The specific activity of a multicomponent vaccine made from the antigens of opportunistic microorganisms

Multicomponent vaccine prepared from the antigens of 4 representatives of opportunistic microflora possesses high specific activity. The passive hemagglutination (PHA) test with the use of associated diagnosticum showed that antibody titers in the sera of immunized rabbits increased 10- to 10(4)-fold in comparison with the titers observed prior to immunization. The PHA test with the use of the antigens contained in the vaccine revealed the accumulation of antibodies to each of the 4 components of the preparation in the blood sera of immunized rabbits. When stored at 4 degrees C, the vaccine was shown to retain its specific activity for 5 years (the term of observation).     

A comparative evaluation of the sensitivity of different methods for the serological diagnosis of leptospirosis

In this work the comparative evaluation of the sensitivity and serological specificity of the microcapsular agglutination (MCA) test, the passive hemagglutination (PHA) test and the microagglutination (MA) test are presented. In the MCA test leptospiral antigens, adsorbed on synthetic carrier capsules produced by Japan Lyophilization Laboratory, were used and the PHA test was made with the use of polyvalent erythrocyte diagnosticum. The study of blood serum samples from 46 leptospirosis patients revealed that the values of antibody titers in the PHA and MCA tests were 5.5-8.1 times higher than the traditional MA test. In the MCA and PHA tests antileptospiral antibodies could be detected as early as on days 1-3 of the disease when the results of the MA test were negative or very low. The maximum values of antibody titers in the MCA and PHA tests were detected on days 11-15 of the disease and in the MA test, on days 21-25. The MCA and PHA tests are genus-specific and permit the detection of antileptospiral antibodies irrespective of the serogroup of the infective agent. In the study of the blood sera of 40 patients with diseases of nonleptospiral etiology the MCA and MA tests yielded false positive results in 7.5% and the PHA test, in 12.5% of cases in titers below the diagnostic level. These data are indicative of high sensitivity and specificity of the serological tests used in this study.     

The characteristics of the antibody response of animals immunized with components of the surface structures of Francisella tularensis

Antibody formation in animals immunized with one of the components of F. tularensis surface structures was studied. The time course of antibody formation in 20 hamadryas baboons was studied in the passive hemagglutination (PHA) test, microagglutination (MA) test, and indirect enzyme immunoassay, used for the determination of IgG, IgA and IgM antibodies. The character of antibody response in the animals immunized with components of F. tularensis surface structures (S-complex) and with live tularemia vaccine was compared. The study revealed that immunization with the S-complex induced the formation of antibodies detected by all three methods. Antibody formation to the S-complex was found to be dose-dependent. With the increase of the injected dose of the S-complex, antibody titers determined in the PHA test decreased and those determined in the MA test increased, which was seemingly due to the induction of antibodies differing in their isotypes. After immunization with the S-complex the levels of IgG antibodies were lower and the levels of IgM antibodies by day 28 after immunization higher than after the injection of live tularemia vaccine.     

A survey of Clostridium perfringens enterotoxin antibody in human and animal sera in western Canada

Sera from human, cattle, sheep, swine, and horse populations in western Canada were tested for the presence of Clostridium perfringens enterotoxin antibody by the passive hemagglutination (PHA) test, supplemented by an immunodiffusion test and by counterimmunoelectrophoresis. A total of 224 human, 345 cattle, 165 sheep, 620 swine, and 768 horse serum samples were examined. Low-titer reactions in the PHA test were detected in human, cattle, horse, and swine sera, in that order, with no titers demonstrated in sheep. The titers in human sera ranged up to 1:128 and three of these samples were also positive in the other two serological tests. The significance of this antibody is not clear, but it is suggested that the low prevalence of the antibody may reflect a low prevalence of enterotoxigenic C. perfringens strains in western Canada. Such serological surveys may be applicable to epidemiological studies involving enterotoxigenic C perfringens.     

Adenovirus antibody measured by the passive hemagglutination test

Lefkowitz, Stanley S. (Variety Children's Research Foundation, Miami, Fla.), Julia A. Williams, Bernard E. Howard, and M. Michael Sigel. Adenovirus antibody measured by the passive hemagglutination test. J. Bacteriol. 91:205-212. 1966.-Rabbits immunized intravenously with adenovirus type 5 antigen were tested for antibody titers by use of the passive hemagglutination test (PHA). Primary and secondary responses were studied, and the class of antibody was determined by means of density gradient centrifugation and reduction with 2-mercaptoethanol (ME). It was found that the PHA was 10 to 100 times more sensitive than complement-fixation and neutralization tests for the detection of antibodies to adenovirus. The immunological response to primary immunization was dependent on the dose of antigen, with antibody appearing in as early as 3 days. After secondary stimulation with the same antigen, there was a rapid response which appeared to be less dose-dependent. It was found that a heavy 19S antibody sensitive to ME was produced early and was followed by a lighter, presumably 7S, ME-resistant antibody. Upon secondary stimulation, both 7S and 19S antibody increased to levels greater than those of the primary injection.     

