Immune Response to Conjugates of Fragments of the Type K9 Capsular Polysaccharide of Acinetobacter baumannii with Carrier Proteins.

The clonal bacterial species Acinetobacter baumannii is an emerging multidrug-resistant pathogen which causes high-lethality infections. Cells of A. baumannii are surrounded by the type-specific capsular polysaccharide (CPS), which provides resistance to the protective mechanisms of the host and is considered a target for immunization. The conjugates of three inert carrier proteins and A. baumannii type K9 CPS fragments, which contained various numbers of oligosaccharide repeats (K-units), were synthesized by periodate oxidation and squaric acid chemistry. The conjugates were applied to immunize mice, and chemical synthesis by squaric acid was shown to significantly improve the immunogenic properties of glycoconjugate. In BALB/c mice, IgG antibodies were predominant among type K9 CPS reactive antibodies, and their total content was several times higher than that of IgM. Immune sera were characterized by their opsonization ability during practically the entire lives of the experimental mice. The sera were cross-reactive, but the highest specificity was observed against the antigen (type K9 CPS) used for immunization. The immunization of BALB/c and ICR-1 mice with a glycoconjugate without adjuvants led to varying degrees of stimulation of IL-10, IL-17A, and TNF-α production, but not IL-4 production in the ICR-1 mice. This is in contrast to the BALB/c mice, in which γ-IFN production was also activated. The protective effectiveness of the glycoconjugates obtained by squaric acid chemistry was demonstrated by experiments that involved challenging immunized and nonimmunized animals with a lethal dose of A. baumannii K9. IMPORTANCE Immunization by glycoconjugates with A. baumannii type K9 CPS fragments induced a high level of antibodies (predominantly IgG) in sera, which reacted specifically with the CPS of A. baumannii type K9, as well as a long immunological memory. The sera of immunized animals efficiently opsonized A. baumannii type K9. Immunization resulted in the balanced production of pro/anti-inflammatory lymphokines and protective antibodies to ensure the survival of the mice infected with A. baumannii. The level of specific antibodies was sufficient to provide protective immunity against the challenge by A. baumannii, making this approach applicable in the development of vaccine preparations.

The zinc ions stabilize the three-dimensional structure and are required for the binding of staphylococcal enterotoxin-like protein P (SEIP) with MHC-II receptors.

Staphylococcus aureus is a common human and animal pathogen. These bacteria have various pathogenicity factors, including enterotoxin-like proteins. SElP (staphylococcal enterotoxin-like protein P) has potential zinc ion-binding sites and is able to interact with major histocompatibility complex class II (MHCII) and T-cell receptor (TCR). A method for the expression and isolation of the enterotoxin-like protein of Staphylococcus aureus (SElP) was developed. The expression was carried out in E. coli cells, and the protein was isolated by affinity chromatography on a NiNTA column. The endotoxins were separated by affinity chromatography on Affi-Prep® polymyxin. It was shown by gel filtration that the resulting protein had a monomeric form. The protein in zinc-bound and zinc-free forms was characterized by protein melting using fluorescence method and it was shown that zinc stabilizes the spatial structure of SElP. The functional activity of SElP was investigated by the ability to interact with the histocompatibility antigen class II receptor (MHC-II) exposed on the B cell line Raji by flow cytofluorometry. The zinc-bound and zinc-free forms were shown to differ in their interaction with MHC-II. The localization of the zinc-binding site was confirmed by the introduction of the H225 and D227 mutations. The mutant protein was characterized by melting, and its propensity to form aggregates was shown.

Imbalances in cellular immunological parameters in blood predetermine tumor onset in a natural mouse model of breast cancer.

The development of new approaches to breast cancer (BC) early diagnosis is an important objective of modern oncology. Although the role of the immune system in cancer initiation process was experimentally well established, the prognostic value of cellular blood immunological parameters (CBIPs) for BC onset prediction was not demonstrated either in clinics or in mouse models. In this study, we focused on revealing informative CBIPs for mammary cancer (MC) onset prediction in the BLRB/BYRB mouse model with a high incidence of natural MC development. Blood samples were collected from 80 aging females of these original mouse strains, 12 basic CBIPs were estimated by flow cytometry. Then mice were followed up for 28 weeks, and the outcome of females (MC diagnosis, death without MC or MC-free survival) was registered. We estimated the patterns of changes in CBIPs with age and in accordance with the outcome. An increasing imbalance in 11 CBIPs during natural aging of females clearly resembled human immunosenescence phenomenon and several patterns corresponded to the results obtained on cancer-free members of BC-affected families. We stratified heterogeneous female population into middle-aged and old subgroups. Low NK-cell levels in middle-aged mice and low B-cell along with high T-helper levels in old mice distinguished females with developed MC from the other groups. We found a reliable correlation of several CBIPs with age at MC diagnosis and survival of cancer-bearing females. Thus, we demonstrated the predictive potential of CBIPs as a basis for the development of prognostic models for BC onset in clinics.

Local Interleukin-2 Immunotherapy of Breast Cancer: Benefit and Risk in a Spontaneous Mouse Model.

Earlier, naturally arising mammary cancer in BLRB female mice was shown to reproduce some key pathological characteristics of the familial set of human breast cancer. Then we advanced a novel 3S-paradigm of anticancer research that helped to develop selection criteria and to estimate benefit/risk of local interleukin-2 (IL-2) effects in this spontaneous mouse model. In this paper, the efficacy of single and triple local IL-2 doses is compared using properly selected murine BLRB females based on our previously published data. Only BLRB females bearing spontaneous mammary tumors without subclinical period were used. The tumor growth rate and recipient survival of single and triple IL-2 applications were compared with corresponding parameter values of untreated control. Tumor growth rate was decreased in both experimental groups versus control parameter values. Single IL-2 application resulted in a significant prolongation of the average survival time while triple application caused acute tumor rejection in some females decreasing the survival time of the rest of the recipients. As a result, proper treatment protocol in accurately selected females allowed increasing the complete response rate to 14% in spontaneous mouse model of breast cancer. In conclusion, our approaches may demonstrate the principle methodology developing preselection procedure for breast cancer patients for local IL-2 therapy application.