Recombinant Antigen Research Articles

Determination of spontaneous and stimulated cytokine production in cell culture in patients with COVID-19 associated multisystem inflammatory syndrome

To date, the pathogenesis of COVID-19 associated multisystem inflammatory syndrome (MIS-C) remains unclear. Despite this, it becomes obvious that the pathogenesis of MIS-C is directly related to a certain immune dysregulation, however, a clear understanding of the mechanisms of this dysregulation has not yet been formulated. In order to identify the cytokine profile in patients with MIS-C, spontaneous and stimulated production of certain cytokines in cell culture was identified. Materials and methods. The study was conducted in the following study groups: group 1 — patients with MIS-C (n = 52); group 2 (comparison group) — patients with COVID-19 associated pneumonia (n = 15); group 3 (control group) — conditionally healthy patients (n = 23). The following stimulating agents were used: S58 — recombinant antigen Spike_SARS-Cov-2; NP is a recombinant NP antigen of the coronavirus SARS-CoV-2 and a standard mitogen. Results. Тhe absence of the initially expected hyperproduction of the main pro-inflammatory cytokines (IL-6, IL-8, TNF-α, etc.) was recorded. Statistically significant developments were recorded between patients of the study groups in the spontaneous production of MCP-1, in particular, the indicated indicator was 40010.82 (19698.1; 64812.1); 643.7 (214.6; 1695.4) and 622.7 (214.6; 1068.1), respectively. The indicated spontaneous hyperproduction of MCP-1 in patients with MIS-C allows us to consider as a probable completely new theory of the pathogenesis of MIS-C associated with dysregulation of the type 2 immune response. The presence of statistically significant differences, primarily in the spontaneous production of this cytokine, can apparently be explained by the presence of genetically determined determinants associated with subsequent dysfunction of the Th2 helper immune response, a potential trigger for which is a previous COVID-19 infection. Thus, further study of the immunopathogenesis of COVID-19 associated MIS-C is required.

Performance Assessment of ELISA Using the Trypanosoma cruzi-Specific Antigen Tc323 for the Diagnosis of Chronic Chagas Disease.

In the chronic phase of Chagas disease (CCD), diagnosis relies on detecting specific IgG antibodies due to the low or absent presence of the parasiteTrypanosoma cruzi in human blood. However, the performance of current serological tests is highly variable, lacking a "gold standard" assay with 100% sensitivity and specificity, which challenges the exploration of new biomarkers. In the present study, we evaluated the diagnostic accuracy of an optimized ELISA using the predicted immunogenic domains (called TcD3 and TcD6) of Tc323, a protein highly conserved among T.cruzi strains but absent in other clinically significant parasites such as Leishmania spp. This study was conducted using plasma or serum samples from CCD individuals with different clinical manifestations and living in endemic regions in Latin America, subjects with unrelated infectious diseases, and noninfected donors. The sensitivity and specificity of recombinant TcD3 were 90.8% and 92.6%, respectively, while rTcD6 displayed values of 93.1% and 93.6% for the same parameters. Area under curve (AUC) values were 0.949 for rTcD3 and 0.954 for rTcD6. The receiver operative characteristic (ROC) curve showed a highly significant difference between CCD individuals and noninfected donors. Cross-reactivity was 10.2% for rTcD3 and 8.2% for rTcD6 in subjects infected with leishmaniasis or with toxoplasmosis. In addition, the reactivity against rTcD3 differed among some geographical areas while no significant difference was found using both domains for the detection of T.cruzi-infected individuals with or without cardiac symptoms. Our findings show that the recombinant antigens rTcD3 and rTcD6 could be used as highly potential biomarkers for the serological diagnosis of CCD.

Antibody isotyping and cytokine profiling in natural cases of Burkholderia mallei infection (glanders) in equines.