Serological method of detecting antirabies antibodies]

The possibility of the determination of rabies antibodies by the serological method has been demonstrated, involving the use of culture rabies virus, strain Vnukovo-32, concentrated and purified by high speed centrifugation, for the sensitization of anserine erythrocytes. The neutralization test in mice and the passive hemagglutination test showed a correlation in rabies antibody titers in the sera of animlas immunized with rabies vaccines. The advantages of the passive hemagglutination test consist in the rapidity of obtaining the results, in a simple and economic method of carrying out the test.     

Humoral response to the injection of acellular Pertussis vaccine

The humoral response of mice and rabbits to the injection of whole-cell pertussis vaccine (PV) and acellular pertussis vaccine (APV), developed at the Mechnikov Research Institute for Vaccines and Sera (Russian Acad. Med. Sci.) in Moscow, was studied. In the sera of immunized animals antibodies to the antigenic complex were determined in the direct hemagglutination (DHA) test, specific antibodies to filamentous hemagglutinin (FHA) and pertussis toxin (PT)--in the enzyme immunoassay (EIA) and antibodies neutralizing PT in a cytopathogenic dose (CPD)--in neutralization test on Chinese hamster ovary (CHO) cells. In mice and rabbits immunized with APV the antibody titers determined in the DHA test were higher than those in the animals immunized with PV. Specific antibodies titers to FHA and PT in the sera of rabbits immunized with APV were also higher than those in the sera of rabbits immunized with PV. High dilutions of sera taken from the animals immunized with APC neutralized 4-16 doses of PT in the neutralization test on CHO cells. The most important result of this study was the detection of a more pronounced immune response in the animals immunized with APV in comparison with that induced by PV according to the results obtained in EIA and in the test of PT CPD neutralization on CHO cells.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity. II. Suppression of cutaneous delayed-type hypersensitivity by macrophage disappearance reaction

Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 micrograms of EA was injected into these animals and the animals were divided into two groups. One group of animals was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine gamma-globulin (DNP-BGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGG-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supernatants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. I. Organ differences of tolerogen susceptibility and cellular sites responsible for the resistance.

Cellular sites of the tolerogen resistance in DDD mice against human IgG (HGG) were examined by reconstitution experiments in which cells of various lymphoid organs from tolerized mice were transferred into lethally irradiated syngeneic recipients with or without the supplement of an excess number of untreated T or B cells. It was shown that T cells but not B cells in the spleen and bone marrow-locating B cells were tolerogen resistant. Kinetic profiles of tolerance induction were compared among thymus, lymph node, and spleen T cells. Thymus cells fall into unresponsive state as early as 2 days after the tolerogen (tHGG) injection when only partial tolerance was observed in lymph node T cells. By 1 week of tolerogen treatment, the tolerant state was completed in both thymus cells and lymph node T cells, while spleen T cells showed marked resistance. Tolerance induced in thymus cells and spleen T cells was of relatively short duration and responsiveness was completely recovered by 5 weeks after the injection of tHGG. At this time lymph node T cells still showed hyporesponsiveness. The differences in tolerance inducibility were also shown among different lymphoid organs in tolerogen dose response. Lymph node T cells were very sensitive to tolerance induction, giving no response even by the injection of 0.01 mg of tHGG. Thymus cells were much less sensitive with the gradual loss of responsiveness by increasing the amount of tHGG. In contrast, spleen T cells showed gradual resistance with increasing amount of tHGG, indicating that some positive response was evoked in spleen T cells by a relatively high dose of tHGG. These results seem to suggest that the tolerogen resistance of spleen T cells may be due to their capability of showing positive response against the tolerogenic material. This was also suggested by the fact that the treatment with cyclophosphamide following the tolerogen injection diminished completely the responsiveness against the subsequent challenge immunization.     

Evolution of alloantibodies and suppressor cells in allografted mice treated for passive enhancement

