Protective action of a vaccine made from Salmonella minnesota R 595 in the intranasal infection of mice with P. aeruginosa

The comparative study of heated corpuscular vaccines prepared from S. minnesota mutant R 595, chemotype Re, from S. minnesota strain SF 1111 with defective lipopolysaccharide and from P. aeruginosa strain PA 103 has been carried out. The vaccine prepared from the chemotype Re mutant, in contrast to the vaccine prepared from S. minnesota strain SF 1111, has been found to induce the development of active immunity (and the corresponding antiserum, passive immunity) to P. aeruginosa introduced intranasally into mice, as well as to stimulate the elimination of the cells of P. aeruginosa infective strain from the lungs of the mice. The potency of the vaccine prepared from the chemotype Re mutant has been found to be significantly no different from that of the vaccine prepared from P. aeruginosa strain PA 103.     

Immunological activity and toxicity of a peroral vaccine made from mutant Re Salmonella minnesota

The possibility of the oral use of heated corpuscular vaccine prepared from S. minnesota mutant R 595 (chemotype Re) for protection against Pseudomonas aeruginosa has been studied. Oral immunization in 3 doses, each containing 10(9) cells of the vaccine strain, has been shown to protect mice from death after the intravenous injection of P. aeruginosa culture in a dose of 5 LD50 and induce a rise in the titers of antibodies to Re-glycolipid (Re-hemagglutinins). After multiple oral administration Re-vaccine shows low acute and chronic toxicity and induces local and systemic immunological transformation.     

Different antiamebic antibody isotype patterns in the large and small intestine after local and systemic immunization of mice with glutaraldehyde fixed Entamoeba histolytica trophozoites

We have determined the major immunoglobulin isotypes (IgG, IgA, IgM) of antiamebic antibodies induced in the serum and in the large and small intestine after local (oral and rectal) or systemic (intraperitoneal and intramuscular) immunization of mice with glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). IgA predominated in the small intestine after immunization through all routes, whereas in the large intestine similar antibody levels of the major isotypes were induced by rectal, intraperitoneal and intramuscular immunization. The intramuscular route elicited intestinal responses lower than those induced by the rectal and intraperitoneal routes, but higher than the slight IgA antibody increase observed after oral immunization. The differences in antiamebic antibody response patterns at the large and small intestine suggest that there are different mucosal effector compartments. They also indicate that isotype analysis of mucosal antibodies from the sites where an infectious agent resides is needed to evaluate whether a vaccine candidate induces responses of higher protective value in the appropriate site, and that the study of antibody responses must not be limited to sampling the serum or mucosal sites distant to the relevant one.     

Evaluation of the immunological activity of a vaccine from mutant Salmonella minnesota R 595 on a model of experimental Pseudomonas aeruginosa infection in mice

The comparative study of heated corpuscular vaccines prepared from S. minnesota mutant R 595 with defective lipopolysaccharide (LPS), chemotype Re, derived from S. minnesota strain SF 1111 with unchanged LPS, and from P. aeruginosa strain PA 103, was carried out. In contrast to the vaccine from S. minnesota strain SF 1111, the vaccine prepared from the mutant with chemotype Re induced the development of cell-mediated and humoral immunity to P. aeruginosa, and its immunogenicity was close to that of the vaccine from P. aeruginosa strain PA 103.     

Increased sensitivity of immune macrophages to the cytotoxic action of virulent shigellae

The in vitro interaction of live bacteria belonging to virulent and avirulent Shigella and Salmonella strains with peritoneal macrophages obtained from mice immunized by the intragastric administration of these bacteria has been studied. In contrast to Salmonella-activated macrophages capable of resisting the intracellular proliferation and the cytopathic action of homologous bacteria, Shigella-activated macrophages become more sensitive to the cytopathic action of virulent shigellae. The ability of shigellae to render an aggravating cytopathic effect on the activated macrophages correlates with the virulence of dysentery bacilli and is practically absent in avirulent strains, including S. flexneri 2a No. 516 M vaccine strain.     