Immunological aspects of B. mallei infection were rarely studied in natural cases of equines. The present study was conducted to determine IgG, IgM, IgA and IgG (T) titre against recombinant Hcp1, TssA and TssB proteins and PilA-Hcp1-TssN-BipD and BpaB-BpaC- BMAA0553 chimeras and cytokine responses in glanders affected equine serum. The study was conducted on serum samples collected from 151 glanders positive equines which include horses (n=134), mules (n=16), and donkeys (n=01). The IgM, IgG, IgA and IgG (T) titre were determined against recombinant antigens by indirect ELISA and interleukin(IL)-1β, IL-17, IL-6, tumor necrosis factor alpha(TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interferon gamma (IFN-γ) responses were measured by commercial ELISA kits. The study showed that glanders affected equines elicited strong antibody response against Hcp1, moderate responses against TssA and TssB, and weak responses against two chimeras. Among the cytokines, IL-1β, MCP-1, IL-17 and IL-6 concentration were significantly higher in glanders affected equine serum. We found that IgG antibody titre was higher than IgM, IgG (T) and IgA isotypes and Hcp1 was most predominant antibody inducers in comparison to TssA, TssB, PilA-Hcp1-TssN-BipD and BpaB-BpaC-T2SS proteins. The elevated level of IL-1β, MCP-1, IL-17, IL-6, IFN-γ and TNF-α observed in this study support the important role of this cytokines for augmenting cellular defence by recruitment of macrophages, neutrophil and dendritic cells against B. mallei infection. Further studies should be conducted to determine memory cell responses in natural cases of equine glanders using recombinant B. mallei proteins for identifying well-characterized immuno-protective vaccine candidates.

Approaching onchocerciasis elimination in Equatorial Guinea: Near zero transmission and public health implication.

Onchocerciasis and lymphatic filariasis (LF) are endemic in Equatorial Guinea with notable variations in disease incidence between island and mainland regions. Historically, efforts to control and map these diseases were concentrated in Bioko Island, where loiasis is absent, allowing for targeted onchocerciasis interruption strategies. With the cessation of onchocerciasis transmission on Bioko and no reported cases on Annobon island, assessing the transmission status in the previously unaddressed mainland region has become imperative. Mapping efforts in mainland Equatorial Guinea have proven low to moderate level of transmission for LF and onchocerciasis, although the results so far have not been very conclusive. The current study aims to update the prevalence estimates for onchocerciasis and LF in mainland Equatorial Guinea using various diagnostic techniques. This is the first cross-sectional study carried out to estimate the prevalence of onchocerciasis and LF in the mainland area of Equatorial Guinea, from September to December 2019, based on the combination of skin snip biopsies, thick blood smears, laboratory serological tests (ELISA tests for the detection of IgG4 antibodies against Onchocerca volvulus recombinant antigen Ov16 and Wuchereria bancrofti recombinant antigen Wb123) and molecular laboratory tests. Frequencies and prevalence rates, along with 95% confidence intervals for interval estimation of a binomial proportion, were computed. The overall onchocerciasis seroprevalence calculated for the study was 0.3% (95% CI: 0.1 to 0.5%). Microscopic examination of skin biopsies from the eight individuals seropositive for Ov16, out of the 3951 individuals initially tested, revealed no O. volvulus microfilariae. However, DNA extracted from one skin snip was successfully amplified, with subsequent sequencing confirming the presence of O. volvulus. Among the 3951 individuals, 182 were found to have anti-Wb123 antibodies, suggesting exposure to W. bancrofti, with an estimated seroprevalence of 4.6% (95% CI: 4.0 to 5.3%). Microscopy and Filaria-real time-PCR (F-RT-PCR) analysis for W. bancrofti were negative across all samples. The findings indicate that onchocerciasis may no longer constitutes a public health problem in Equatorial Guinea, positioning the country on the verge of achieving elimination. Additionally, the mapped prevalence of LF will facilitate the formulation of national strategies aimed at eradicating filariases countrywide.

