Ivana Gabriela Reidel

Osteosarcoma is an aggressive primary malignant bone tumor of poorly defined etiology that predominantly affects adolescents and young adults. A viral cause has long been proposed, and a recent study from Xinjiang, China, reported frequent detection of Kaposi sarcoma–associated herpesvirus (KSHV) in Uyghur osteosarcoma cases, suggesting a possible association in this KSHV‐endemic population. Whether this association extends to broader populations remains unknown. Our study investigated the presence of KSHV in osteosarcoma specimens from KSHV‐endemic African countries (Cameroon, Kenya, South Africa, Zambia) and the non‐endemic United States. A total of 356 formalin‐fixed paraffin‐embedded and fresh‐frozen specimens were retrieved or prospectively collected. In 77 selected high‐quality specimens, KSHV infection was assessed by immunohistochemistry for LANA1 and by qPCR targeting 5 viral open reading frames (ORF25, ORF26, ORF37, ORF65, and ORF73). LANA1 expression was undetectable in all tumors. Using qPCR, 75/77 specimens were negative for all targets, 1/77 excluded due to insufficient remaining DNA quantity to perform the assay, and 1/77 positive across all five targets. Additionally, we studied the KSHV seroprevalence in a separate cohort comprised of 49 sera obtained from individuals with osteosarcoma from Zambia ( n = 39) and the United States ( n = 10). We measured by ELISA the presence of specific antibodies against four KSHV antigens: K8.1, KCP, VCA, and LANA1. KSHV seropositivity was detected in 15/39 individuals from Zambia and none from the United States. In the absence of compelling evidence, our findings could not support an association between KSHV infection and osteosarcoma in our study population.

Epstein-Barr virus (EBV) is implicated in multiple epithelial and lymphoid malignancies worldwide; however, no licensed prophylactic vaccine exists, partly due to the lack of an adequate preclinical animal model to test candidate vaccines. Rhesus lymphocryptovirus (rhLCV) faithfully recapitulates EBV infection, persistence, and pathogenesis in rhesus macaques, offering an invaluable surrogate model. While recombinant EBV expressing enhanced green fluorescent protein (eGFP) enables swift identification of infected cells in vitro and ex vivo , an analogous tool for rhLCV has been lacking. Here, employing en passant recombination technology, we engineered a recombinant rhLCV expressing eGFP (rhLCV.eGFP) from an rhLCV bacterial artificial chromosome derived from the B-lymphoblastoid rhesus macaque cell line LCL 8664. Whole-genome sequencing confirmed sequence integrity, and functional assays demonstrated infectivity in peripheral blood mononuclear cells (PBMCs) from multiple primate species and EBV-susceptible human cell lines, and transformation competence in rhesus macaque PBMCs. To define the role of gp350/220 (gp350) in viral entry, we generated a glycoprotein gp350-deficient mutant (rhLCV.eGFPΔgp350), verified by Sanger sequencing and immunoblotting with a novel anti-gp350 monoclonal antibody (4A6). This mutant exhibited markedly reduced infectivity in rhLCV.eGFP-susceptible human B and epithelial cell lines, underscoring gp350 as a key determinant of B cell tropism, analogous to EBV gp350. Our successful engineering of rhLCV.eGFP provides a powerful platform for visualizing infection, dissecting viral entry mechanisms and cellular tropism, and advancing rhLCV as a surrogate EBV model for vaccine design, defining correlates of protection, studies of latency and reactivation, and preclinical testing of candidate antivirals and immunotherapies. IMPORTANCE: Epstein-Barr virus (EBV) infection is associated with various epithelial and lymphoid diseases. The absence of a suitable animal study model for EBV infection has hindered mechanistic studies of pathogenesis and preclinical testing of vaccines and EBV‑targeted therapies. Rhesus lymphocryptovirus (rhLCV), an EBV homolog, accurately recapitulates EBV infection and pathogenesis in rhesus macaques, offering a promising EBV surrogate model. In this study, we engineered a stable recombinant rhLCV expressing enhanced green fluorescent protein (eGFP), rhLCV.eGFP, for easy identification of infected or transformed cells in vitro and ex vivo . Further illustrating the utility of this platform, we also generated a gp350-deficient mutant rhLCV.eGFP and demonstrated markedly reduced infectivity in otherwise susceptible B and epithelial cells, confirming the EBV gp350-homologous role of rhLCV gp350 in viral entry. Thus, rhLCV.eGFP will serve as an invaluable resource for studying EBV biology and advancing the development of EBV prophylactic and therapeutic strategies using the rhLCV surrogate model.

The development of effective adjuvant systems remains a critical challenge in veterinary vaccine design, particularly for infectious diseases causing significant economic losses in dairy cattle. This study evaluated the immunological profile induced by a mixture of recombinant antigens, formulated with cationic liposomes containing different combinations of gemini lipopeptide AG2‐C16, bovine‐specific CpG oligodeoxynucleotide, and S. cerevisiae mannans, in dairy heifers. Thirty‐five dairy heifers aged 8–10 months were randomly assigned to seven groups and immunized with recombinant Staphylococcus aureus antigens (α‐toxin, β‐toxin, clumping factor A, and fibronectin‐binding protein A) formulated with different adjuvant combinations. Animals received two primary doses 21 days apart, followed by a booster dose 8 months later. Humoral immune responses were assessed through ELISA measuring specific immunoglobulin G (IgG), IgG1, and IgG2 levels, while cytokine production was evaluated via whole‐blood assays. Results demonstrate that liposomal formulations combining AG2‐C16, CpG‐ODN, and mannans induce a significant immune response, in heifers, with IFN‐γ production. These findings provide valuable insights for the development of new vaccine strategies in cattle.

Modified vaccinia Ankara (MVA) virus is a widely used vaccine platform, making accurate titration essential for vaccination studies. However, the current plaque forming unit (PFU) assay, the standard for MVA titration, is prone to observer bias and other limitations that affect accuracy and precision. To address these challenges, we developed a new flow cytometry-based quantification method using a highly specific monoclonal antibody (mAb) for the detection of MVA-infected cells, as a more accurate titration assay. Through previous work, we serendipitously identified three MVA-specific hybridoma antibody clones, which we characterized through ELISA, immunoblot, and flow cytometry, confirming their specificity for MVA. Sequencing confirmed that each antibody was monoclonal, and mass spectrometry results revealed that all mAbs target the MVA cell surface binding protein (CSBP, MVA105L). We next optimized the titration protocol using the most effective mAb, 33C7 by refining culture conditions and staining protocols to enhance sensitivity and minimize background. Our optimized method demonstrated superior sensitivity, reliability, and reduced processing time when compared with the traditional PFU assay, establishing it as a more accurate and efficient approach for MVA titration.

The design of subunit vaccines requires new adjuvant systems. We designed and synthesized new lipopeptides (cysteine-based) of low molecular weight with different hydrophobic chains that dimerize becoming gemini lipopeptides. They were characterized and their adjuvant capacity was tested in mice by the inoculation of a protein antigen formulated with the lipopeptides, with and without the addition of CpG-oligodeoxynucleotides. Formulations were able to induce an immune response and produced no adverse effects. An adjuvant ability is described for the first time for this type of molecules.