Anti-bacterial and anti-endotoxic immunity, induced by the administration of an enterobacterial vaccine

The work demonstrates that the sera of animals immunized with enterobacterial vaccine, when adsorbed on sheep red blood cells sensitized with glycolipid Re, lose their capacity of decreasing the lethal effect of Shigella sonnei endotoxin, which is indicative of the antiendotoxic action of antibodies. At the same time, immune sera obtained after immunization with enterobacterial vaccine contain antibodies having also other specificity and thus ensuring antibacterial immunity.     

The effect of Shigella sonnei strains opposite in their virulence on different links in the immune system]

For the first time different action of S. sonnei strains, opposite in their virulence, on hematopoiesis and the functional activity of T- and B-lymphocytes has been shown. The hematopoiesis-disturbing action of virulent shigellae is manifested by their capacity, more pronounced than similar capacity of an avirulent (vaccine) strain, for stimulating the processes of endo- and exocolony formation, the proliferation of hematopoietic stem cells and their migration to the blood. The effect produced by shigellae on T-cell-mediated immune response is manifested by the suppression of macrophage migration and its subsequent activation, whose manifestations and duration depend on the virulence of S. sonnei strains under study. The modulating effect of S. sonnei on B-cell-mediated immune reactions is manifested by the inhibiting action of S. sonnei virulent strain and the stimulating action of S. sonnei vaccine strain on the formation of antibody-producing cells synthesizing S. sonnei lipopolysaccharide antibodies shortly after the injection of shigellae. The results of this study indicate that S. sonnei virulent and avirulent (vaccine) produce multifunctional and differing effects on cell-mediated immune reactions, these processes being dependent on the virulence of shigellae and their individual specific antigens.     

Experimental study of vaccines against Salmonella typhimurium infection.

Serological and protective activities of vaccines from S. typhimurium and S. minnesota were studied. It has been demonstrated that active protection against infection in experimental salmonellosis in mice can only be obtained by immunization of the animals using vaccines from complete antigenic complexes isolated from S-strains. It has been found that expressed anti-infection immunity (unlike anti-endotoxic immunity) is induced to the same extent by either high-molecular components (2,000,000 daltons and more), showing great serological activity, or components with relatively low molecular weight (15,000--20,000 daltons) and minimum serological activity. Vaccines from Ra- and Re-strains of S. minnesota do not induce resistance to S. typhimurium infection in mice in either active protection tests or passive protection tests.     

Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.     

Passive protection of mice with the 19 S and 7 S fractions of anti-pertussis sera in experiment.

Protective activity of anti-persussis rabbit and mouse sera and 19 S and 7 S fractions obtained from these sera was investigated in the test of passive protection of mice on a model of pertussis meningoencephalitis. The method of simultaneous intracerebral administration of the serum or fraction with live culture of a virulent B. pertussis strain was used. Hyperimmune rabbit serum containing mercaptoethanol-resistant agglutinins in a high titre was found to have the most pronounced protective effect. Serum of mice, collected 14 days after single immunization of the animals, did not show any protective properties. A small amount of protective activity was observed in the serum collected on the 30th day after a single administration of the vaccine. A sharp increase in the protective activity of the serum was observed after double immunization of mice. Correlation was found between the increase in the titre of agglutinins (in particular of 7 S antibodies) and the protective activity of the serum. Protective properties of 19 S and 7 S fractions isolated from immune rabbit and mouse sera by the method of gel filtration were investigated. Both fractions were found to possess protective properties, but fraction 7 S was more active than fraction 19 S.     

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.     

The immunization of adults against respiratory syncytial virus infection

The results of the comparative study of the immunological effectiveness of experimental samples of respiratory syncytial (RS) viral vaccine, prepared from a live attenuated strain and introduced in a single administration to young adults by the intranasal, intradermal and combined intranasal-intradermal) routes, are presented. The effectiveness of intranasal immunization was inversely related to the level of previously existing humoral (serum, secretory) antibodies. Intradermal immunization enhanced the frequency of the formation of serum antibodies in persons having had such antibodies before the introduction of RS vaccine. The most active formation of serum and secretory antibodies was ensured by the combined (intranasal-intradermal) method of the administration of live RS vaccine which proved to be particularly effective in persons having had antibodies in the blood and secretions prior to immunization.     