The kinetics and quality of the alloimmune reaction were studied in CBA (H-2k) mice treated for passive enhancement of tumor allografts (Sa 1 indigenous of A/J (H-2a or H-2k/d) mice). Serum samples of treated animals were tested for their biological properties relevant to different antibody isotypes in vitro (hemagglutination, complement-dependent cytotoxicity, and anaphylaxis, i.e., mast cell degranulation involving all main Ig isotypes; IgM, IgG2, and IgG1, IgE, respectively) as well as in vivo (allograft enhancement). Spleen cells from these treated animals were examined for their capacity to interfere with the rejection of tumor allografts by adoptive transfers into syngeneic recipients. In vitro, 51Cr release cytolysis assays were performed in order to test their cytolytic and regulatory activities in comparison to rejecting control animals. It has been shown that: grafted mice, pretreated for passive enhancement, kept their grafts longer and synthetized anaphylactic antibodies (mainly IgG1) earlier and at higher titers than normal serum controls, which rejected the same Sa 1 allografts. Mice with enhanced tumors synthetized cytotoxic antibodies (mainly IgG2) later than rejecting controls. Serum samples from treated and control animals, harvested 10 days (early sera) and 30 days (late sera) after grafting, were injected with a "normal dose" (0.2 ml) and a "high" dose (0.4 ml) to new CBA recipients grafted with Sa 1. Early immune sera were only enhancing at high doses when derived from animals previously treated for enhancement (at the low dose both immune sera were enhancing). Late sera, presenting both complement-fixing, cytotoxic (predominantly IgG2), and IgG1 anaphylactic alloantibodies in the two groups, induced enhancement in all cases, but more strongly when derived from the group treated for Sa 1 enhancement. Adoptive transfer of spleen cells from animals treated for passive enhancement were able either to inhibit the accelerated rejection (Day 10) or to promote enhancement of Sa 1 allogeneic cells (Day 30) while similar cells taken (Day 10 and Day 30) from control graft-rejecting mice transferred accelerated rejection. Among the transferred T-cell sub-populations, the suppressive effect was mediated by Lyt 2 T cells. In vitro, these spleen cells showed a weaker cytolytic activity than those of allograft-rejecting mice. Moreover, they were able to regulate the cytolytic activity of cytotoxic effector cells from specifically immunized CBA mice.     

Persistent activity of suppressor cells in T cell-mediated immune response.

Tolerance to dinitrochlorobenzene contact sensitivity induced i.v. injection of dinitrobenzenesulfonic acid in guinea pigs is a long-lasting phenomenon (up to 1 year). The tolerogen, however, was traceable in the circulation only up to 3 months after its application. In spite of that, tolerance was adoptively transferred by parabiosis 6 months after being induced. Moreover, active suppressor cells eliminated by cyclophosphamide treatment are able to regenerate in those adoptively tolerized animals. These results indicate that the tolerogenic injection stimulates precursors of suppressor cells to generate active suppressor cells and memory cells of suppression. The further formation of active suppressor cells from memory cells seems to be tolerogen independent, but the existence of specific stimulator cells for suppression may be considered. These cells may bind undetectable small amounts of tolerogen. The recovery of suppression might, however, be also due to recovery of suppressor cells which were temporarily inactivated but not destroyed by cyclophosphamide treatment.     

Prevalence of antibody to Toxoplasma gondii in the moose (Alces alces americana Clinton) of Nova Scotia, Canada

The prevalence of antibodies to Toxoplasma gondii was investigated in the sera of 125 moose taken by hunters in 5 countries of Nova Scotia. Nineteen of these sera (15%) were positive by the indirect passive hemagglutination test, with titers above 1:64. This study adds further evidence to the prevalence of antibodies to T. gondii in the wildlife, extending this evidence to eastern Canada. The possibility that humans may acquire toxoplasmosis by ingesting undercooked infected meat from game animals is supported by the results of this investigation; the implications of these findings to public health are obvious.     

Elimination of Klebsiella pneumoniae and humoral immunity in immunization with the IS-4 immunostimulating mixture

The present investigation has revealed that in mice, immunized with preparation HC-4 (an immunostimulating agent consisting of water-soluble antigenic complexes obtained from 4 opportunistic microorganisms: Klebsiella pneumoniae, staphylococcus, Proteus and Escherichia coli K-100 having a common antigen with Haemophilus influenzae) and challenged with K. pneumoniae culture on day 7 after immunization, the complete elimination of K. pneumoniae from the blood occurs within 24 hours. The subcutaneous immunization of rabbits with the above preparation leads to a significant increase in antibody titers, determined in the passive hemagglutination test with Klebsiella diagnosticum. The test of the passive protection of mice from Klebsiella sepsis has revealed a rise in the preventive activity of the sera of rabbits immunized with this preparation.     

Factors Affecting the Passive Hemagglutination Titration: Dilution Loops, Titration Trays, Vibration, Diluents

Serial dilution with Takatsy loops resulted in exaggerated passive hemagglutination titers with most of the anti-bovine serum albumin sera tested. It appears that certain types of agglutinins adhere to the loop surface and are released only gradually. This adherence, or carry-over effect, was prevented by presoaking loops in gelatin or gelatin-rabbit serum-albumin solutions. Hemolysins did not adhere to loops. In general, hemagglutination reactions performed on plastic trays gave higher titers than those performed in glass test tubes. The quality of the hemagglutination pattern was dependent to a great extent on the type of plastic tray used. As much as a 100-fold difference in titers was obtained depending on the composition of the antiserum diluent. The increase in vibration, in terms of linear displacement (approximately twofold), resulted in an eightfold decrease in titers.     