Current vaccines, experimental immunization trials, and new perspectives to control selected vector borne blood parasites of veterinary importance

Parasite infections transmitted by vectors such as ticks and blood-sucking arthropods pose a significant threat to both human and animal health worldwide and have a substantial economic impact, particularly in the context of worsening environmental conditions. These infections can manifest in a variety of symptoms, including fever, anemia, jaundice, enlarged spleen, neurological disorders, and lymphatic issues, and can have varying mortality rates. In this review, we will focus on the current state of available vaccines, vaccine research approaches, and trials for diseases caused by vector-borne blood parasites, such as Babesia, Theileria, Anaplasma, and Trypanosoma, in farm animals. Control measures for these infections primarily rely on vector control, parasiticidal drug treatments, and vaccinations for disease prevention. However, many of these approaches have limitations, such as environmental concerns associated with the use of parasiticides, acaricides, and insecticides. Additionally, while some vaccines for blood parasites are already available, they still have several drawbacks, including practicality issues, unsuitability in non-endemic areas, and concerns about spreading other infectious agents, particularly in the case of live vaccines. This article highlights recent efforts to develop vaccines for controlling blood parasites in animals. The focus is on vaccine development approaches that show promise, including those based on recombinant antigens, vectored vaccines, and live attenuated or genetically modified parasites. Despite intensive research, developing effective subunit vaccines against blood stage parasites remains a challenge. By learning from previous vaccine development efforts and using emerging technologies to define immune mechanisms of protection, appropriate adjuvants, and protective antigens, we can expand our toolkit for controlling these burdensome diseases.

Local and systemic humoral immune responses to Histophilus somni recombinant antigens administered intranasally and subcutaneously to dairy calves

AbstractBovine respiratory disease (BRD) causes significant economic losses in dairy calves. Induction of an early immune response via parenteral vaccination is complicated by the interference of colostral immunity. In this study, we investigated early immunization against selected conserved bacterial antigens. Calves were vaccinated twice intranasally and then subcutaneously with Histophilus somni recombinant proteins (rOMP40 or rHsp60) mixed with one of two adjuvants: CpG ODN2007 or MPLA. The control group (Con) was treated with PBS. The first immunization was done between 24 and 48 h of life and then twice in two weeks intervals. Blood, nasal, and saliva secretion samples were collected directly before vaccination (S1–S3) and then on 42–44 (S4) and 59–61 (S5) day of life. Antibodies (IgG1/IgG2/IgM/IgA in serum; IgG1/IgA in secretions) against both vaccine antigens were quantified in all samples. Intranasal and subcutaneous vaccinations using the described formulas did not increase antibody reactivity against the tested proteins. The reactivity of serum IgG1, IgM, and IgA anti-rOMP40 antibodies was significantly higher in S1 in all groups than that in the other samplings (p˂0.01). Significant differences in the reactivity of serum anti-rOMP40 antibodies between groups were identified in S1 (IgA reactivity was higher in the CpG vs. MPLA group; p < 0.05), S4 (IgM reactivity was higher in Con vs. CpG group; p < 0.05), and S5 (IgG1 reactivity was higher in MPLA vs. Con group; p < 0.05). The lack of consistent changes in antibodies after immunization (S4 and S5) hinders the drawing of conclusions regarding the effect of immunization on antibody reactivity. In the future, establishing a proper immunization window and adjuvants for nasal vaccines against bacterial pathogens causing BRD in calves remains to be determined.

Oceanic Breakthroughs: Marine-Derived Innovations in Vaccination, Therapy, and Immune Health

The vast, untapped potential of the world’s oceans is revealing groundbreaking advancements in human health and vaccination. Microalgae such as Nannochloropsis spp. and Dunaliella salina are emerging as resources for recombinant vaccine development with specific and heterologous genetic tools used to boost production of functional recombinant antigens in Dunaliella salina and Nannochloropsis spp. to induce immunoprotection. In humans, several antigens produced in microalgae have shown potential in combating diseases caused by the human papillomavirus, human immunodeficiency virus, hepatitis B virus, influenza virus, Zika virus, Zaire Ebola virus, Plasmodium falciparum, and Staphylococcus aureus. For animals, microalgae-derived vaccine prototypes have been developed to fight against the foot-and-mouth disease virus, classical swine fever virus, vibriosis, white spot syndrome virus, and Histophilus somni. Marine organisms offer unique advantages, including the ability to express complex antigens and sustainable production. Additionally, the oceans provide an array of bioactive compounds that serve as therapeutics, potent adjuvants, delivery systems, and immunomodulatory agents. These innovations from the sea not only enhance vaccine efficacy but also contribute to broader immunological and general health. This review explores the transformative role of marine-derived substances in modern medicine, emphasizing their importance in the ongoing battle against infectious diseases.