The role of mucosal immunity in prevention of HIV transmission

Vaccines designed to prevent mucosal transmission of HIV should establish multiple immune effectors in vaccine recipients, including antibodies which are capable of blocking HIV entry at mucosal epithelial barriers and of preventing initial infection of target cells in the mucosa. Immunological analyses of HIV-resistant humans and data obtained in nonhuman primate vaccine studies indicate that both secretory and serum antibodies may play an important role in protection against mucosal transmission of HIV or SIV, whereas cytotoxic T cells are required for clearance of mucosal infection and prevention of systemic spread. This review summarizes the roles of IgA and IgG antibodies in preventing mucosal infection by other viral and bacterial pathogens, and then discusses the various mechanisms by which antibodies might contribute to protection against HIV at mucosal surfaces. These include prevention of mucosal contact, blocking attachment of virus or infected cells to epithelial cells, interception of virus during transepithelial transport, neutralization of virus in the mucosa, and elimination of locally infected cells through antibody-dependent cell-mediated cytotoxic reactions. The regional nature of mucosal immune responses is reviewed in light of its relevance to HIV vaccine development. We conclude that mucosal immunization should be considered a component of vaccine strategies against HIV.     

Intranasal immunization with a lipooligosaccharide-based conjugate vaccine from nontypeable Haemophilus influenzae enhances bacterial clearance in mouse nasopharynx

Nontypeable Haemophilus influenzae (NTHi) is a major cause of otitis media in children. We investigated whether intranasal immunization with a detoxified lipooligosaccharide-tetanus toxoid (dLOS-TT) conjugate vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal clearance. The results demonstrated that intranasal immunization with dLOS-TT plus adjuvant cholera toxin (CT) significantly induced LOS-specific IgA antibodies in mouse external secretions, especially in nasal wash (90-fold), bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (three-fold). LOS-specific IgA antibody-forming cells were also found in mucosal and lymphoid tissues with their highest numbers in the nasal passage (528 per 10(6) cells). In addition, the intranasal immunization elicited a significant rise in LOS-specific IgG (32-fold) and IgA (13-fold) in serum. For the immunized mice which had been challenged through the nose with 10(7) live NTHi strain 9274 cells, the vaccine group showed a significant reduction (74-77%) of NTHi, compared to that of control groups with CT alone or dLOS plus CT (P<0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA and serum IgG. The clearance of five heterologous strains was investigated and revealed a significant clearance of strains 3198, 5657 and 7502 but not of strains 1479 and 2019. These data suggest that intranasal immunization with dLOS-TT vaccine elicits both mucosal and systemic immunity against NTHi and enhances bacterial clearance from nasopharynx in mice. Such a vaccine and vaccination regime may be applicable to humans with an appropriate formulation.     

The dynamics of antibody formation to a vaccinal strain of Francisella tularensis in different immunization regimens

The use of different schemes of albino mice immunization either by living or by killed preparations of the vaccine strain of Francisella tularensis when obtaining monoclonal antibodies to the tularemia microbe made it possible to reveal definite regularities in the dynamics of antibody formation. The highest titres of antibodies in sera of animals-donors of splenocytes were obtained during the daily (for 3 days) intraperitoneal immunization of mice with living vaccine or with its thrice administration to the spleen thrice with the interval of 10 days. Revaccination against a background of high titres of antibodies decreased their quantity in blood serum of mice, while that against a background of low titres increased them.     

Optimization of Mucosal Responses after Intramuscular Immunization with Integrase Defective Lentiviral Vector

Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.     

Protective efficacy and immunogenicity of Vi-porin conjugate against Salmonella typhi.

