Vi Antibody Research Articles

Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: use of a nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with Western blotting

Previously, a panel of monoclonal antibodies (MCAb) was used to define specific epitopes of herpes simplex virus glycoprotein D (gD) (R. J. Eisenberg et al., J. Virol. 53:634-644, 1985). Three groups of antibodies recognized continuous epitopes; group VII reacted with residues 11 to 19 of the mature protein (residues 36 to 44 of the predicted sequence), group II reacted with residues 272 to 279, and group V reacted with residues 340 to 356. Four additional antibody groups recognized discontinuous epitopes of gD, since their reactivity was lost when the glycoprotein was denatured by reduction and alkylation. Our goal in this study was to localize more precisely the discontinuous epitopes of gD. Using a nondenaturing system of polyacrylamide gel electrophoresis ("native" gel electrophoresis) coupled to Western blotting, we analyzed the antigenic activity of truncated forms of gD. These fragments were generated either by recombinant DNA methods or by cleavage of purified native gD-1 (gD obtained from herpes simplex virus type 1) and gD-2 (gD obtained from herpes simplex virus type 2) with Staphylococcus aureus protease V8. Antibodies in groups III, IV, and VI recognized three truncated forms of gD-1 produced by recombinant DNA methods, residues 1 to 287, 1 to 275, and 1 to 233. Antibodies in group I recognized the two larger forms but did not react with the gD-1 fragment of residues 1 to 233. On the basis of these and previous results, we concluded that a protion of epitope I was located within residues 233 to 259 and that epitopes III, IV, and VI were upstream of residue 233. Antibodies to continuous epitopes identified protease V8 fragments of gD-1 and gD-2 that contained portions of either the amino or carboxy regions of the proteins. None of the V8 fragments, including a 34K polypeptide containing residues 227 to 369, reacted with group I antibodies. This result indicated that a second portion of epitope I was located upstream of residue 227. Two amino-terminal fragments of gD-1, 33K and 30K, reacted with group III, IV, and VI antibodies. A 33K fragment of gD-2 reacted with group III antibodies. Based on their size and reactivity with endo-beta-N-acetylglycosaminidase F, we hypothesized that the 33K and 30K molecules represented residues 1 to 226 and 1 to 182 of gD-1, respectively. These results suggest that epitopes III, IV, and VI are located within the first 182 residues of gD.(ABSTRACT TRUNCATED AT 400 WORDS)

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy.

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.

Identification of a carrier by using Vi enzyme-linked immunosorbent assay serology in an outbreak of typhoid fever on an Indian reservation.

In May 1981 an outbreak of typhoid fever occurred in a small village on a southwestern United States Indian reservation. Five of the six culture-proven cases, but only 2 of 15 community, age-matched controls, had eaten food prepared for a party held in the village on 20 April (chi-square = 4.3; P less than 0.05). Food histories obtained from 16 persons who ate food at the party suggested that chicken with chili (P = 0.03) and potato salad (P = 0.09) were possible vehicles. Eleven adults who attended the party, 5 of whom helped prepare an implicated food, were studied with one or more stool cultures and serum for Vi antibody by using enzyme-linked immunosorbent assay (ELISA) and hemagglutination techniques. All initial stool cultures were negative for Salmonella typhi; however, one subject, a 70-year-old female foodhandler, had a Vi antibody titer of 1:320 by ELISA. Subsequent cultures from this subject were positive for S. typhi. ELISA for Vi antibody directed the investigators to a single individual as the most probable carrier source and obviated the need for multiple fecal cultures from the other potential carriers identified by the epidemiological investigation.

Detection of urinary Vi antigen as a diagnostic test for typhoid fever.

Since Vi antigen is limited primarily to Salmonella typhi, it has been thought that detection of the antigen may be a useful method for diagnosing acute typhoid fever. The slide coagglutination method and enzyme-linked immunosorbent assay have recently been suggested as ways to detect small quantities of Vi antigen in urine. In Santiago, Chile, we compared the results of these two methods in patients with acute typhoid fever, paratyphoid fever, and other febrile illnesses and in afebrile control subjects. Using a cut-off value that maximally separated typhoid patients from controls, the enzyme-linked immunosorbent assay was positive in 62.4% of 141 patients with culture-proven typhoid infections and in 13.2% of 159 afebrile control subjects. The enzyme-linked immunosorbent assay was false positive in 64.7% of 34 culture-proven paratyphoid A or B patients and 47.1% of 21 patients with other nontyphoidal febrile illnesses. The coagglutination test was positive in 34% of typhoid patients, 14% of afebrile control subjects, and 46% of febrile control subjects. We conclude that these tests when performed with the Vi antibodies employed in this study are of little value for the diagnosis of typhoid fever in this setting.

