The antibody response in the bovine mammary gland is influenced by the adjuvant and the site of subcutaneous vaccination.

Intramammary infections in cattle resulting in mastitis have detrimental effects on cows' well-being, lifespan and milk production. In the host defense against S. aureus mastitis antibodies are thought to play an important role. To explore potential ways to increase antibody titers in the bovine mammary gland the effects of various adjuvants on the magnitude, isotype, and neutralizing capacity of antibodies produced following subcutaneous vaccine administration at different immunization sites were analyzed. In this study, α-toxoid was used as a model antigen and formulated in three different alum-based adjuvants: Alum-Saponin, Alum-Oil, and Alum-Saponin-Oil. Vaccines were administered near the suspensory ligament of the udder or in the lateral triangular area of the neck. At both immunization sites, immunization with α-toxoid in Alum-Saponin-Oil resulted in higher specific antibody titers in milk and serum as compared with Alum-Oil and Alum-Saponin, without favoring an IgG1, IgG2, or IgA response. Furthermore, the neutralizing capacity of milk serum and serum following immunization near the udder and in the neck was higher when Alum-Saponin-Oil was used as adjuvant compared with Alum-Oil and Alum-Saponin. Prime immunizations near the udder effectively increased both antibody isotype titers and neutralization titers, while prime plus boost immunizations were required to induce similar effects following immunization in the neck. Results indicate that subcutaneous administration of an Alum-Saponin-Oil based vaccine near the udder could be further explored for the development of a one-shot vaccination strategy to efficiently increase intramammary antibody responses.

Immunity to Staphylococcus aureus secreted proteins protects rabbits from serious illnesses.

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. This study investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. Importantly, 20% of humans and 50% of rabbits in our TSS model cannot generate antibody responses to native superantigens. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (ß-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins ß-toxin and γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.

Toxicokinetics of mercury after long-term repeated exposure to thimerosal-containing vaccine.

The preservative thimerosal contains ethyl mercury (EtHg). Concerns over possible toxicity have re-emerged recently due to its presence in (swine and other) flu vaccines. We examined the potential accumulation of mercury in adults given repeated injections of a thimerosal-preserved vaccine for many years. Fifteen female patients were recruited from an outpatient clinic running a clinical trial with repeated injections (1 ml every 3-4 weeks) of a staphylococcus toxoid vaccine containing 0.01% thimerosal to treat chronic fatigue syndrome. Fifteen untreated female patients with the same diagnoses served as controls. Blood samples were taken before injecting the vaccine, 1 day later, about 2 weeks later, and just before the next injection. In the 15 controls, samples were taken twice. Blood was analyzed for total mercury and EtHg. The toxicokinetics were assessed for each patient separately as well as with a population-based pharmacokinetic model. Total mercury in blood increased on Day 1 in all treated patients (median: 0.33, range: 0.17-1.3 µg/l), as did EtHg (median: 0.14 µg/l, range: 0.06-0.43 µg/l). After a few weeks, levels were back to normal and similar to those in controls. Levels of methyl mercury (MeHg; from fish consumption) were much higher than those of EtHg. After exclusion of an outlier, the mean half-life in a population-based model was 5.6 (95% CI: 4.8-6.3) days. The results indicate that mercury from thimerosal is not accumulated in blood in adults. This is in accordance with short half-lives and rapid metabolism of EtHg to inorganic mercury.

[Adverse immunologic effects of immunomodulators revealed in experiment and ways to their surmounting].

Immunomodulators licopid (synthetic analogue of muramylpeptide) and purified staphylococcal toxoid (PST) in some variants of experiments on mice caused adverse immunologic effects: enhancement of virus-induced immunosupression, shift from latent immunosupression (revealed only by low, but not standard, doses of test-antigen) to manifested one. Described adverse effects are not a contraindication for use of the studied drugs in practice. Their adverse effects were revealed only after single inoculation, whereas in clinical conditions PST and licopid are used by courses with duration of 5-7 and 10 days respectively. Our experiments show that inoculation of suppressive doses of the preparations repeated by 2-4 times can prevent the shift from latent to manifested immunosupression. Enhancement of immunosupression was not observed in case of combined administration of suppressive doses of PST and adjuvant dose of licopid.