Primary and secondary immune response of mice to polysaccharide antigens of meningococcal serogroups A and C

The peculiarities of the primary immune response, the formation of immunological memory and the secondary immune response to serogroup A and C meningococcal polysaccharides were studied in 7 strains of inbred mice, hybrids F1 and in noninbred animals. The passive local hemolysis test and the passive hemagglutination test indicated that the intensity of immune response to A and C polysacchardies depended on the genotype of the animals: both antigens induced the most intense response in CBA and BALB/c mice. The primary immune response to the both antigens was characterized by a short latent period, a rapid (by days 4-5) increase in the amount of antibody-producing cells in the spleen and in antibody titer in the blood serum to the maximum level, and a pronounced decrease inantibody formation by days 6-7 followed by a gradual extinction of the response. A single injection of A and C polysaccharides in a dose of 0.5 microgram induced the formation of immunological memory in mice, persisting for at least 4 weeks and manifesting after reimmunization as the increased or more prolonged synthesis of IgM and IgG.     

Establishment of unresponsiveness in primed B lymphocytes in vivo

As an approach to examine the influence of the state of cellular activation on the ability to tolerize B cells, the induction of unresponsiveness in human gamma-globulin-(HGG) primed B lymphocytes was studied in an adoptive transfer system. In contrast to transferred normal spleen cells, spleen cells from HGG-primed mice are not readily rendered unresponsive when exposed to the tolerogen, deaggregated HGG (DHGG), in irradiated recipients. A kinetic study showed that unfractionated primed spleen cells do not respond to an antigenic challenge given between 6 and 10 days after cell transfer and injection of DHGG, indicating that they are transiently depressed. In contrast, isolated primed B cells are tolerized when transferred to recipients and treated with DHGG in the absence of T cells. Furthermore, primed B cells exposed to tolerogen in the recipients do not recover the ability to respond to HGG either after a secondary challenge with AHGG given up to 14 days after transfer, or after 2 consecutive challenges given on days 14 and 24 after transfer. The presence of primed T cells at the time of tolerization interferes with the induction of unresponsiveness in these primed B cells. These studies suggest that the presence of primed T cells is responsible for the inability to tolerize unfractionated primed spleen cells populations and that primed B cells themselves are not intrinsically resistant to the induction of unresponsiveness.     

Comparative study of resistance to tetanus and indices of the passive hemagglutination reaction in experimental animals

The article presents the results of the study of resistance to tetanus in 450 guinea pigs immunized against tetanus in a single injection and having antitoxin titers in their blood, as determined in the passive hemagglutination test, from less than or equal to 0.01 IU/ml to 1.6 IU/ml and more. The degree of protection in the immunized animals was determined by their challenge with Clostridium tetani spores in DCL and LD50.     

Comparative evaluation of methods for detecting HBsAg in sera

The results of the analysis of 1209 serum samples, made with a view to detecting those containing HBsAg, are presented. This analysis was made by the radioimmunoassay (RIA) on a polyethylene film, by the standard RIA technique with the use of a diagnostic kit obtained from Abbott Laboratories (USA) and by the passive hemagglutination (PHA) test. The RIA film technique was found to have the sensitivity of about 2 ng/ml HBsAg, which is similar to the sensitivity of the kit from Abbott Laboratories and exceeds the sensitivity of the PHA test approximately 50-fold. The percentage of detected HBsAg-positive sera, yielded by analysis with the use of the RIA film technique and the standard RIA technique, is the same. The RIA technique make it possible to detect more positive sera than the PHA test by about 2.5%.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. III. Development of the resistance with age and cellular events.

Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.     

Trypanosoma musculi: Passive hemagglutination technique to measure antibody in mice

A passive hemagglutination assay was developed to measure Trypanosoma musculi-specific antibody in mice. Indicator-erythrocyte donor mice received 550 rad ⁶⁰Co 24 hr before intraperitoneal injection of 3 × 10⁴T. musculi. T. musculi antigen-coated erythrocytes were obtained from these mice on Day 9 postinfection. T. musculi antigen-coated erythrocytes obtained in this manner were used as indicator erythrocytes in a passive hemagglutination procedure. Serum from hyperimmunized mice (three consecutive infections at 21-day intervals) gave titers as high as 1:1024. Titers of 1:256 and 1:512 were obtained from singly infected mice on Days 18 and 28 postinfection, respectively. In marked contrast, nude mice infected with T. musculi did not produce a detectable agglutinating antibody response. Erythrocytes obtained from either irradiated (550 rad ⁶⁰Co) uninfected mice, nonirradiated infected mice, or normal mice did not agglutinate when combined with any of the sera tested. These data suggest the usefulness of this passive hemagglutination assay for the measurement of antibody to T. musculi in the serum of infected mice.