Mechanofluorochromic behaviors and data security protection properties of salicylaldimine-based difluoroboron complexes with different aryl substituents.

To further explore the relationship between aryl substituents and mechanofluorochromic (MFC) behaviors, four salicylaldimine-based difluoroboron complexes (ts-Ph BF2, ts-Ph-NA BF2, ts-2NA BF2, and ts-triphenylamine [TPA] BF2), including aromatic substituents with different steric hindrance effects, were designed and successfully synthesized. Four complexes with twisted molecular conformation displayed intramolecular charge transfer and aggregation-induced emission properties. Under external mechanical stimuli, the as-synthesized powders of ts-Ph BF2, ts-Ph-NA BF2, and ts-TPA BF2 exhibited redshift fluorescence emission behaviors, and ts-Ph BF2 and ts-TPA BF2 could be recovered to original shifts by fuming, but ts-Ph-NA BF2 displayed irreversible switching. ts-2NA BF2 had no change during the grinding and fuming processes. The results indicated that the MFC behaviors could be attributed to the phase transformation between the well-defined crystalline and disordered amorphous states by X-ray diffraction measurement. Further research illustrated that ts-TPA BF2 with the most significant MFC phenomenon could be applied in data security protection in ink-free rewritable paper.

Discordance in the Epithelial Cell-Dendritic Cell Major Histocompatibility Complex Class II Immunoproteome: Implications for Chlamydia Vaccine Development.

BACKGROUND: Chlamydia trachomatis and Chlamydia muridarum are intracellular bacterial pathogens of mucosal epithelial cells. CD4 T cells and major histocompatibility complex (MHC) class II molecules are essential for protective immunity against them. Antigens presented by dendritic cells (DCs) expand naive pathogen-specific T cells (inductive phase), whereas antigens presented by epithelial cells identify infected epithelial cells as targets during the effector phase. We previously showed that DCs infected by C trachomatis or C muridarum present epitopes from a limited spectrum of chlamydial proteins recognized by Chlamydia-specific CD4 T cells from immune mice. METHODS: We hypothesized that Chlamydia-infected DCs and epithelial cells present overlapping sets of Chlamydia-MHC class II epitopes to link inductive and effector phases to generate protective immunity. We tested that hypothesis by infecting an oviductal epithelial cell line with C muridarum, followed by immunoaffinity isolation and sequencing of MHC class I- and II-bound peptides. RESULTS: We identified 26 class I-bound and 4 class II-bound Chlamydia-derived peptides from infected epithelial cells. We were surprised to find that none of the epithelial cell class I- and class II-bound chlamydial peptides overlapped with peptides presented by DCs. CONCLUSIONS: We suggest the discordance between the DC and epithelial cell immunoproteomes has implications for delayed clearance of Chlamydia and design of a Chlamydia vaccine.

Identification of MHC-Bound Peptides from Dendritic Cells Infected with Salmonella enterica Strain SL1344: Implications for a Nontyphoidal Salmonella Vaccine.

Worldwide Salmonella enterica infections result in substantial morbidity and mortality and are the major cause of infant bacteremia in Sub-Saharan Africa. Diseases caused by Salmonella are treatable with antibiotics, but successful antibiotic treatment has become difficult due to antimicrobial resistance and collateral effects on the microbiome. An effective vaccine together with public health efforts may be a better strategy to control these infections. Protective immunity against Salmonella depends primarily on CD4 T-cell-mediated immune responses; therefore, identifying relevant T-cell antigens is necessary for Salmonella vaccine development. We previously used a dendritic-cell-based immunoproteomics approach in our laboratory to identify T-cell antigens. The testing of these antigens as vaccine candidates against Chlamydia infection in mice yielded positive results. We applied this technology in the present study by infecting murine bone-marrow-derived dendritic cells from C57BL/6 mice with Salmonella enterica strain SL1344, followed by immunoaffinity isolation of MHC class I and II molecules and elution of bound peptides. The sequences of the peptides were identified using tandem mass spectrometry. We identified 87 MHC class-II- and 23 MHC class-I-binding Salmonella-derived peptides. Four of the 12 highest scoring class-II-binding Salmonella peptides stimulated IFN-γ production by CD4+ T cells from the spleens of mice with persistent Salmonella infection. We conclude that antigens identified by MHC immunoproteomics will be useful for Salmonella immunobiology studies and are potential Salmonella vaccine candidates. Data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD004451.