Epidemiological surveillance of Neospora caninum in sheep (Ovis aries) and European mouflon (Ovis aries musimon) in southern Spain.

Neospora caninum is a protozoan with a facultative heteroxenous life cycle, with canids as the definitive hosts and other mammals, mainly ruminants, acting as intermediate hosts. This parasite is recognized as one of the major abortifacient pathogens in cattle. Although reproductive disorders have also been reported in other domestic and wild ruminants, epidemiological data on N. caninum in ruminant species other than cattle is still limited. Here, we evaluate the seroprevalence and risk factors associated with N. caninum exposure in sheep (Ovis aries) and European mouflon (Ovis aries musimon) in southern Spain. Serum samples from 390 sheep and 387 free-ranging mouflons were tested for antibodies against N. caninum using in-house time-resolved fluorescence immunoassay based on NcGRA7 as a recombinant antigen. The individual seroprevalence was 26.2% (95%CI: 22.0-30.7) in sheep and 5.7% (95%CI: 3.8-8.5) in mouflons. At least one seropositive animal was detected in all the 26 sheep farms (100%) and in eight of the 18 (44.4%) hunting estates sampled. The presence of dogs on the farm (≥3) and the sheep breed (purebred) were potential risk factors associated with exposure to N. caninum in sheep. Our results indicate a high circulation of N. caninum in sheep farms in southern Spain. Control measures should be implemented to limit the exposure to this protozoan in sheep flocks. Although we have confirmed for the first time the presence of anti- N. caninum antibodies in European mouflon in Spain, and, despite the relatively low seroprevalence found, the impact of this parasite on mouflon populations should be further studied.

The immune responses elicited by six recombinant antigens of Mycoplasma hyopneumoniae in mice

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causative agent of swine enzootic pneumonia, resulting in substantial economic losses in global pig farming. Although vaccination is the primary strategy for controlling M. hyopneumoniae infection, current vaccines fall short in preventing transmission of this pathogen or protecting the body from secondary infection. This study aimed to assess the immunogenicity of six recombinant antigens (P97R1, P46, GAPDH, PdhA, DnaK, and EF-Tu) of M. hyopneumoniae through intramuscular immunization in mice. The results showed that the six antigens elicited high levels of serum IgG. Among them, P97R1, P46, PdhA, and DnaK stimulated robust antigen-specific IgA mucosal immune responses. CCK-8 assays revealed that both P97R1 and DnaK significantly increased the proliferation of mononuclear cells from spleen and lung, and DnaK also promoted the proliferation of blood mononuclear cells. Additionally, PdhA induced Th17-type immune response with a high level of IL-17 level in serum. Flow cytometry analysis indicated that P97R1 and PdhA increased the ratio of CD8+/CD4+ T lymphocyte, favoring cytotoxic T lymphocyte (CTL) immune responses. Notably, P97R1 immunization significantly decreased the percentages of CD4+ T cells while increased the percentages of CD8+ T cells. The present findings demonstrate that the candidate antigens P97R1, PdhA, and DnaK of M. hyopneumoniae induce specific humoral and mucosal immunity; P97R1 and DnaK also stimulated intense cellular immunity, and PdhA induced CTL and Th17-type immune responses. In conclusion, P97R1, PdhA, and DnaK emerge as potential candidate antigens for the future development of a more effective subunit vaccine against M. hyopneumoniae.

Exploring Potential Application of Anti-Dengue NS1 Human Polyclonal Antibodies for Detection of Dengue Virus Infection

Dengue, a vector-borne disease, affects nearly 400 million people annually. Although commercially available dengue NS1 antigen-based ELISA kits are simple and rapid, they are expensive as monoclonal antibodies are used in these tests, and also, they have short expiry dates. As an alternative, the polyclonal antibodies generated against dengue NS1 antigen from the individuals who recovered from the dengue infection (human polyclonal antibodies against dengue; HuPA-D) can be explored for the detection of NS1 antigen in the dengue virus (DENV) infected patients’ sera. In this study, blood samples were collected from the dengue-recovered patients after obtaining the IHEC approval. The anti-NS1 HuPA-D (IgG) was purified using NAb™ Spin Column kit and tested on SDS-PAGE. HuPA-D ELISA was developed to test the sensitivity and specificity of the antibodies using the recombinant NS1 antigens of dengue serotypes and flaviviruses. Further, the HuPA-D were used to detect the sera of the dengue patients. The assay was found to be sensitive to detect all the serotypes of recombinant dengue NS1 antigen and also NS1 antigen from the sera of DENV infected patients.

