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Cytomegalovirus UL44 Protein as a new target of Humoral Immune Response in Solid Organ Transplant Recipients

ABSTRACT: Due to the severity of CMV infection and the high number of deaths associated to this pathogen, the American Institute of Medicine proposed the development of a vaccine as a priority. Numerous attempts have been made to develop vaccines against CMV however, the complex interaction between CMV and the immune system and the absence of an animal model able to reproduce human physiology associated to CMV infection have hindered the development of a vaccine. Based on the long history of unsuccessful trials, it is necessary to keep looking for new CMV antigens that can contribute to induce a wide immune response. We designed a proteomic approach to find, select and characterize CMV antigens that induce a CMV-specific immune response, using serum samples from patients that have developed a protective CMV-specific response after transplantation. Serum samples from 28 kidney transplant recipients that exhibit a high neutralizing antibody response and CMV-specific immune response collected at the University Hospital 12 Octubre in Spain were tested. One sample was collected at transplantation with no or low antibody titers (baseline) and the second sample (post-immunization) after acquiring high neutralizing antibody titers (>1/640). Using serum samples from immunized patients as primary antibody, we detected by western blot CMV antigens that were specifically recognized by the post-immunization serum compared with the baseline serum. Excised proteins were further characterized and identified using liquid chromatography coupled with tandem mass spectrometry. To prove the recognition of the identified protein, the open reading frames were cloned and individually expressed in HEK 293T cells and further detected by western blot using serum samples. To characterize the in vivo immune response elicited against UL44, groups of mice were immunized using the pcDNA-UL44 construct and PBS control. We identified and validated three proteins (UL83, UL55 and UL44). Proteins UL83 (pp65) and UL55 (gB) have previously shown to elicit a potent antibody response, while UL44 was newly identified to elicit a neutralizing antibody immune response. In conclusion, we demonstrate that using serum of immunized patients is an excellent strategy to identify CMV proteins that may be involved on stimulating a wide and protective CMV-specific immunity.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Cytomegalovirus Sp.

TISSUE(S): Blood Serum

DISEASE(S): Viral Infectious Disease

SUBMITTER: Luz Valero

LAB HEAD: Pilar Pérez-Romero

PROVIDER: PXD028655 | Pride | 2026-04-14

REPOSITORIES: Pride

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Project description:Natural antibodies, which arise without known immune exposure, have been described that specifically recognize cells dying from apoptosis but their role in innate immunity remains poorly understood. Herein, we show that the immune response to neo-antigenic determinants on apoptotic thymocytes is dominated by antibodies to oxidation-associated antigens, phosphorylcholine (PC), a head group that becomes exposed during programmed-cell death, and malondialdehyde (MDA), a reactive aldehyde degradation product of polyunsaturated lipids produced following exposure to reactive-oxidation species. While natural antibodies to apoptotic cells in naM-CM-/ve adult mice were dominated by PC and MDA specificities, the amounts of these antibodies were substantially boosted by treatment of mice with apoptotic cells. Moreover, the relative amounts of PC and MDA antibodies was affected by VH gene inheritance. Antibody interactions with apoptotic-cells also mediated the recruitment of C1q, which alone can promote apoptotic-cell phagocytosis by immature dendritic cells. Significantly, IgM-antibodies to both PC and MDA were primary factors in determining the efficiency of serum-dependent apoptotic-cell phagocytosis. Hence, we demonstrate a mechanism by which certain natural antibodies that recognize neo-antigens on apoptotic cells, in naM-CM-/ve mice and those induced by immune exposure to apoptotic-cells, can enhance the functional capabilities of immature dendritic cells for phagocytic engulfment of apoptotic cells. Keywords: Natural antibodies, Inflammation, Complement, Apoptosis In the study presented here, a total of 11 custom-spotted protein slides were hybridized with a T15 IgM monoclonal antibody at three different concentrations, an isotype control IgM, 2 slides were hybridized with serum from naM-QM-^Wve mice, 2 slides incubated with serum from mice immunized with apoptotic cells, 2 slides were incubated with serum from mice immunized with saline, and a negative control slide with no antibody hybridized

Project description:The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.

			Action	DRS
	20525-PPR-J3.mgf	Mgf
	20525-PPR-J3.wiff	Wiff
	20525-PPR-J3.wiff.scan	Wiff
	20525-PPR-J3__FDR.xlsx	Xlsx
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Cytomegalovirus UL44 Protein as a new target of Humoral Immune Response in Solid Organ Transplant Recipients

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