Mechanofluorochromic behaviors and latent fingerprint detection of triphenylamine-based compounds with mono-/bis-BF2 fluorophores.

To better understand the relationship between molecular structure of the mono-/bis-BF2-core compounds and mechanofluoroboron behaviors, two pyridine-based difluoroboron compounds with triphenylamine group (TPA-ts-BF2 and TPA-ts-2BF2) were designed and successfully synthesized, which TPA-ts-BF2 including a BF2 fluorophore and TPA-ts-2BF2 containing the bisBF2 fluorophores. Based on the photophysical properties measurements results, it was found that TPA-ts-2BF2 had more excellent intramolecular charge transfer characteristics than that of TPA-ts-BF2, and exhibited significant aggregation-induced emission activity, however, TPA-ts-BF2 displayed typical aggregation-caused quenching phenomenon. Meanwhile, the emission spectrum of the solid powders of TPA-ts-2BF2 was red-shifted 52 nm after grinding, that of TPA-ts-BF2 was red-shifted 46 nm, which was resulted from crystalline state switching to amorphous state. According to the theoretical calculations, we conjectured that TPA-ts-BF2 with uncoordinated amide linkage moiety had a tendency to forming a more twisted conformance and higher molecular polarity, which made that mechanofluorochromic behavior was worse than that of TPA-ts-2BF2. Additionally, TPA-ts-2BF2 was applied to latent fingerprint detection due to its prime aggregation-induced emission property.

Immunoproteomic discovery of Mycobacterium bovis antigens, including the surface lipoprotein Mpt83 as a T cell antigen useful for vaccine development.

Tuberculosis (TB) is one of the leading causes of death from infectious diseases, killing approximately 1.3 million people worldwide in 2022 alone. The current vaccine for TB contains a live attenuated bacterium, Mycobacterium bovis BCG (Bacille Calmette-Guérin). The BCG vaccine is highly effective in preventing severe forms of childhood TB but does not protect against latent infection or disease in older age groups. A new or improved BCG vaccine for prevention of pulmonary TB is urgently needed. In this study, we infected murine bone marrow derived dendritic cells from C57BL/6 mice with M. bovis BCG followed by elution and identification of BCG-derived MHC class I and class II-bound peptides using tandem mass spectrometry. We identified 1436 MHC-bound peptides of which 94 were derived from BCG. Fifty-five peptides were derived from MHC class I molecules and 39 from class II molecules. We tested the 94 peptides for their immunogenicity using IFN- γ ELISPOT assay with splenocytes purified from BCG immunized mice and 10 showed positive responses. Seven peptides were derived from MHC II and three from MHC class I. In particular, MHC class II binding peptides derived from the mycobacterial surface lipoprotein Mpt83 were highly antigenic. Further evaluations of these immunogenic BCG peptides may identify proteins useful as new TB vaccine candidates.

Immunization with live and dead Chlamydia muridarum induces different levels of protective immunity in a murine genital tract model: correlation with MHC class II peptide presentation and multifunctional Th1 cells.

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.

Pin-assisted retraction technique in unilateral biportal endoscopic discectomy: a retrospective cohort study.