[Modulation of antigen-presenting functions in peritoneal macrophages and changes in production of several cytokines in mice caused by purified staphylococcal toxoid].

With adaptive transfer method it has been shown that immunomodulator purified staphylococcal toxoid (PST) changed (stimulate or suppress) antigen-presenting function (APF) of mice peritoneal macrophages (MF) in vivo. This phenomenon was registered during assessment of ability of peritoneal MF to present heterologous (with regard to PST) antigen--sheep erythrocytes (SE). Modulation vector depended from time interval between PST and SE inoculations. Inoculation of PST 1.5 h before SE resulted in stimulation of APF. When SE were inoculated to donor mice 24 h after PST, suppression of APF was developed. Suppression of APF was observed along with suppression of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) as well as B-lymphocytes growth factor (IL-4). When cytokine profile was assessed 4 h after PST injection, the suppression of synthesis of these cytokines was not observed. Production of IL-12 increased in 9-12 times in 4 and 24 h after PST injection.

[Immunomodulators of microbial origin enhance cytotoxicity of human mononuclear leukocytes and reduce metastatic progression of Lewis lung carcinoma in mice].

Effect of immunomodulators for microbial origin on innate immunity and antitumor system was continued to study. Immunomodificator Immunovac VP-4, purified staphylococcal toxoid and glucosaminyl muramyl dipeptide (GMDP) equally enhanced cytotoxicity of mononuclear leukocytes of peripheral blood of healthy donors. Index of cytotoxicity was 2.78, 2.77 and 2.70 respectively. Reduced metastatic progression of Lewis lung carcinoma in mice was observed after Immunovac VP-4 and GMDP administration. Effectiveness was seen when preparations administered according to schedules including their administration before implantation of the tumor. If preparations were administered number of metastases reduced in 4.4-5.6 times and size of metastases reduced in 7-10 times. Interplay between antitumor activity of studied immunomodulators and cytotoxic activity of NK-cells, which are base effectors of antitumor immune response, are discussed.

[The mechanisms of the impact of preliminary immunization with staphylococcus anatoxin on postresuscitation pathology of the myocardium].

Chronic experiments lasting for 5 weeks were carried out in 40 nonpedigree mature dogs. It has been detected that preliminary immunization with staphylococcus anatoxin potentiates the compensation mechanisms in the course of postresuscitation period, stimulates cell proliferation by increasing cardiac output that is an adaptive reaction of the cardiovascular system in postresuscitation period and provides optimal metabolism of the organism.

[Immunomodulation activity of new vaccines for pertussis prophylaxis--acellular pertussis vaccine and adsorbed DPT vaccine with acellular component]. / Otsenka immunomoduliruiushchei aktivnosti novykh preparatov dlia profilaktiki kokliusha--beskletochnoi kokliushnoi vaktsiny i AKDS-vaktsiny s beskletochnym kokliushnym komponentom.

The immunomodulating activity of acellular pertussis vaccine (APV) and adsorbed DPT vaccine with acellular pertussis component (DPTA vaccine) was studied. The study revealed that only large doses of APV, 10 immunizing doses (ID), suppressed humoral and cell-mediated response to sheep red blood cells (SRBC). 1 ID produced no influence on the formation of antibody producing cells, but increased the development of delayed hypersensitivity (DH) to SRBC. The modulation of cell-mediated immune response, induced by APV, returned to normal after the injection of purified staphylococcal toxoid, used as immunomodulator, in doses of 0.15 BU per mouse and 1.5 BU per mouse. DPTA vaccine containing 1 ID, as well as 10 ID, produced no immunomodulating effect. This was established by the evaluation of humoral response to SRBC in CBA mice and the study of the formation of DH to SRBC in BALB/c mice. As indicated by the total of the presented data, the inclusion of APV into DPTA vaccine enhanced the immunological safety of its pertussis component.

