivated measles vaccine caused a production of HI antibodies and in some cases NC-CF antibodies. Non-HI HLI antibodies were not detectable. The combined immunization with inactivated vaccine followed by further attenuated live vaccine caused the appearance of high titers of HI antibodies but in contrast to the situation of administration of only live vaccine non-HI HLI antibodies were not detectable. The significance of non-HI HLI antibodies in protection against disease also was indicated by observations on the reactions of individuals immunized with inactivated vaccine and subsequently exposed to wild measles virus. The non-HI HLI antibody response was poorer in the vaccinees who contracted a complicated infection as compared to those who had only a subclinical or a mitigated infection. An accentuated non-HI HLI antibody and NC-CF antibody response was seen in patients with subacute sclerosing panencephalitis. It is proposed that the failure of hitherto used inactivated measles vaccines is due to the absence of certain envelope antigens in the preparations which leads to a deficiency as concerns the capacity to induce a production of non-HI HLI antibodies.     

Induction of V3-Specific Cytotoxic T Lymphocyte Responses by HIVgagParticles Carrying Multiple Immunodominant V3 Epitopes of gp120

Efforts to develop a vaccine to prevent infection of human immunodeficiency virus (HIV) have focused on the induction of neutralizing antibodies. In our previous study, we reported that chimericgag–envvirus-like particles (VLPs) induce neutralizing antibodies which block HIV infection. In addition to the neutralizing antibodies, the cytotoxic T-lymphocyte (CTL) response is considered to be another major immune defense mechanism required for recovery from many different viral infections. In the present study, we have constructed chimeric fusion proteins using HIV-2gagprecursor protein with (1) four neutralizing epitopes from HIV-1 gp160; (2) three tandem copies of consensus V3 domain, which have been derived from 245 different isolates of HIV-1 and carries both the principal neutralizing determinant (PND) and CTL epitopes; and (3) V3 domains from HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and HIV-1_SF2. These chimeric fusion proteins were expressed in a large quantity within insect cells, and released as VLPs into the cell culture medium. The purifiedgag–envVLPs from all three constructs appear to be spherical particles similar to immature HIV but slightly larger than thegagVLPs. Immunoprecipitation analysis showed that the chimeric proteins were recognized not only by HIV-1 positive patient sera, but also by monoclonal and polyclonal antisera raised against V3 peptides of HIV-1_IIIB, HIV-1_MN, HIV-1_RF, and the gp120 antiserum against HIV-1_SF2. Balb/C mice immunized with these chimeric VLPs successfully induced CTL activity against V3 peptide-stimulated target cells. In addition, a high degree of cross-reactivity was observed among the four different strains of HIV-1 V3 domain, indicating that the tandem multiple consensus V3 peptide sequence carried by HIV-2gagcan be used as a potential HIV vaccine against various HIVs.     

The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting

The technique of SDS-polyacrylamide gel electrophoresis and immunoblotting was used to study the evolution of the IgG antibody response to Toxoplasma gondii antigens in sequential sera of acutely infected patients. The results show that the IgG immunoblot pattern can be a useful marker for the stage of T. gondii infection. A 35-kD antigen elicited the first IgG response soon after exposure. During the course of infection additional bands appeared consecutively, following a constant sequence, to evolve to a late-stage-specific pattern with about 13–15 major bands. This pattern, which was reached at least 4 months after infection, was also found in the immunoblots of 28 patients with a chronic (latent) T. gondii infection.     

Immunoglobulin-class-specific immune response to respiratory syncytial virus structural proteins in infants, children, and adults

The protein specificities of IgG, IgM, and IgA antibodies induced during respiratory syncytial virus (RSV) infection in 74 patients (4 weeks to 81 years of age) were investigated using the technique of immunoblotting. Although the pattern of antibody reactivity varied among patients, most of the humoral immune response in all age groups was directed against the 48, 42, 35, and 27 K proteins. An infant's own antibody response was discernible in 55 of the 57 children below 1 year of age, despite the presence of maternally derived antibodies. Antibody against the 90 K surface glycoprotein was not detectable in those less than 1 year of age. Primary RSV infection induced antibodies only against a subset of RSV proteins. Although a broadening of the antibody response occurred with increasing age and in the course of reinfection, an immune response to all the viral structural proteins was observed rarely.     