OBJECTIVE: Unilateral biportal endoscopic (UBE) discectomy is a reliable endoscopic technique in the treatment of lumbar disc herniation. However, UBE discectomy involves a single-handed manipulation, which may compromise the utility of the procedure. The present study was performed to examine the efficacy and safety of a novel pin-assisted retraction technique. METHODS: This single-center retrospective cohort study involved 57 consecutive patients who underwent UBE lumbar discectomy from July 2021 to May 2022. The patients were randomly divided into the pin-assisted UBE discectomy group (P-UBE group) and the traditional UBE discectomy group (T-UBE group). The patients' perioperative data, clinical outcomes, and radiologic outcomes were collected and compared between the two groups. RESULTS: The operative time, intraoperative blood loss, endoscopic irrigation volume, and overall complication rate were significantly lower in the P-UBE group than in the T-UBE group. There were no significant differences in the clinical outcome data between the two groups. CONCLUSION: P-UBE discectomy may have superior safety and efficacy over the traditional technique, and it has the potential to serve as an optional method in UBE lumbar surgery.

Chlamydia muridarum T cell antigens and adjuvants that induce protective immunity in mice.

Major impediments to a Chlamydia vaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model against Chlamydia infection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for a Chlamydia vaccine, we evaluated 10 new Chlamydia T cell antigens discovered via immunoproteomics in addition to the 3 antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6'-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720-CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4(+) T cells coexpressing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), and reduced infection by more than 3 logs. Using DDA-MPL as an adjuvant, we found that 7 of 13 Chlamydia T cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly defined Chlamydia vaccine.

Thy28 partially prevents apoptosis induction following engagement of membrane immunoglobulin in WEHI-231 B lymphoma cells.

Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects induction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential (ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27(Kip1). The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide (H(2)O(2)) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H(2)O(2) activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27(Kip1) in WEHI-231 B lymphoma cells.

Novel Chlamydia muridarum T cell antigens induce protective immunity against lung and genital tract infection in murine models.

Using a combination of affinity chromatography and tandem mass spectrometry, we recently identified 8 MHC class II (I-A(b)) -bound Chlamydia peptides eluted from dendritic cells (DCs) infected with Chlamydia muridarum. In this study we cloned and purified the source proteins that contained each of these peptides and determined that three of the eight peptide/protein Ags were immunodominant (PmpG-1, RplF, and PmpE/F-2) as identified by IFN-gamma ELISPOT assay using splenocytes from C57BL/6 mice recovered from C. muridarum infection. To evaluate whether the three immunodominant Chlamydia protein Ags were also able to protect mice against Chlamydia infection in vivo, we adoptively transferred LPS-matured DCs transfected ex vivo with the cationic liposome DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate) and individual PmpG-1(25-500aa), RplF, or PmpE/F-2 (25-575 aa) proteins. The results showed that the transfected Chlamydia proteins were efficiently delivered intracellularly into DCs. Mice vaccinated with DCs transfected with individual Chlamydia protein PmpG-1(25-500), RplF, or PmpE/F-2(25-575) exhibited significant resistance to challenge infection as indicated by reduction in the median Chlamydia inclusion forming units in both the lung and genital tract models. The major outer membrane protein was used as a reference Ag but conferred significant protection only in the genital tract model. Overall, vaccination with DCs transfected with PmpG-1(25-500) exhibited the greatest degree of protective immunity among the four Chlamydia Ags tested. This study demonstrates that T cell peptide Ags identified by immunoproteomics can be successfully exploited as T cell protein-based subunit vaccines and that PmpG-1(25-500) protein may be a suitable vaccine candidate for further evaluation.

Chlamydia muridarum T-cell antigens formulated with the adjuvant DDA/TDB induce immunity against infection that correlates with a high frequency of gamma interferon (IFN-gamma)/tumor necrosis factor alpha and IFN-gamma/interleukin-17 double-positive CD4+ T cells.