Vi SEROLOGY IN DETECTION OF CHRONIC SALMONELLA TYPHI CARRIERS IN AN ENDEMIC AREA

A passive haemagglutination assay measuring antibody to highly purified Vi antigen, known to be sensitive and specific for the detection of chronic Salmonella typhi carriers in a non-endemic area, was assessed in an endemic area. A reciprocal serum Vi antibody titre of 160 was found to have a sensitivity of 75%, specificity of 92%, and a high predictive value in screening for chronic S typhi carriers in high-risk population groups (eg, women over 40 years). This simple assay can screen for chronic S typhi carriers even in areas where typhoid fever is highly endemic.

Enzyme-linked immunosorbent assay for detection of human antibodies to Salmonella typhi Vi antigen.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to Salmonella typhi Vi antigen in human serum, and the results were compared with those from a previously described hemagglutination assay (HA). The ELISA detected Vi antibodies at a titer of greater than or equal to 20 in 40 (52%) of 77 sera from typhoid fever patients, whereas the HA gave titers of greater than or equal to 20 in 35 (47%). Determination of titers of serum specimens from 170 persons without typhoid fever revealed Vi antibody titers of greater than or equal to 20 in 4 (2.3%) by the ELISA and 3 (1.7%) by the HA. Unlike the sensitized erythrocytes used in the HA, the ELISA reagents have a shelf life of greater than or equal to 1 year. The ELISA may be preferred by some laboratories, especially those already performing other ELISA tests.

Immunity in typhoid fever. II. Antigens and immune response in systemic salmonellosis

Os resultados dos trabalhos acumulados a partir do início do século permitem concluir que, apesar de numerosos progressos e estudos dos pontos de vista químico, imunoquímico, genético e biológico, o (s) antígeno (s) responsável (is) pela imunidade nas salmoneloses sistêmicas ainda não está (ao) definido (s). As mais diversas preparações têm sido propostas como possiveis imunogenos, bem como uma ampla variedade de modelos experimentais e de métodos de avaliação da resposta imune. Em relacao ao primeiro aspecto, sendo a Salmonella typhi, em condições naturais, um patógeno exclusivo do homem, os resultados obtidos em animais de laboratório frequentemente não se correlacionaram com os obtidos em seres humanos. Salienta-se que a pesquisa da resposta imune tem sido limitada na maioria das vezes a avaliação do titulo de aglutininas para os antigenos O, H e Vi; ou a testes de proteção passiva ou ativa em camundongos. A imunidade celular tem sido definida, principalmente por testes cutâneos, com preparações de natureza proteica, de composição variável e estrutura quimica mal-definida. Todos os dados revistos levam a conclusão de que até o presente, ignora-se o, ou os mecanismos de imunoproteção nas salmoneloses sistemicas.

Vi SEROLOGY IN THE DETECTION OF TYPHOID CARRIERS

A new haemagglutination assay for Vi antibodies was evaluated in searches for symptom-free carriers of Salmonella typhi associated with sporadic cases of typhoid fever. The assay differs from previous ones in that a purified (instead of crude) Vi antigen from Citrobacter was used to sensitise the red blood cells. In ten sporadic outbreaks of typhoid stool culture identified seven enteric carriers of S. typhi among the patients' families or other close contacts. All seven carriers had Vi antibodies in titres ranging from 1:40 to 1:2560. Moreover, among thirty- seven stool-culture-negative contacts of patients, only one had Vi antibodies, in a titre of 1:10. Thus, the new assay for Vi antibodies was as sensitive and as specific as faecal culture in detecting symptom-free typhoid carriers. It could become a convenient screening test.