[Correlation between Staphylococcus carriage, specific antibody-production and AB0-blood grouping in plasma donors]. / Vzaiemozv'iazok bakterionosiistva stafilokokiv, spetsyfichnoï antytiloproduktsiï ta AB0-hrupovoï nalezhnosti donoriv plasmy.

Interaction peculiarities of three components of the immune human homeostasis-antigens of blood groups AB0, staphylococcus antigens and antistaphylococcus antibodies have been investigated. Donors (85) of antistaphylococcus plasma immunized by staphylococcus anatoxin have been investigated. It is found that the nasal staphylococcus carriage in donors depends on the level of specific and natural antibodies and on the coincidence between the staphylococcus antigen structure and the protein substance of the specific blood group factors.

[Homeostasis indexes of staphylococcal anatoxin immunized blood donors, previously immunized with tetanus toxoid]. / Pokaznyky homeostasu imunizovanykh stafilokokovym anatoksynom donoriv, ranishe revaktsynovannykh proty pravtsia.

On the 50 volunteers from 20 to 40 years old gomeostasis indexes of inoculating by staphylococci cleaning adsorbing anatoxini donors, before have been revactinated against tetanus, were studied. It was established, that attract of plasmaferes donors to consistent immunization by tetani and staphylococci anatoxini don't call negative alterations in their gomeostasis indexes. Results of study demonstrate, that it's possibly to attract for immunization by staphylococci anatoxini donors, before have been revactinated against tetanus.

[Correction of immune response using purified staphylococcal toxoid and likopid in the secondary immunodeficiency induced by Coxsackie virus B3]. / Korrektsiia immunogo otveta s pomoshch'iu anatoksina stafilokokkovogo ochishchennogo i likopida pri vtorichnom immunodefitsite, indutsirovannom virusom Koksaki B3.

The action of immunomodulators, purified staphylococcal toxoid (PST) and lycopid, on secondary immunodeficiency state developing during infection caused by Coxsackie virus B3 was studied. This defect was manifested by delayed hypersensitivity to sheep red blood cells (SRBC) and the suppression of neutralizing antibodies to poliomyelitis virus. Depending on the scheme of the experiment, PST normalized the defects of immune response to SRBC or poliovirus, increased suppression or showed no activity. Lycopid corrected the defects of humoral response to SRBC. The combination of PST and lycopid was found to produce no increase of suppression. The suggestion was made on the expediency of combination of two (and probably more) immunomodulators for increasing the efficiency of correction of secondary immunodeficiency.

Immune response with biodegradable nanospheres and alum: studies in rabbits using staphylococcal enterotoxin B-toxoid.

In this study, the adjuvant effect of the sustained release biodegradable nanospheres (100-150 nm in diameter) has been compared with alum. Nanospheres were formulated using a biodegradable polylactic polyglycolic acid copolymer (PLGA, 50:50) containing Staphylococcal Enterotoxin B (SEB) toxoid as a model vaccine antigen. Systemic immune response of the nanospheres containing toxoid was studied in rabbits by subcutaneous immunization. The data demonstrated that approximately 30% of the toxoid activity was lost following its encapsulation into nanospheres. Under in vitro conditions, nanospheres demonstrated sustained release of the toxoid. However, only 20% of the antigenic toxoid was released over the first 2 weeks of the release study. Immunization of animals with equal doses of toxoid, either using nanospheres or alum induced a comparable systemic immune response (IgG, IgM and IgA). The immune response reached a maximum level at 7 weeks post-immunization, which then gradually declined with time. The booster dose of toxoid at 19 weeks, either using alum or nanospheres induced similar immune response in both the groups, but was greater than the primary immune response. The studies, thus, suggest that biodegradable nanospheres could be used as a vaccine adjuvant.

[Staphylococcal anatoxin, an alternative in the treatment of endogenous uveitis]. / Anatoxina stafilococica, o alternativa în tratamentul uveitei endogene.