Major impediments to developing a Chlamydia vaccine lie in identifying immunologically relevant T-cell antigens and delivery in a manner to stimulate protective immunity. Using an immunoproteomic approach, we previously identified three immunodominant Chlamydia T-cell antigens (PmpG-1, PmpE/F-2, and RplF). Because RplF has high homology to a human ortholog, it may not be suitable for human vaccine development. Therefore, in this study, we evaluated protection against Chlamydia infection in the genital tract in C57BL/6 mice immunized with Chlamydia-specific membrane proteins PmpG-1, PmpE/F-2, and major outer membrane protein (MOMP; as a reference) or a combination of them formulated with one of three adjuvants, CpG oligodeoxynucleotide (CpG-ODN), AbISCO-100 (AbISCO), or DDA/TDB (dimethyldioctadecylammonium bromide/D-(+)-trehalose 6,6'-dibehenate). The results show that immunization with the CpG-ODN formulation failed to provide protection against Chlamydia infection; the AbISCO formulation conferred moderate protection, and the DDA/TDB formulation showed the highest degree of protective efficacy. The combination of PmpG-1, PmpE/F-2, and MOMP proteins formulated with DDA/TDB exhibited the greatest degree of protection among all vaccine groups studied. Moreover, this vaccine combination also engendered significant protection in BALB/c mice, which have a different major histocompatibility complex (MHC) background. We measured cell-mediated immune cytokine responses in mice immunized with PmpG-1 mixed with each of the three adjuvants. The results demonstrate that mice immunized with the DDA/TDB formulation induced the strongest gamma interferon (IFN-gamma) and interleukin-17 (IL-17) responses, characterized by the highest frequency of IFN-gamma/tumor necrosis factor alpha (TNF-alpha) and IFN-gamma/IL-17 double-positive CD4(+) T cells. In conclusion, a Chlamydia vaccine based on the recombinant proteins PmpG-1, PmpE/F-2, and MOMP delivered in a DDA/TDB adjuvant conferred protection against infection that correlated with IFN-gamma/TNF-alpha and IFN-gamma/IL-17 double-positive CD4(+) T cells.

Differences in innate immune responses correlate with differences in murine susceptibility to Chlamydia muridarum pulmonary infection.

We investigated the phenotypic basis for genetically determined differences in susceptibility and resistance to Chlamydia muridarum pulmonary infection using BALB/c and C57BL/6 mice. Following C. muridarum intranasal inoculation, the intensity of infection was very different between BALB/c and C57BL/6 beginning as early as 3 days post-infection. Intrapulmonary cytokine patterns also differed at early time-points (days 2 and 4) between these two strains of mice. The early recruitment of neutrophils to lung tissue was greater in BALB/c than in C57BL/6 mice and correlated with a higher number of inclusion forming units (IFU) of C. muridarum. At day 12 post-infection, BALB/c mice continued to demonstrate a greater burden of infection, significantly higher lung cytokine levels for tumour necrosis factor-alpha and interleukin-17 (IL-17) and a significantly lower level for interferon-gamma than did C57BL/6 mice. In vitro, bone-marrow-derived dendritic cells (BMDCs) from BALB/c mice underwent less functional maturation in response to C. muridarum infection than did BMDCs from C57BL/6 mice. The BMDCs of BALB/c mice expressed lower levels of activation markers (CD80, CD86, CD40 and major histocompatibility complex class II) and secreted less IL-12 and more IL-23 than BMDCs from C57BL/6 mice. Overall, the data demonstrate that the differences exhibited by BALB/c and C57BL/6 mice following C. muridarum pulmonary infection are associated with differences in early innate cytokine and cellular responses that are correlated with late differences in T helper type 17 versus type 1 adaptive immune responses.

Comparison of Chlamydia outer membrane complex to recombinant outer membrane proteins as vaccine.

The Chlamydial outer membrane complex (COMC) from the elementary body (EB) is a protein rich insoluble outer membrane shell from which cytosolic proteins have been extracted with detergent. In this study we conducted mass spectrometry experiments to detect proteins in the COMC prepared from C. muridarum EB. Proteomic analysis showed that the COMC contained 75 proteins that included 10 outer membrane proteins (OMPs) such as the major outer membrane protein (MOMP) and polymorphic membrane proteins (Pmps) that were previously identified as CD4 T cell vaccine candidates. We tested the vaccine efficacy of COMC in comparison to individual or combination of recombinant OMPs formulated with Th1 polarizing adjuvant DDA/MPL in two murine genital tract models (C. muridarum and C. trachomatis) by measuring organismal shedding, tubal pathology and immune responses including neutralizing antibodies. In the C. muridarum model, the COMC vaccine generated broadly reactive immune responses against multiple outer membrane proteins, high levels of EB binding and neutralizing antibody and exhibited superior protection against genital infection and pathology when compared to the recombinant PmpG vaccine. Denaturing the COMC by boiling significantly reduced protection. In the C. trachomatis model, the COMC vaccine also conferred greater protection compared to individual or multiple recombinant outer membrane proteins. Immunization with multiple COMCs from C. trachomatis serovars D, F and J generated neutralizing antibodies against multiple C. trachomatis serovars. We conclude that broader immunogenicity and generation of neutralizing antibody may explain the superior efficacy of COMC vaccine. The study suggests that conformationally intact proteins will be necessary for a successful recombinant OMP vaccine.

