Pilar Pérez-Romero

Development of vaccines against Klebsiella pneumoniae has potential to prevent infections in both community and hospital settings and reduce associated mortality and morbidity worldwide. DNA vaccines represent a promising approach given their stability, safety, design flexibility and cost‐effectiveness for rapid production. In this study, two plasmid backbones, namely the commercial pVAX1 and the newly designed PLASMIVAX (containing additional CpG motifs), were tested to develop two DNA vaccines targeting the MrkA fimbrial subunit from K. pneumoniae . MrkA is highly conserved among K. pneumoniae and was expressed and secreted in vitro, fused to immunostimulatory elements, by both DNA vaccines following transfection of HEK‐293T cells. Immunogenicity was assessed in mice by measuring antibody levels (IgM, IgG, IgG1, IgG2a and IgG3) after intramuscular immunization with three doses of 50 μg of each DNA vaccine. Efficacy of protection was established in a sepsis infection model. The pVAX1‐MrkA DNA vaccine elicited the most robust antibody response and protection against K. pneumoniae infection (80%, p = 0.0373), while the PLASMIVAX‐MrkA DNA vaccine protected at a lower rate (30%, p = 0.0163), consistent with lower post‐immunization antibody titers elicited by this vaccine. This study reveals that DNA plasmid properties strongly influence immunogenicity and vaccine efficacy and highlights the potential role of MrkA as a vaccine antigen and DNA platforms as a valuable tool for developing vaccines against multidrug‐resistant bacteria.

Human cytomegalovirus (CMV), a herpesvirus that infects most of the global population remains without a licensed vaccine. Although natural infection elicits an immune response that confers partial protection, the development of an effective CMV vaccine has proven challenging. Despite multiple vaccine attempts, the complex interactions between CMV and the host immune system have hindered successful vaccine design. In this study, we utilize the naturally acquired CMV-specific immunity of solid organ transplant (SOT) recipients to identify viral antigens recognized by antibodies present in patient sera. Using Western blot and ELISA in combination with proteomic analyses, we identified five highly immunogenic CMV proteins: UL44, UL57, UL83, UL132 and RL11. While UL44, UL57 and UL83 are well-established immunodominant antigens, hereby validating our strategy, UL132 and RL11 emerged as additional highly immunogenic targets. The detection of antibodies against these CMV antigens in patients with a protective CMV-specific immune response underscores their immunogenicity and supports their potential relevance for vaccine development. Taken together, these findings identify CMV antigens of potential interest for vaccine research.

Human cytomegalovirus (HCMV) poses serious health risks, particularly for immunocompromised individuals. However, the current FDA-approved anti-HCMV drugs face challenges such as drug resistance and significant side effects, underscoring the need for alternative treatment options. Essential oil components (EOCs), including eugenol, thymol and vanillin, are recognized for their therapeutic potential. This study evaluates their antiviral effects against HCMV in epithelial (ARPE-19) and fibroblast (MRC-5) cell lines. Among the EOCs, vanillin demonstrated the highest efficacy, characterized by low toxicity and a high selectivity index in both cell types. Mechanistic differences were noted between the cell lines. In ARPE-19 cells, eugenol showed virucidal activity, inhibited viral entry and suppressed early gene expression (IE-1). Conversely, in MRC-5 cells, eugenol mainly blocked viral entry and exhibited virucidal effects. Thymol was most effective in ARPE-19 cells, where it completely suppressed IE-1 expression as a result of both inhibition of viral entry and a direct disruptive effect on IE-1 expression. In addition, thymol showed an effect on viral replication. In MRC-5 cells, thymol primarily inhibited viral entry and attachment. Vanillin exhibited dual inhibitory activity in both cell lines, blocking viral attachment and entry. In MRC-5, vanillin also appears to affect intermediate processes. Notably, combining EOCs with ganciclovir resulted in synergistic effects. The eugenol/ganciclovir combination was particularly effective in ARPE-19 cells, while thymol/ganciclovir showed enhanced efficacy in MRC-5 cells. These findings suggest that EOCs have significant potential as adjunct therapies to improve antiviral outcomes and address drug-resistant HCMV strains.

