Subcutaneous Route Of Immunization Research Articles

Assessing the efficiency of SBA-15 as a nanocarrier for diphtheria anatoxin

Abstract SBA-15 ordered mesoporous silica can be considered a promising inorganic nanocarrier with emerging potential as an oral vaccine adjuvant. In this study, we investigated its application in the encapsulation of the diphtheria anatoxin (dANA). We observed a considerable preservation of dANA secondary and tertiary structures, even after the drying process by means of Circular Dichroism (CD) and fluorescence spectroscopies. Antigen loading was assessed at a number of different ratios of adjuvant-to-antigen using a combination of nitrogen adsorption porosimetry and Small Angle X-ray Scattering (SAXS). Our data showed that the mass ratio of 1:10 (dANA:SBA-15) is recommended for total encapsulation of dANA in the mesopores, considering that at this relative mass concentration antigen clustering was avoided, which is deleterious effect for immunization purposes. dANA release in mimetic intestine fluid, at a pH equal to 6.8, was followed by in-situ SAXS measurements and shown to be slow, being more pronounced after 6 h and continuous up to 35 h. Finally, the immunogenic complex was tested in isogenic Balb C mice by oral and subcutaneous immunization routes, including a comparison with the only permitted adjuvant for human use, aluminum hydroxide. A higher antibody titer was obtained by subcutaneous and oral administration routes using SBA-15 as the vehicle of dANA, compared with the conventional aluminum hydroxide, demonstrating the viability to use this ordered mesoporous silica in the formulation of oral vaccines, as already proved for the Virus Like Particles (VLP) Hepatitis B (HBsAg) case.

Evaluation of intranasal and subcutaneous route of immunization in neonatal mice using DODAB-BF as adjuvant with outer membrane vesicles of Neisseria meningitis B

BackgroundThe Neisseria meningitidis bacterium is a Gram-negative diplococcus that can be classified into different serogroups according to the capsular structure. Six of them (A, B, C, W, X, Y) are responsible for causing Invasive Meningococcal Disease (IMD). The strategies for the development of a vaccine for serogroup B have been directed to the use of outer membrane vesicles (OMVs). The aim of this study was to evaluate the immunogenicity of antigenic determinants from OMVs of N. meningitidis B complexed with two different adjuvants: DODAB-BF and aluminum hydroxide (alum), comparing the evaluation of intranasal and subcutaneous route of immunization. MethodsWe used prime-boost immunization for the first time in outbred neonatal mice evaluating the cellular and humoral immune response. ResultsImmunoblot, ELISA DOT-ELISA and ELISpot were used universal methods of antibody detection, in order to detect the humoral and cellular immune response in male and female mice. Immunoblot analyzes the specificity of antibodies with the homologous N. meningitidis strain. ELISA served to quantify and compare the titers of antibodies in the serum of mice immunized with DODAB-BF + OMVs and alum + OMVs for IgG, IgG1, and IgG2a. Intranasal immunization produced a mixed response in the T helper cells Th1 and Th2, while subcutaneous immunization exhibited a Th1 profile. The DOT-ELISA identified cross-reactivity with DODAB-BF to different serogroups of N. meningitidis (B, C, W, and Y) that was not observed with alum. ELISpot analyzed IFN-γ- and IL-4 and the results showed the response directly to Th1 and Th2 profile. ConclusionOur findings indicate that DODAB-BF can be an alternative adjuvant for mucosal cell activation with OMVs of N. meningitidis B and that DODAB-BF was similar to aluminum hydroxide as an adjuvant for subcutaneous immunization.

Production of polyclonal antibodies in ascitic fluid of mice: time and dose relationships.

Further studies of the humoral immune response have been undertaken using ovine immunoglobulin G as the immunogen and BALB/C mice as the recipients. Mice immunized by the intraperitoneal route weekly for five weeks with an emulsion of the immunogen in Freund's complete adjuvant and given an intraperitoneal injection of pristane on day 14 produce relatively large volumes of ascitic fluid. A simple fluoroimmunoassay is used to determine specific antibody titres. The mice do not lose condition, remain active and eat normally throughout, although all show extensive granuloma formation within the peritoneal cavity. Tapping is performed before ascitic fluid production is excessive and each study is complete within 49 days, with a maximum of 5 taps. Only a single immunization site is required, since no higher antibody titres are obtained using combined intraperitoneal plus intramuscular plus subcutaneous immunization routes. A dose response study has shown that relatively large amounts (100 micrograms) of ovine immunoglobulin G must be injected to evoke a maximum response. Studies are in progress of various adjuvants and potential modulators of the immune response and of ways in which the immunogenicity of macromolecules can be changed. This approach can also be used to obtain antibodies for diagnostic purposes more rapidly than in rabbits and sheep.