Characterization of murine dendritic cell line JAWS II and primary bone marrow-derived dendritic cells in Chlamydia muridarum antigen presentation and induction of protective immunity.

Dendritic cells (DCs) appear to orchestrate much of the immunobiology of Chlamydia infection, but most studies of Chlamydia-DC interaction have been limited by the availability and heterogeneity of primary bone marrow-derived DCs (BMDCs). We therefore evaluated the immunobiology of Chlamydia muridarum infection in an immortal DC line termed JAWS II derived from BMDCs of a C57BL/6 p53-knockout mouse. JAWS II cells were permissive to the developmental cycle of Chlamydia. Infection-induced cell death was 50 to 80% less in JAWS II cells than in BMDCs. Chlamydia infected JAWS II cells and yielded infectious progeny 10-fold greater than that with primary BMDCs. JAWS II cells showed an expression pattern of cell activation markers and cytokine secretion following Chlamydia infection similar to that of primary BMDCs by up-regulating the expression of CD86, CD40, and major histocompatibility complex class II and secreting significant amounts of interleukin-12 (IL-12) but not IL-10. JAWS II cells pulsed with Chlamydia stimulated immune CD4(+) T cells to secrete gamma interferon. Adoptive transfer of ex vivo Chlamydia-pulsed JAWS II cells conferred levels of immunity on C57BL/6 mice similar to those conferred by primary BMDCs. Taken together, the data show that JAWS II cells exhibit immunobiological characteristics and functions similar to those of primary BMDCs in terms of Chlamydia antigen presentation in vitro and antigen delivery in vivo. We conclude that the JAWS II cell line can substitute for primary BMDCs in Chlamydia immunobiological studies.

Subunit vaccines for the prevention of mucosal infection with Chlamydia trachomatis.

Chlamydia trachomatis is the most common preventable cause of tubal infertility in women. In high-income countries, despite public health control efforts, C. trachomatis case rates continue to rise. Most medium and low-income countries lack any Chlamydia control program; therefore, a vaccine is essential for the control of Chlamydia infections. A rationally designed Chlamydia vaccine requires understanding of the immunological correlates of protective immunity, pathological responses to this mucosal pathogen, identification of optimal vaccine antigens and selection of suitable adjuvant delivery systems that engender protective immunity. Fortunately, Chlamydia vaccinology is facilitated by genomic knowledge and by murine models that reproduce many of the features of human C. trachomatis infection. This article reviews recent progress in these areas with a focus on subunit vaccine development.

Knockdown of asporin affects transforming growth factor-ß1-induced matrix synthesis in human intervertebral annulus cells.