Diagnostic value of antibody immunoblotting detection in rheumatoid arthritis patients

Objective. To assess the diagnostic value of antinuclear antibody (ANA) profiling in rheumatoid arthritis by immunoblotting.Materials and methods. In total, 46 patients with rheumatoid arthritis (RA) with a mean age of 34.6 years (21.3–63.2) were observed. The disease duration was 11.2 years (3.7–19.8); the activity according to DAS 28 was 3.15 ± 1.36 (3.05–3.61) points. The RA diagnosis was based on generally accepted clinical guidelines. The control group included 28 patients with osteoarthritis. Laboratory examinations were conducted using a set of reagents to determine IgG antibodies to nuclear antigens by immunoblotting. Results. The RA patients showed an increased level of antibodies to the RNP/Sm antigen, with its frequency being significantly higher than in the control group (p = 0.007). The most specific testomes for diagnosing RA were anti-RNP/Sm (specificity 96%, sensitivity 25%) and antibodies to the recombinant antigen (sensitivity 25%, specificity 88.1%). The method of ROC analysis found that the value of anti-RNP/Sm = 0 is the point corresponding to the optimal ratio of sensitivity/specificity. This value corresponds to a sensitivity of 50% and a specificity of 73.8%.Conclusion. The studied laboratory tests, as a rule, showed high specificity, but rather low sensitivity. The most specific test for RA is anti-RNP/Sm. The conducted ROC analysis showed that the anti-RNP/Sm test has an average quality index for diagnosing RA.

Comparison of Four Chimeric Antigens and Commercial Serological Assays for the Diagnosis of Trypanosoma cruzi Infection

Chagas disease (CD), a neglected tropical disease caused by Trypanosoma cruzi, is a significant public health issue particularly in Latin America, affecting millions worldwide. Diagnosis is a challenge owing to the genetic diversity of T. cruzi and the complexities involved in selecting antigens for the detection of anti–T. cruzi antibodies. This study evaluated four chimeric recombinant antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) designed to enhance diagnostic accuracy by addressing assay variability. We compared the diagnostic performance of these chimeric antigens using indirect ELISA as a diagnostic platform, with three commercial serological assays in Brazil, analyzing 100 serum samples from individuals with confirmed CD and 86 from non-infected controls. The results revealed that all assays and antigens demonstrated an area under the receiver operating characteristic curve of 100%, signifying their exceptional ability to distinguish between CD-positive and CD-negative samples. Notably, the chimeric antigens achieved 100% sensitivity, specificity, accuracy, and kappa index, equaling or surpassing the commercial assays. This research highlights the efficacy of IBMP chimeric antigens as reliable diagnostic tools for CD, suggesting their potential integration into commercial diagnostic platforms to enhance the accuracy and reliability of CD detection.

Evaluation of the Use of Sub-Immunodominant Antigens of Babesia bovis with Flagellin C Adjuvant in Subunit Vaccine Development