Studies on reovirus-antigens

Summary Reovirus immune serum prepared by intravenous immunization of rabbits with the concentrated L cell culture fluid does not only contain virus-neutralizing and haemagglutination-inhibiting antibodies, but also complement fixing antibodies and precipitins against both reovirus antigens and cellular antigens.Active immunization by application of concentrated reovirus-containing cell culture fluid on the scarified skin results in the development of comparable antibody levels against reovirus, whereas demonstrable antibody to cellular antigens fails to develop. Hence, the complement fixation and immunoprecipitation tests are reovirus-specific. In contrast to the fairly typespecific neutralizing and haemagglutination-inhibiting antibodies, the complement fixing antibodies and the precipitins are group-specific.     

Serological comparison among various strains of equine infectious anemia virus

Summary The serological relationship between 8 strains of equine infectious anemia (EIA) virus was investigated by means of complement fixation and neutralization tests. All strains had a common complement fixing antigen, although there were some differences in the intensity of CF reaction in certain antigen-antiserum combinations.Cross neutralization tests revealed that all strains were only neutralized by homologous antisera. The respective antibody titers recorded ranged from 32 to 512. When horses were infected with the virus strains, neutralizing antibodies could be detected as early as 32 to 87 days after inoculation and maximum titers were demonstrable after 42 to 148 days. Of 34 horses infected with 6 strains of EIA virus, 33 horses had neutralizing antibody when they were tested 52 to 127 days after inoculation. Antibodies were only produced against the virus strains inoculated, and not against heterologous strains.     

Characterization of the humoral immune response of experimentally infected and vaccinated pigs to swine influenza viral proteins

Summary. The value of serologic tests for diagnosis of swine influenza virus (SIV) infection has been diminished by the emergence of new subtypes and by antigenic drift within subtype. The intensive use of vaccination also has complicated interpretation of serology results. Serologic assays are needed that can detect infection regardless of subtype or antigenic variation and that can differentiate antibody induced by infection from that induced by vaccination. In this study, the antibody responses to specific viral proteins in pigs infected by or vaccinated for SIV were characterized by Western immunoblot. Both IgM and IgG against hemagglutinin, nucleoprotein, NS1 and NS2 were detected in experimentally infected pigs by 7 days post inoculation (DPI). IgG against these proteins was still detectable at the end of the study (28 DPI). In contrast, IgG against neuraminidase and M1 was not detected until 14 DPI and no IgM against these proteins was detected. In vaccinated pigs, no antibody against NS1 was detected while antibody responses to other proteins were identical to those in exposed pigs. In conclusion, nucleoprotein may be a suitable antigen for use in a subtype-unrestricted serologic assay. NS1 protein may be suitable for a serologic assay that differentiates between infected and vaccinated pigs.     

First documented case of human immunodeficiency virus type 2 infection in an asymptomatic Swiss subject

Few cases of human immunodeficiency virus type 2 (HIV-2) infection have been reported in individuals other than of West African origin. The first well documented case of HIV-2 infection observed in a Swiss subject is presented here. The 50-year-old woman had a sexual relationship with a Senegalese man, who was later shown to be HIV seropositive. Initially, the subject's serum was tested using a routine screening assay for the detection of HIV-1 antibodies. This assay elicited a borderline positive result. A confirmatory competitive EIA and a Western blot test for anti-HIV-1 antibodies showed a positive reaction withgag andpol proteins of HIV-1, but not withenv proteins. Thus, HIV-2 infection was suspected and subsequently confirmed by three different methods, including Western blot analysis and an HIV-1/HIV-2 differentiation test. This case emphasizes the need for screening with combined HIV-1/HIV-2 tests.     

Antibody specificity for human immunodeficiency virus type 1 in serum and cerebrospinal fluid from patients with AIDS and AIDS-related complex

Since little information has been reported about the specificity of antibodies to human immunodeficiency virus type 1 (HIV-1) found in the cerebrospinal fluid (CSF), 21 CSF and serum specimens were examined from 19 patients with clinical AIDS, AIDS-related complex, or asymptomatic HIV-1 infection. The predominant specificity of antibodies using Western blot analysis in both serum (100 %) and CSF (100 %) was directed towardenv gene products. The next most common antibody specificities were to thepol gene products (serum 95 %; CSF 62 %). Less commonly found was antibody to thegag-encoded proteins (serum 71 %; CSF 38 %). The level of antibody to HIV-1 in CSF could not be predicted from the level found in serum. Also, the spectrum of antibodies seen did not correlate with disease stage or with the quantity of antibody present. The serum/CSF pairs were also examined for the presence of HIV-1 antigen by commercial enzyme immunoassay. HIV-1 antigen was present in eight of 19 (43 %) of the serum samples and five of 20 (25 %) of the CSF samples tested.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Immune response of rats to subcellular fractions of isologous brain and liver

The titres of complement fixing antibodies in the sera of rats injected with the soluble fraction of rat brain emulsified in Freunds' complete adjuvant (FCA) were usually below 10.