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes asymptomatic infections in immunocompetent individuals but can cause severe or even life-threatening symptoms in immunocompromised patients. HCMV can replicate in a wide variety of cells through the engagement of diverse cell factors with the viral envelope protein gH/gL/gO (trimer) or gH/gL/UL128/UL130/UL131a (pentamer), allowing for systemic spread within the human host. This study explores HCMV infection tropism and dynamics in human microglia, demonstrating the susceptibility of microglia to both clinical and laboratory HCMV strains, albeit with lower efficacy for the laboratory strain, implying that both the gH/gL-trimer and -pentamer can mediate virus entry in microglia. The importance of the gH/gL pentamer for virus entry was demonstrated by the inhibition of virus infection upon pre-incubation with a soluble neuropilin-2 (NRP-2) entry factor. Further, we demonstrated that HCMV infection can be effectively inhibited by monoclonal antibodies specific for the gH/gL complexes and HCMV hyperimmunoglobulin. Lastly, we report that microglia infection can be prevented by newly characterized chemical entry inhibitors. Altogether, these findings underscore the potential of microglia as valuable models for studying HCMV neurotropism and strategies to block virus infection in cells that can impact neurological disorders.

Background: Kidney transplant (KT) recipients at intermediate risk for cytomegalovirus (CMV) infection constitute a potential target for individualized prevention strategies informed by the CMV‐specific cell‐mediated immunity (CMV‐CMI). The optimal method for the functional assessment of CMV‐CMI in this group remains unclear. Methods: We included 74 CMV‐seropositive KT recipients that did not receive T‐cell‐depleting induction and were managed by preemptive therapy. CMV‐CMI was monitored at baseline and months 1, 3, 6, and 12 by intracellular cytokine staining (ICS) and a interferon (IFN)‐γ‐release assay (QuantiFERON‐CMV [QTF‐CMV]). Both methods were compared for discriminative capacity (areas under the receiving operating characteristic curve [auROCs]) and diagnostic accuracy to predict protection against high‐level (≥1000 IU/mL) CMV DNAemia and/or disease. Results: Eighteen patients (24.3%) experienced high‐level CMV DNAemia or disease. There were no significant differences in the discriminative capacity to predict protection of CMV‐specific CD8+ (auROC: 0.719) and CD4+ T‐cell counts (auROC: 0.664) enumerated by ICS and IFN‐γ production measured by QTF‐CMV (auROC: 0.666). Optimal cutoff values of ≥9.8 CMV‐specific CD4+ T‐cells/µL and ≥5.7 CD8+ T‐cells/µL by ICS yielded excellent specificity (95.7% and 86.9%, respectively) and positive predictive values (PPVs) (>98.0%), but a sensitivity below 60%. A reactive QTF‐CMV (IFN‐γ ≥0.2 IU/mL) provided good sensitivity (81.6%) and PPV (92.5%), at the expense of a poor specificity (22.2%). Conclusions: The discriminative capacity to predict immune protection against clinically relevant CMV infection among intermediate‐risk KT recipients was comparable for ICS and QTF‐CMV. A selected ICS threshold may provide better specificity than the interpretative cut‐off values currently recommended for QTF‐CMV. image

We measured cytomegalovirus (CMV)-specific antibodies that neutralize epithelial cell infection (CMV-AbNEIs) in 101 CMV-seropositive kidney transplant recipients (KTRs) at baseline and posttransplant months 3 and 6. All the patients received antithymocyte globulin and 3-month valganciclovir prophylaxis. There were no significant differences in pretransplant AbNEIs titers between KTRs that developed or did not develop any-level CMV infection or the composite of high-level infection and/or disease. One-year CMV infection-free survival was comparable between KTRs with or without pretransplant CMV-AbNEIs. No differences were observed by months 3 and 6. We observed no protective role for CMV-AbNEIs among CMV-seropositive KTRs undergoing T-cell–depleting induction.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200–300 nm) compared to fibroblast-derived DB (100–600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.