A conjugate vaccine against Salmonella typhi was prepared by covalently binding capsular polysaccharide (Vi) with porin, both isolated from S. typhi. First, Vi and porins were extracted. The Vi was purified from S. typhi Ty2. The purified Vi conformed to the requirements of the World Health Organization. Porins were purified from S. typhi 0901. The Vi was bound to the porins by a heterobifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyl dithio)-propionate (SPDP). After preparing the Vi-porin conjugate, its protective ability and immunogenicity were studied in mice following systemic immunization. The results showed that the conjugate is 6.5-fold more protective than Vi alone against S. typhi. The mice immunized with conjugate elicited higher anti-Vi antibody (IgG) levels (P < 0.01) than the mice immunized with Vi alone. Anti-porin antibodies were also induced by the conjugate. To study the mucosal immune responses, secretory IgA (sIgA) in the intestinal fluid was measured. Conjugate-immunized mice showed the induction of sIgA as compared to Vi alone. The results showed that when Vi is bound to porins, both isolated from same organism, the resultant conjugate induced both systemic and mucosal immune responses and provided better protection against S. typhi than Vi alone.     

结核分枝杆菌EsxV脂质体纳米颗粒亚单位疫苗的制备及免疫学特性

本研究旨在制备脂质体纳米颗粒（lipid nanoparticles，LNP）为载体的结核分枝杆菌（Mycobacterium tuberculosis，Mtb）抗原EsxV亚单位疫苗，明确该疫苗经黏膜免疫诱导的免疫应答水平。采用薄膜分散法制备包裹蛋白EsxV和c-di-AMP的LNP （EsxV：C：L），并对其包封率、LNP形态、粒径、表位电荷及多相分散指数进行了检测。EsxV：C：L滴鼻免疫BALB/c小鼠，检测免疫后血清和黏膜抗体、肺或脾细胞因子转录及分泌水平、肺T细胞亚群细胞比例。结果成功获得大小均一、呈球状、带负电的EsxV：C：L LNP亚单位疫苗。与EsxV：C相比，EsxV：C：L鼻黏膜接种可诱导小鼠呼吸道黏膜sIgA水平增加，脾细胞因子IL-2分泌水平升高，提高中央记忆T细和组织驻留T细胞的比例。综上，EsxV：C：L经黏膜免疫，可诱导更强的黏膜免疫和记忆性T细胞免疫应答，可能提供更好的抗Mtb感染的保护作用。     

Biological characteristics of strains of lactobacilli, promising for use as eubiotics]

In the course of in vitro studies 3 Lactobacillus strains with pronounced antagonistic activity against some pathogenic and opportunistic bacteria (shigellae, enteropathogenic Escherichia, Proteus, staphylococci) were selected. In experiments on germ-free rats faint colonization by L. plantarum 37 was observed in the small intestine, as well as in the large intestine when low doses of these bacilli were introduced into the gastrointestinal tract of the animals. In vitro experiments demonstrated the decreased growth rate of this strain. The prophylactic administration of two eubiotic strains, L. plantarum 37 and L. fermentum 39, simultaneously with chloramphenicol to primates inhibited the growth of opportunistic bacteria, though L. fermentum 39 excessively suppressed the content of Escherichia coli in the enterobacterial population. The optimum biological effect was achieved with the therapeutic use of these three strains for the correction of dysbiotic disturbances caused by the administration of tetracycline in volunteers.     

Protective activity of a cell-free Klebsiella vaccine in infection in mice caused by Streptococcus pneumoniae serotypes

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial mass with hydroxylamine, for protecting mice from pneumococcal infection caused by S. pneumoniae, serotypes 3, 4 and 9N, has been studied. Klebsiella vaccine has been found to possess immunostimulating potency with respect to the S. pneumoniae serotypes under study. On day 5 this potency is manifested to a greater extent than 24 hours after immunization. The combination of Klebsiella vaccine with Proteus vulgaris, Staphylococcus aureus and Escherichia coli K-100 antigens enhances the stimulation of nonspecific resistance.