BACKGROUND/OBJECTIVE: Asporin is associated with osteoarthritis and lumbar disk degeneration. Previous studies in chondrocytes showed that asporin can bind to transforming growth factor-ß1 (TGF-ß1) and downregulate matrix biosynthesis. However, this has not been studied in intervertebral disk (IVD) cells. This study aimed to inspect the expression of asporin under TGF-ß1 stimulation and its effect on TGF-ß1-induced matrix biosynthesis in human intervertebral annulus cells. METHODS: Human intervertebral annulus cells were obtained from the pathological region of IVD in eight patients. After primary culture and redifferentiation in alginate beads, cells were reseeded and treated with different concentrations (5 ng/mL, 10 ng/mL, and 15 ng/mL) of TGF-ß1 for up to 24 hours. Total RNA extracted from the cells and those with asporin knockdown were subjected to real-time polymerase chain reaction analysis to examine the expression of asporin and extracellular matrix genes. RESULTS: TGF-ß1 stimulation induces asporin transcription significantly in a dose- and time-dependent manner. Knockdown of endogenous asporin leads to the upregulated expression of collagen II alpha 1 and aggrecan. CONCLUSION: Our results have verified a functional feedback loop between TGF-ß1 and asporin in human intervertebral annulus cells indicating that TGF-ß1-induced annulus matrix biosynthesis can be significantly upregulated by knockdown of asporin. Therefore, asporin could be a potential new therapeutic target and inhibition of asporin could be adopted to enhance the anabolic effect of TGF-ß1 in human intervertebral annulus cells in degenerative IVD diseases.

Outer membrane proteins preferentially load MHC class II peptides: implications for a Chlamydia trachomatis T cell vaccine.

CD4 T cell immune responses such as interferon-γ and tumor necrosis factor-α secretion are necessary for Chlamydia immunity. We used an immunoproteomic approach in which Chlamydia trachomatis and Chlamydia muridarum-derived peptides presented by MHC class II molecules on the surface of infected dendritic cells (DCs) were identified by tandem mass spectrometry using bone marrow derived DCs (BMDCs) from mice of different MHC background. We first compared the C. muridarum immunoproteome in C3H mice to that previously identified in C57BL/6 mice. Fourteen MHC class II binding peptides from 11 Chlamydia proteins were identified from C3H infected BMDCs. Two C. muridarum proteins overlapped between C3H and C57B/6 mice and both were polymorphic membrane proteins (Pmps) which presented distinct class II binding peptides. Next we studied DCs from C57BL/6 mice infected with the human strain, C. trachomatis serovar D. Sixty MHC class II binding peptides derived from 27 C. trachomatis proteins were identified. Nine proteins were orthologous T cell antigens between C. trachomatis and C. muridarum and 2 of the nine were Pmps which generated MHC class II binding epitopes at distinct sequences within the proteins. As determined by antigen specific splenocyte responses outer membrane proteins PmpF, -G and -H and the major outer membrane protein (MOMP) were antigenic in mice previously infected with C. muridarum or C. trachomatis. Furthermore a recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with MOMP formulated with a Th1 polarizing adjuvant significantly accelerated (p<0.001) clearance in the C57BL/6 mice C. trachomatis transcervical infection model. We conclude that Chlamydia outer membrane proteins are important T cell antigens useful in the development of a C. trachomatis subunit vaccine.

Modulation of mThy28 nuclear protein expression during thymocyte development.

We have recently shown that down-regulation of mouse Thy28 (mThy28) protein expression appears to be accompanied by apoptotic processes. Thymocytes from mice contain moderate amounts of mThy28 protein and undergo proliferation, differentiation, or apoptosis during murine thymic maturation. As a first step to examine the potential role of the mThy28 protein in the thymocyte development, such as positive-negative selection, the expression of mThy28 protein in the thymocyte subsets was examined. Thymocytes are separated into four subpopulations by the expression levels of CD4 and CD8: CD4-CD8- (DN), CD4+CD8+ (DP), and CD4+CD8- or CD4-CD8+ (SP). Flow cytometry analysis using three-color staining demonstrated that the mThy28 expression in immature DP cells is lower than that in DN and SP cells. The down-regulation of the mThy28 expression in the DP stage was also detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The immunostaining method also showed that mThy28 protein was expressed in the medulla containing mature thymocytes, but not the cortex having immature thymocytes. The mThy28 protein in the thymocytes was mainly localized in the nucleus, as recently demonstrated in lymphoma cells, indicating that the mThy28 protein resides in the nucleus, irrespective of the cyclic or resting stage of the cell cycle. Together, the observation that mThy28 expression is down-modulated during the DP stage suggests that mThy28 protein might play some role in the positive-negative selection step in thymic maturation.

Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice.

An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.