(1) Background: Lyme borreliosis (LB) is a tick-borne disease known for its diagnostic challenges. Conventional two-tiered testing (CTTT) for antibodies is time-consuming, has low sensitivity in the early stages of disease, and sometimes generates false-positive IgM immunoblots. To tackle this issue, modified two-tiered testing (MTTT) was introduced, incorporating recombinant VlsE and C6 antigens to enhance diagnostic accuracy. (2) Methods: In this prospective study, we enrolled children exhibiting symptoms indicative of LB. We collected serum samples at various intervals and subjected them to analysis using standard enzyme immunoassays. We then compared these results with the outcomes from the VlsE and C6 assays. (3) Results: In our study, all 33 patients displaying erythema migrans (EM), a characteristic symptom of LB, exhibited positive responses to the C6 antigen. This finding underscores the potential utility of the C6 antigen as a reliable diagnostic tool for LB. Additionally, we observed a significant reduction in anti-VlsE antibody levels following antibiotic treatment in EM patients. (4) Conclusions: The utilization of recombinant VlsE and C6 antigens in LB diagnostics and monitoring has yielded promising results. Nonetheless, it is imperative for clinicians to exercise caution and interpret results in conjunction with clinical findings, considering the dynamic nature of medical guidelines. Even with recombinant antigen tests, some children with EM tested negative, highlighting the importance of clinical diagnosis for treatment decisions. Furthermore, clinicians should be mindful of the possibility of persistently positive VlsE/C6 test results during LB treatment monitoring.

UNASSIGNED: Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares.
UNASSIGNED: Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA.
UNASSIGNED: After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares.
UNASSIGNED: Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.

UNASSIGNED: Campylobacteriosis causes gastrointestinal tract lesions in adults and children and may result in severe complications. The primary sources of infection are infected animals and animal products. Immunochemical methods effectively diagnose intestinal infections but require highly specific antigens to detect their antibodies. This study aimed to obtain two recombinant immunogenic antigens of Campylobacter jejuni, an outer membrane protein with a molecular weight of 18 kDa (Omp18) and the major outer membrane protein (MOMP) with a molecular weight of 45 kDa, and evaluate their suitability for the serological diagnosis of campylobacteriosis using immunochromatographic assay (ICA).
UNASSIGNED: The C. jejuni Omp18 and MOMP gene sequences were synthesized de novo (Macrogen, Korea) and cloned into the pET32 expression plasmid. Using these genetic constructs, electrocompetent cells of the Escherichia coli BL21 strain were transformed and cultured under various conditions. Antigens were purified and refolded using metal affinity chromatography. The properties of the purified proteins were studied by western blotting, liquid chromatography with tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA).
UNASSIGNED: We developed two recombinant E. coli BL21 cells producing rOmp18 and Recombinant MOMP (rMOMP) antigens with molecular weights of 36 and 64 kDa, respectively. Amino acid sequence analysis of the obtained antigens showed complete homology with the reference sequences in the PubMed NCBI database. Western blotting using positive-control sera demonstrated the specificity of the recombinant antigens. The results of ELISA with 94 bovine sera showed the interaction of recombinant antigens with specific antibodies.
UNASSIGNED: The obtained rOmp18 and rMOMP antigens can detect antibodies in the serum of infected or recovered animals and can be used to develop ICA.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is an important problem of public health even in regions where it is not endemic. Spain ranks second worldwide in terms of imported cases of T. cruzi infection in the chronic phase. The diagnosis in this stage is made via the detection of antibodies against T. cruzi. Therefore, we aimed to evaluate the sensitivity and specificity of two fully automated chemiluminescence immunoassays, Chagas VirClia® (CHR), which uses a mixture of recombinant antigens, and Chagas TESA VirClia® (TESA), the first chemiluminescence assay based on excretion-secretion antigens of trypomastigotes, both designed in monotest format. A retrospective case-control study was performed using 105 well-characterized samples: 49 from patients with CD, 22 from uninfected individuals, and 32 from patients with other pathologies. Sensitivity was 98% for CHR and 92% for TESA. In contrast, the specificity in both was 100%. Cross-reactivity was observed in leishmaniasis (2/10). CHR meets the criteria to become a tool for serological screening, while TESA has the potential for confirmation and cross-reaction discrimination. The monotest format allows its application in laboratories with a small number of samples. The high specificity of both assays is useful in areas where leishmaniasis is endemic.

Visceral leishmaniasis (VL) is a fatal manifestation of an infection caused by intracellular protozoa of the Leishmania genus. In New World countries, VL is classified as a zoonotic disease with domestic dogs acting as its main reservoir. Asymptomatic dogs are as competent to transmit Leishmania to the vectors as symptomatic dogs, however current diagnostic tests are limited and present low sensitivity for this important group. The development of accurate tests is fundamental to the early diagnosis, treatment, and control of canine leishmaniasis. In this study, we investigated the use of a recombinant protein (dynamin-1-like protein, Dyn-1) from L. infantum, as a potential target antigen for leishmaniasis serodiagnosis in both symptomatic and asymptomatic dogs. The antigenic performance of the protein was evaluated by means of ELISA assays using sera from symptomatic (n = 25), asymptomatic (n = 34) and non-infected dogs (n = 36) using ELISA. In addition, sera from dogs experimentally infected with Trypanosoma cruzi (n = 49) and naturally infected with Babesia sp. (n = 8) were tested to evaluate possible cross-reactivity. A crude soluble antigen (CSA) of Leishmania was used as an antigen control and K39 and K26 were used as reference antigens because they are already widely used in commercial tests. rDyn-1-based assay showed the highest sensitivity (97%) compared to the antigens K39 (88%), K26 (86%) and crude extract (95%). The highest specificity among the tests was also obtained with the protein rDyn-1 (94%), compared with the other antigens K39 (81%), K26 (87%), and crude extract (77%). This study showed that the rDyn-1 ELISA assay was able to identify 100% of asymptomatic dogs, establishing its potential as a target for the diagnosis of canine leishmaniasis.