In contrast, injections of the nuclear, mitochondrial or microsomal fractions of rat brain in FCA were followed by the appearances of heat stable complement fixing and haemagglutinating antibodies within a few days, the highest antibody levels being attained about 6 days after the first injection of a particulate fraction. The microsomal fraction was the most efficient particulate antigen. After 2 weeks of immunization about 60 per cent of the complement fixing activity of the antisera was due to 19S antibody and the remainder to 7S antibody.

The response to injections of the nuclear and microsomal fractions of rat liver followed a similar time course but produced levels of complement fixing antibodies that were consistently lower than those engendered by the corresponding brain antigens.

     

Immunoblot detection of antibodies to Toscana virus

Sera from patients with sandfly fever caused by Toscana virus (TOSV) infection were tested by immunoblot for specific antibody response to TOSV derived from infected Vero-E6 cells. The 28 kDa TOSV nucleoprotein (N) was identified as the major immunodominant protein recognized by immunoblot. In sera of patients with acute TOSV infection, specific antibodies of the IgM, IgA, and IgG class were detected. Using sandfly fever virus, serotypes Sicilian (SFSV) and Naples (SFNV), as antigens for immunoblot, TOSV antibody-positive sera cross-reacted with the corresponding N proteins. These sera reacted for IgM and IgG by SFSV immunoblot, and for IgM by SFNV immunoblot. The diagnosis of sandfly fever may be confirmed by TOSV immunoblot. © 1996 Wiley-Liss, Inc.     

Acute retinal necrosis six years after herpes simplex encephalitis: an elusive immune deficit suggested by insufficient test sensitivity

A patient presented with acute retinal necrosis of the left eye. Demonstration of herpes simplex virus (HSV) DNA in the aqueous humour confirmed the diagnosis. Negative results of HSV type-specific antibody tests based on gG antigens suggested a primary HSV infection. However, the patient had a past history of laboratory-confirmed herpes simplex encephalitis 6 years ago. Using antibody tests based on whole viral lysate antigens, he was seropositive from the onset, and immunoblot testing confirmed a lack of anti-gG reactivity. To be able to assess whether this might be related to the apparent inability of his immune system to suppress clinically symptomatic HSV infection, serial samples were tested by an HSV neutralisation test and a whole-blood flow cytometric assay to determine the frequency of HSV-specific CD4 lymphocytes. However, this did not yield evidence of obvious immunodeficiency; the patient reacted similarly to known positive controls by both assays. Although type-specific HSV serological tests based on gG are generally more specific than those based on whole viral lysate antigens, they have a somewhat lower sensitivity, as a certain percentage of HSV-infected individuals do not develop antibodies against gG, and others may suffer a secondary loss of anti-gG reactivity. Thus there is a risk of missing individual infected patients. Unless this potential problem is recognised, serious consequences might possibly result. We therefore urge virologists and clinicians to exercise great care if highly specific antibody assays based on recombinant proteins are employed.     

Humoral immune responses during experimental infection with <Emphasis Type="Italic">Fascioloides magna</Emphasis> and <Emphasis Type="Italic">Fasciola hepatica</Emphasis> in goats and comparison of their excretory/secretory products

This study investigated the humoral immune responses of goats experimentally infected with Fascioloides magna and Fasciola hepatica to F. magna excretory/secretory products (FmESP) or F. hepatica excretory/secretory products (FhESP), respectively. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum antibody responses and for possible discrimination of F. magna and F. hepatica infections in goats. Comparison of ESPs of both flukes and evaluation of ESP antigenicity was also studied applying immunoblotting techniques. In all infected goats, antibody level was significantly increased (against negative control) since 2 weeks post infection (WPI). However, the dynamics of antibodies varied between F. magna and F. hepatica groups during the course of the infection. The cross-reaction of antibodies developed against F. magna and F. hepatica with ESP proteins was recorded by ELISA. The species-specific proteins 40, 120 kDa from FmESP and 80, 160 kDa from FhESP (with no antibody cross-reaction) were detected by two dimensional electrophoresis and immunoblot as the potential immunodiagnostic markers. Our results suggest that F. magna and F. hepatica infection could be distinguished by common immunological techniques based on species-specific antigen–antibodies interaction.