Fluorescent Vi Antibody Test in the Screening of Typhoid Carriers

The comparative efficacies of direct bacterial agglutination and immunofluorescent antibody for the estimation of serum Vi antibody were determined in the detection of typhoid carriers. Sera from all 12 typhoid carriers gave significant titers of 1/10 or more in the direct bacterial agglutination test; however, 26 of 119 (21.8%) sera from culture-negative individuals were also falsely positive. In the fluorescent Vi antibody test, 11 of 12 typhoid carriers showed significant serum antibody levels, while only two of 119 (1.7%) culture-negative subjects had significant antibody titers. In view of the much lower false-positive rate, the immunofluorescent Vi antibody test is considered to be superior to the direct bacterial agglutination test in the screening of typhoid carriers.

Studies on cellular immunity to Salmonella typhi in vitro.

Salmonella typhi (strain Ty2—4446) cultivatedin vitro within the macrophage of mice immunised twice specifically with a high dose of killed vaccine, multiplied less intensively than in the cells of non-immunised control animals during 24 hours cultivation. In addition, a certain suppression of growth ofSalmonella enteritidis andSalmonella suis var. Kunzendorf was observed in the macrophages of mice immunised withSalmonella typhi vaccine. Double immunisation of mice with high doses of killed vaccine fromSalmonella enteritidis andSalmonella suis led to the mice macrophages being able to suppress multiplication of both homologous microbes andSalmonella typhi. The formation of such cross resistance in mice immunised with salmonella vaccines excludes the possible participation of O, H, and Vi antibodies in this phenomenon.

Immunological response of the rabbit to Vi antigen.

Vi antibody response of rabbits varied depending on whether Vi antigen was administered in particulate or soluble state. Vi antigen in particulate form induced hemagglutinins, bacterial agglutinins, and passive cutaneous anaphylaxis (PCA) antibodies, whereas soluble Vi antigen induced only hemagglutinins. Guinea pigs passively sensitized with antisera against particulate Vi antigen gave PCA reactions when challenged with either soluble or cellular Vi antigen; antisera against soluble Vi antigen were negative for PCA. The specificity of PCA was demonstrated by its dependence on the Vi concentration and by absorption of PCA activity from antisera with V-form cells of Salmonella typhosa.

Agar plaque formation by mouse spleen cells in response to vaccination with Vi antigen and typhoid vaccines.

Vi-containing Salmonella typhosa organisms were resistant to killing by immune spleen cells in the presence of complement even when the cells were producing Vi antibody.

Factors Affecting Formation of Incomplete Vi Antibody in Mice

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Factors affecting formation of incomplete Vi antibody in mice. J. Bacteriol. 90:635-642. 1965.-Single immunizing doses of purified Vi antigen elicited complete and incomplete Vi antibodies in BALB/c mice, but only incomplete antibody in Cinnamon mice. Three of six other mouse strains tested responded like BALB/c mice; the remaining three, like Cinnamon mice. Varying the quantity of antigen injected or the route of administration failed to stimulate the production of detectable complete Vi antibody in Cinnamon mice. Such antibody was evoked in these animals by multiple injections of Vi antigen or by inoculating them with Vi-containing bacilli or Vi-coated erythrocytes. The early protection afforded by serum from Vi-immunized BALB/c mice coincided with the appearance of incomplete Vi antibody, 1 day prior to the advent of complete antibody. Persistence of incomplete as well as complete antibody in the serum of immunized mice was demonstrated for at least 56 days after injection of 10 mug of Vi antigen. Incomplete Vi antibody was shown to have blocking ability, in vitro bactericidal activity, and the capability of protecting mice against intracerebral as well as intraperitoneal challenge with virulent typhoid bacilli. Production of incomplete and complete Vi antibodies was adversely affected by immunization with partially depolymerized Vi antigens.

DETECTION AND PERSISTENCE OF VI ANTIGEN IN TISSUES OF ACTIVELY IMMUNIZED MICE.

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Detection and persistence of Vi antigen in tissues of actively immunized mice. J. Bacteriol. 89:776-781. 1965.-The presence, distribution, and persistence of Vi antigen in mouse tissue was determined by means of active immunization tests with tissue extracts. Mice were injected intraperitoneally with purified Vi antigen or Vi-containing bacilli. At appropriate intervals, animals were killed, and saline extracts of their tissues were prepared. Mice were immunized with these extracts and challenged 6 days later with 10 ld(50) of Salmonella typhosa Ty2. Protection was afforded by tissue extracts from Vi-injected mice, but not by normal tissue extracts. That the immunizing capacity of tissue extracts from Vi-injected mice was attributable to Vi antigen was affirmed by the demonstration that these extracts stimulated the production of Vi antibody in mice, coated erythrocytes for agglutination by Vi antiserum, and inhibited agglutination of Vi-sensitized red blood cells by known Vi antisera. Vi antigen could be detected in the liver and spleen of mice injected with as little as 1 mug. In mice given 150 mug, the antigen was still present in liver tissue 231 days later.