BACKGROUND: Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work.
METHODS: Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus.
RESULTS: Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A+ cells among CD8+ T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment.
CONCLUSIONS: The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.

Human toxocariasis is one of the neglected helminthiases and it is caused by the zoonotic roundworm species Toxocara canis and Toxocara cati. Diagnosis of human toxocariasis is based on the combination of clinical, parasitological, and epidemiological criteria, as well as serology tests that detect anti-Toxocara antibodies. Notwithstanding, due to the absence of pathognomonic symptoms and signs of the disease, serology is the key evidence to support a conclusive diagnosis. TES-ELISA is the most widely used serological test for diagnosis. However, cross-reaction of TES antigens with antibodies produced to other helminth antigens is a major drawback for its application in countries with high parasitic prevalence. T. canis recombinant antigens have been described as an alternative to native TES for diagnosis. Nevertheless, the selection of antigenic proteins is a complex process that requires validation. In this paper, we developed an eGFP carrier-based system to express and purify blocks of recombinant polypeptides of T. canis antigenic proteins. Intense cross-reaction polypeptides were detected by Immunoblot and avoided to finally produce a chimeric prototype protein. Additionally, a control chimeric protein that harbors the complete tested proteins was produced. Purified chimeric antigens were tested in ELISA and Immunoblot assays with 310 sera samples of negative and positive control individuals. Our results showed that chimeric rCHITC0 and rCHITC1 antigens (with sensitivities of 62% 58%, 38% and 16% in IB-rCHITC0, ELISA-rCHITC0, ELISA-rCHITC1 and IB-rCHITC1 respectively for OLMS) can perform better in terms of specificity (being 91%, 89%, 87% and 76% for ELISA-rCHITC1, IB-rCHITC1, ELISA-rCHITC0 and IB-rCHITC0 respectively for OLMS) than T. canis TES-ELISA (with 61% specificity), giving a higher signal with serum samples of infected individuals as well the possibility to discriminate false positive cases with other parasitic infections. Our data suggest that T. canis chimeric proteins, represent candidate antigens for phase II studies.

Secretory enzymes from Schistosoma japonicum are promising candidate antigens in the diagnosis of schistosomiasis. Our previous studies have proven that thioredoxin peroxidase-1 (SjTPx-1) is useful for the detection of this parasitic disease in humans, water buffaloes, and dogs. In this study, we evaluated two more secretory enzymes namely phosphoglycerate mutase (SjPGM) and phytochelatin synthase (SjPCS) with SjTPx-1 as the reference antigen. SjPGM was shown to have good diagnostic potentials in animal samples in previous studies, whereas SjPCS was chosen because of its absence in the mammalian hosts. Serum samples including 96 endemic negative controls, 107 schistosomiasis japonica positive samples, and 31 samples positive for other parasitic trematode infections (Clonorchis sinensis, Opisthorchis viverrini, Paragonimus westermani) were tested with the antigens using enzyme-linked immunosorbent assay. Results showed that SjPCS detected more positive samples and had fewer cross-reactions than SjPGM. With 85.05% sensitivity and 93.55% specificity, SjPCS can therefore be used in the detection of human schistosomiasis.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis. It has been shown that the severity of symptoms depends on the functioning of the host immune system. Although T. gondii infection typically does not lead to severe disease in healthy people and after infection, it induces a stable immunity, but it can contribute to severe and even lethal Toxoplasmosis in immunocompromised individuals (AIDS, bone marrow transplant and neoplasia). The antigens that have been proposed to be used in vaccine candidate in various studies include surface antigens and secretory excretions that have been synthesized and evaluated in different studies. In some studies, secretory antigens play an important role in stimulating the host immune response. Various antigens such as SAG, GRA, ROP, ROM, and MAG have been from different strains of T. gondii have been synthesized and their protective effects have been evaluated in animal models in different vaccine platforms including recombinant antigens, nanoparticles, and DNA vaccine. Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and Google Scholar were searched for articles published up to 2020.The current review article focuses on recent studies on the use and usefulness of recombinant antigens, nanoparticles, and DNA vaccines.

Identifying the mechanism of naturally acquired immunity against Plasmodium falciparum malaria could contribute to the design of effective malaria vaccines. Using a recently developed multiplexed FluoroSpot assay, we assessed cross-sectional pre-existing memory B-cells (MBCs) and antibody responses against six well known P. falciparum antigens (MSP-119, MSP-2 (3D7), MSP-2 (FC27), MSP-3, AMA-1 and CSP) and measured their associations with previous infections and time to clinical malaria in the ensuing malaria season in Kenyan children. These children were under active weekly surveillance for malaria as part of a long-term longitudinal malaria immunology cohort study, where they are recruited from birth. After performing Cox regression analysis, we found that children with a breadth of three or more antigen-specific MBC or antibody responses at the baseline had a reduced risk for malaria in the ensuing P. falciparum transmission season. Specifically, MBC responses against AMA-1, MSP-2 (3D7) and MSP-3, as well as antibody responses to MSP-2 (3D7) and MSP-3 were prospectively associated with a reduced risk for malaria. The magnitude or breadth of MBC responses were however not correlated with the cumulative number of malaria episodes since birth. We conclude that increased breadth for merozoite antigen-specific MBC and antibody responses is associated with protection against malaria.