The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the composition of Aeromonas hydrophila OMVs is missing. In this study we analyzed the composition of purified OMVs from A. hydrophila ATCC® 7966TM by proteomics. Also we studied the effect of OMVs on human peripheral blood mononuclear cells (PBMCs). Vesicles were grown in agar plates and then purified through ultracentrifugation steps. Purified vesicles showed an average diameter of 90–170 nm. Moreover, 211 unique proteins were found in OMVs from A. hydrophila; some of them are well-known as virulence factors such as: haemolysin Ahh1, RtxA toxin, extracellular lipase, HcpA protein, among others. OMVs from A. hydrophila ATCC® 7966TM induced lymphocyte activation and apoptosis in monocytes, as well as over-expression of pro-inflammatory cytokines. This work contributed to the knowledge of the composition of the vesicles of A. hydrophila ATCC® 7966TM and their interaction with the host cell

Immune Monitoring of Paediatric Patients Infected with Rickettsia rickettsii, Ehrlichia canis and Coinfected

Abstract: In 2021, 273 Rocky Mountain spotted fever cases were reported nationwide in Mexico. In Chihuahua City, fourteen samples were obtained from children suspected of rickettsial infection. The analysis of samples (January to December 2021) showed prevalence rates of 28.5%, 43%, and 28.5% for Rickettsia rickettsii, Ehrlichia canis, and both pathogens in coinfection, respectively. The analysis of clinical haematological and biochemistry analytes showed alterations; 100% of the children had elevated liver enzymes and coagulation times, 64% showed leukocytosis due to neutrophilia, 55% had thrombocytopenia, lymphopenia, and hypoalbuminemia, and 45% showed normocytic normochromic anaemia. Statistically significant differences were observed in the expression of the chemokines IL-8, RANTES, CXCL9/MIG, and CXCL10/IP-10 across the coinfected and control groups, and the difference in IP-10 expression was significant for patients infected by R. rickettsii compared to the control group. Additionally, significant differences were observed for expression levels of IL-1β, IL-6, IL-17, IFNγ, and TNFα among the R. rickettsii-positive group compared to the control group. On the other hand, the coinfected group exhibited modified levels of IL-6, IL-8, and IL-10 compared with the control group. Finally, significant differences were observed for CD8+ T lymphocyte subpopulations between individuals positive for R. rickettsii and those positive for E. cani

Rickettsia Vaccine Candidate pVAX1-OmpB24 Stimulates TCD4+INF-γ+ and TCD8+INF-γ+ Lymphocytes in Autologous Co-Culture of Human Cells

Background: In recent years, promising vaccination strategies against rickettsiosis have been described in experimental animal models and human cells. OmpB is considered an immunodominant antigen that is recognized by T and B cells. The aim of this study was to identify TCD4+INF-γ+ and TCD8+INF-γ+ lymphocytes in an autologous system with macrophages transfected with the vaccine candidate pVAX1-OmpB24. Lymphocytes and monocytes from 14 patients with Rickettsia were isolated from whole blood. Monocytes were differentiated into macrophages and transfected with the plasmid pVAX1-OmpB24 pVax1. Isolated lymphocytes were cultured with transfected macrophages. IFN-γ-producing TCD4+ and TCD8+ lymphocyte subpopulations were identified by flow cytometry, as was the percentage of macrophages expressing CD40+, CD80+, HLA-I and HLA-II. Also, we analyzed the exhausted condition of the T lymphocyte subpopulation by PD1 expression. Macrophages transfected with pVAX1-OmpB24 stimulated TCD4+INF-γ+ cells in healthy subjects and patients infected with R. typhi. Macrophages stimulated TCD8+INF-γ+ cells in healthy subjects and patients infected with R. rickettsii and R. felis. Cells from healthy donors stimulated with OmpB-24 showed a higher percentage of TCD4+PD1+. Cells from patients infected with R. rickettsii had a higher percentage of TCD8+PD-1+, and for those infected with R. typhi the larger number of cells corresponded to TCD4+PD1+. Human macrophages transfected with pVAX1-OmpB24 activated TCD4+IFN-γ+ and CD8+IFN-γ+ in patients infected with different Rickettsia species. However, PD1 expression played an important role in the inhibition of T lymphocytes with R. felis.</i

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent <i>Mycobacterium tuberculosis</i>

<div><p>Airways infection with <i>Mycobacterium tuberculosis</i> (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205⁺ APCs and to assess its <i>in vivo</i> effects on protection associated responses (IFN-γ production, <i>in vivo</i> CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4⁺ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ⁺ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. <i>In vivo</i> CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205⁺ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study <i>in vivo</i> the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.</p></div

α-DEC-ESAT-treated mice show reduced bacterial burden and increased cellular infiltrate in the lungs.

<p>Pulmonary mycobacterial load (CFUs/lung) and cellular infiltrate (% pneumonic areas) were quantified at two time points during Mtb infection in the different experimental groups. Horizontally stripped bars indicate uninfected non-immunized mice (No-Inf). All other bars represent infected mice with different treatments. Black bars: untreated mice (Inf/No Tx); white bars: α-DEC-ESAT-treated mice (Inf/DE6-Tx); diagonally stripped bars: mice treated with isotype control antibody-ESAT (Inf/IsoE6-Tx). Quantification of the cellular infiltrate during the acute infection is shown in (A) while that of the chronic stage is shown in (C). The bacterial burden expressed as CFUs per lung is shown in (B) for the acute stage of the infection and in (D) for the chronic phase. (E) Representative picture of HE staining of lungs from infected mice which were α-DEC-ESAT-untreated (-) or α-DEC-ESAT-treated (+). Individual lungs were analyzed by duplicate and 3 mice were used per group. Data are presented as mean plus standard error. (*) Represents P<0.05.</p