A RAPID SLIDE TEST FOR THE DETECTION OF VI ANTIBODY IN HUMAN AND ANIMAL SERA.

A rapid slide test for the detection of Vi antibodies, using spheroplasts of Salmonella typhi (Vi I, Bhatnagar strain) as the test antigen, has been described. The spheroplasts, grown in a semisynthetic medium containing 1.5% glycine, maintained their antigenic sensitivity for periods of at least 1 month when stored in the cold in the growth medium preserved with 1:5000 Thimerosal.Comparative tests were carried out with the spheroplast slide technique, agglutination tests using intact Bhatnagar cells and Vi-antigen-sensitized red cells, and flocculation tests of Vi-antigen-sensitized bentonite particles. The slide test proved to be specific and reproducible and was as sensitive as the test tube hemagglutination test for both rabbit and human sera. The intact cell agglutination technique and the bentonite flocculation test were relatively insensitive.

The Bactericidal Actions of O and VI Antibodies against Salmonella Typhosa

Summary Anti-Vi and anti-O are both bactericidal against virulent strains of S. typhosa. With currently used vaccines, however, the anti-Vi response contributed very little to the bactericidal action of immune serums. The bactericidal activity of such serums reflected primarily their anti-O content. The apparent lack of protection afforded by anti-Vi in human subjects may be related to this finding.

Vi-negative strains of Salmonella typhosa: attempts to induce W-V reversion and the use of non-Vi strains in evaluating typhoid vaccines.

Tully, Joseph G. (Walter Reed Army Institute of Research, Washington, D.C.) and Julius A. Currie. Vi-negative strains of Salmonella typhosa: attempts to induce W-V reversion and the use of non-Vi strains in evaluating typhoid vaccines. J. Bacteriol. 84:747-753. 1962.-Repeated attempts have been made to detect reversion of several W-form Salmonella typhosa strains to Vi antigen-containing cultures. Passage of the O-901, H-901, and Ty2W cultures through various mouse strains did not result in the recovery of V-form typhoid bacilli. Rabbits immunized repeatedly with non-Vi strains of typhoid bacilli, or with W cultures successively passed through mice, did not respond with Vi antibody formation. Attempts also were made to detect reversion of non-Vi strains passed in broth and plate cultures to which heat-killed Vi antigen-containing strains had been added. No evidence was obtained that the selection of Vi-containing cultures had been enhanced. The non-Vi typhoid strains employed in this study thus appeared to be stable W-form cultures. Viable-cell vaccines and acetone-killed and dried (AKD) vaccines of V- and W-form S. typhosa were compared in active mouse protection tests against intraperitoneal and intracerebral challenges with V- and W-form typhoid strains. Only Vi-containing cultures effectively protected mice against virulent V-form challenges, regardless of the route utilized. Greater quantities of both viable-cell and AKD vaccines prepared from non-Vi strains were required for protection against V- as well as W-form S. typhosa challenges.

Production of incomplete Vi antibody in mice.

SummaryImmunization with purified Vi antigen elicited incomplete Vi antibody in the Cinnamon strain, and complete Vi antibody in BALB/c mice. Incomplete Vi antibody alone accounted for protection afforded by serum from Cinnamon mice against challenge with fully virulent typhoid bacilli. Absorption tests verified the specificity of incomplete Vi antibody and protection it afforded.

The VI Antigens of the Enterobacteriaceae

Summary Using the Ouchterlony technique, a serologic study was made of native and deacetylated Vi antigen prepared from Paracolobactrum ballerup and mixtures of the two. The data presented indicated that the two zones observed in the reaction of native Vi antigen and antibody were due to the presence of both fully acetylated and partially deacetylated antigens in the preparations studied.

Investigation into the Existence of Functionally Deficient Vi Antibody

Investigation into the Existence of Functionally Deficient Vi Antibody