Immunological response of three mouse strains to typhoid vaccine and Vi antigen.

Vi-agglutinin, active cutaneous anaphylaxis and protective responses (ed(50)) of three mouse strains (CFW, NIH, and Balb/cAnN) to acetone-inactivated typhoid vaccine and soluble Vi antigen were compared. Seven days after immunization with either typhoid vaccine or Vi antigen the three strains of mice differed with respect to Vi-antibody titers. Significant differences were observed in the protective responses. Each mouse strain was significantly better protected by the intraperitoneal than by subcutaneous route of immunization. Active cutaneous anaphylaxis was more pronounced in showing strain differences in response to Vi antigen. The serological responses to Vi antigen of the strains of mice did not correlate with their protective response.

Separation of the Phase I Agglutinogen of H. Pertussis from Toxic Components

Summary A method of separation of the agglutinogen from phase I, H. pertussis is described. About ten per cent of all the original agglutinogen present in the organisms may be concentrated into a fraction which represents but one per cent of the total solid content of the original sonic extract of the organisms. In the purified form the agglutinin-absorbing fraction elicits agglutinins in rabbits; upon intracutaneous testing it yields an immune response in rabbits which have been immunized with whole phase I organisms or the purified fraction. The agglutinogen has been freed of toxins; in normal animals marked primary toxicity is absent as determined by survival of animals after intravenous injection and by intracutaneous testing. This is in contrast with the primary toxicity found present when whole organisms or less pure fractions are used. The purification also results in a fraction showing specificity of intradermal response in rabbits immunized with phase I as contrasted with phases III and IV, H. pertussis and with parapertussis and unrelated organisms. Chemical studies indicate that the agglutinogen is probably protein in nature but one showing unusual characteristics, e.g., a high degree of thermostability over a wide range of pH. The extremely thermolabile toxin of Evans and Maitland has been detected. This thermolabile toxin is precipitated at pH = 4.5 without detectable loss of activity and may thereby be separated from the agglutinogen except that it adsorbs some agglutinogen. The agglutinogen is very readily adsorbed on all precipitates produced by lowering the pH, as well as on bacterial filters. Note: Since going to press a very interesting article has appeared by Dr. Mary Lee Wood, “A Filterable Toxic Substance in Broth Cultures of B. Pertussis,” Jour. Immunol., 1940, 39, 25. From the properties described it would appear that it is non-antigenic TLT which is the toxic component of the filtrate. As suggested by Dr. Wood, this component would appear to exist on the surface of the organism from which it passes into the filtrate. From the quantitative relations of the work of Evans and Maitland and from the fact that we have separated TLT equally well from extracts of organisms that were either unwashed or well washed before disintegration, this toxin must also exist throughout the organism as well as at the surface. More recently, D. G. Evans has reported (Jour. Path. Bact., 1940, 51, 49) this endotoxin to be antigenic when extracted and formolized, but not while in the intact organisms as injected into experimental animals or as in the parasite causing disease in man (no antitoxin found in convalescent serum). He has obtained neutralization of the intracutaneous reaction in rabbits previously injected with the extract. We, like Evans, have not observed neutralization in rabbits immunized with live whole organisms, even following immunization sufficiently intense to yield sera of agglutinative titer of 1:100,000. Our SX-I-immunized rabbits, accordingly, were not so tested. For an answer to this seemingly anomalous situation, where a part would appear to be greater than the whole, further investigation is indicated. If TLT is antigenic, it may be that it is but very slightly so, and that the subcutaneous route of immunization, in this case, is favorable so that antitoxin is produced in amounts which are detectable. Whatever the explanation, however, immunization of children against a toxin would not appear to be the method of choice when agglutinative immunization is available against a non-flagellated organism, occurring as a single type, to halt invasion initially. Toxic immunization would guard merely against one of the actions of the invaders should invasion occur.