The paper presents one clinical study effected by fourty-four patients with endogenous uveitis which beneficiary of a general treatment with staphylococcal anatoxin. The conclusions of the study impose that one effective treatment is obtained at the young patients with anterior, acute uveitis with moderate inflammatory phenomenon and what present one effective answer of the lymphocyte T by interleukin 1. The application of the treatment suppose several exams reminiscent of the immunologic status especially of the cellular immunology.

[The patterns of the corrective action of a natural immunomodulator on the secondary immunological defect caused by a corpuscular pertussis vaccine]. / Zakonomernosti korrigiruiushchego deistviia prirodnogo immunomoduliatora na vtorichnyi immunologicheskii defekt, vyzvannyi korpuskuliarnoi kokliushnoi vaktsinoi.

Purified staphylococcal toxoid (PST) was shown to be capable of preventing the development of secondary antigen-nonspecific immune deficiency in mice, immunized with whole-cell pertussis vaccine. The immunocorrective action of PST was manifested after its injection before (on day -1), simultaneously with and after (on day +1) the injection of whole-cell pertussis vaccine. Correction was either complete or partial, depending on the scheme of the experiment and the dose of PST.

Biodegradable and biocompatible poly(DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies.

Microspheres composed of biocompatible, biodegradable poly(DL-lactide-co-glycolide) (DL-PLG) and staphylococcal enterotoxin B (SEB) toxoid were evaluated as a vaccine delivery system when subcutaneously injected into mice. As measured by circulating immunoglobulin G (IgG) antitoxin titers, the delivery of SEB toxoid via DL-PLG microspheres, 1 to 10 microns in diameter, induced an immune response which was approximately 500 times that seen with nonencapsulated toxoid. The kinetics, magnitude, and duration of the antitoxin response induced with microencapsulated toxoid were similar to those obtained when an equal toxoid dose was administered as an emulsion with complete Freund adjuvant. However, the microspheres did not induce the inflammation and granulomata formation seen with complete Freund adjuvant. The adjuvant activity of the microspheres was not dependent on the superantigenicity of SEB toxin and was equally effective at potentiating circulating IgG antitrinitrophenyl levels in response to microencapsulated trinitrophenyl-keyhole limpet hemocyanin. Empty DL-PLG microspheres were not mitogenic, and SEB toxoid injected as a mixture with empty DL-PLG microspheres was no more effective as an immunogen than toxoid alone. Antigen-containing microspheres 1 to 10 microns in diameter exhibited stronger adjuvant activity than those greater than 10 microns, which correlated with the delivery of the 1- to 10-microns, but not the greater than 10-microns, microspheres into the draining lymph nodes within macrophages. The antibody response induced through immunization with microencapsulated SEB toxoid was protective against the weight loss and splenic V beta 8+ T-cell expansion induced by intravenous toxin administration. These results show that DL-PLG microsphere vaccine delivery systems, which are composed of pharmaceutically acceptable components, possess a strong adjuvant activity for their encapsulated antigens.

[The characteristics of the protective and antigenic activities of purified staphylococcal anatoxin in trials with mice of different genotypes]. / Kharakteristika protektivnoi i antigennoi aktivnosti anatoksina stafilokokkovogo ochishchennogo v opytakh na myshakh raznykh genotipov.

The protective potency of purified staphylococcal toxoid by the survival rate of immunized mice challenged with the culture of Staphylococcus aureus strain L-1726 and the antigenic properties of the toxoid, determined by the level of antitoxin in the blood of mice and by the intensity of cell-mediated immunity in the spleen-cell migration inhibition test, were studied. The experiments were made on CBA and C57BL/6 mice. Purified staphylococcal toxoid was shown to possess antigenic and protective properties in a wide range of doses between 0.15 and 15 binding units per mouse. The protective effect of the toxoid in CBA mice was manifested in the presence of circulating antibodies and cell-mediated reaction or only in the presence of the toxoid. In C57BL/6 mice the protective effect of the toxoid was less pronounced and appeared in combination with the induction of cell-mediated immunity in the presence of an extremely low antibody level (0.062 I.U.).