Bovine babesiosis caused by the tick-borne apicomplexan parasite Babesia bovis remains a threat for cattle worldwide, and new vaccines are needed. We propose using immune-subdominant (ISD) antigens as alternative vaccine candidates. We first determined that RAP-1 NT and RRA are subdominant antigens using recombinant antigens in ELISAs against sera from B. bovis-protected cattle. Protected animals demonstrated high antibody responses against the known immunodominant rRAP-1 CT antigen, but significantly lower levels against the rRAP-1 NT and rRRA antigens. Next, a group of cattle (n = 6) was vaccinated with rRRA and rRAP-1 NT using a FliC–Emulsigen mix as the adjuvant, and there was a control group (n = 6) with the adjuvant mix alone. All but one immunized animal demonstrated elicitation of strong humoral immune responses against the two ISD antigens. Acute babesiosis occurred in both groups of cattle upon a challenge with the virulent B. bovis, but a significant delay in the average rate of decrease in hematocrit in the vaccinated group, and an early monocyte response, was found in half of the vaccinated animals. In conclusion, we confirmed the immune subdominance of rRRA and rRAP-1 NT and the ability of FliC to increase immunogenicity of ISD antigens and generate useful information toward developing future subunit vaccines against B. bovis.

Preparation and Construction of Chimeric Humanized Broadly Reactive Antibody 10H10 to Protein E of Tick-Borne Encephalitis Virus.

A full-length humanized chimeric antibody 10H10ch that specifically interacts with the surface glycoprotein E of flaviviruses was obtained. To construct it, we used variable fragments of the heavy and light chains of the monoclonal antibody 10H10 that form the active center of the antibody and a fragment of the constant part of the heavy chain of the human IgG1 antibody. The resulting full-length chimeric humanized antibody 10H10ch specifically interacted with the E protein of flaviviruses pathogenic to humans, such as tick-borne encephalitis, Zika, West Nile, and dengue viruses. An immunochemical assessment of the interaction constants of the 10H10ch antibody with a panel of native and recombinant flavivirus antigens by ELISA and biolayer interferometry showed that the dissociation constant (Kd) of the chimeric antibody is in the nanomolar region and is comparable to that of the high-affinity mouse monoclonal antibody 10H10. The possibility of using the resulting chimeric humanized antibody 10H10ch for the diagnosis, prevention, and treatment of various flavivirus infections is discussed.

CD4 T cells restricted to DRB1*15:01 recognize two Epstein–Barr virus glycoproteins capable of intracellular antigen presentation

Both genetic and environmental factors contribute to multiple sclerosis (MS) risk. Infection with the Epstein-Barr virus (EBV) is the strongest environmental risk factor, and HLA-DR15 is the strongest genetic risk factor for MS. We employed computational methods and in vitro assays for CD4 T cell activation to investigate the DR15-restricted response to EBV. Using a machine learning-based HLA ligand predictor, the EBV glycoprotein B (gB) was predicted to be enriched in epitopes restricted to presentation by DRB1*15:01. In DR15-positive individuals, two epitopes comprised the major CD4 T cell response to gB. Surprisingly, the expression of recombinant gB in a DR15-homozygous B cell line or primary autologous B cells elicited a CD4 T cell response, indicating that intracellular gB was loaded onto HLA class II molecules. By deleting the signal sequence of gB, we determined that this pathway for direct activation of CD4 T cells was dependent on trafficking to the endoplasmic reticulum (ER) within the B cell. We screened seven recombinant EBV antigens from the ER compartment for immune responses in DR15-negative vs. DR15-homozygous individuals. In addition to gB, gH was a key CD4 T cell target in individuals homozygous for DR15. Compared to non-DR15 controls, DR15-homozygotes had significantly higher T cell responses to both gB and gH but not to EBV latent or lytic antigens overall. Responses to gB and gH were slightly elevated in DR15 homozygotes with MS. Our results link MS environmental and genetic risk factors by demonstrating that HLA-DR15 dictates CD4 T cell immunity to EBV antigens.

Gold Nanoparticles-Based Colorimetric Immunoassay of Carcinoembryonic Antigen with Metal–Organic Framework to Load Quinones for Catalytic Oxidation of Cysteine

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal–organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu2+ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His6) tag in CEA and the unsaturated Cu2+ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01–1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His6 tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers.

Overview of Recombinant Tick Vaccines and Perspectives on the Use of Plant-Made Vaccines to Control Ticks of Veterinary Importance.

Ticks are obligate hematophagous ectoparasites that affect animals, and some of them transmit a wide range of pathogens including viruses, bacteria, and protozoa to both animals and humans. Several vaccines have shown immunogenicity and protective efficacy against ticks in animal models and definitive hosts. After several decades on anti-tick vaccine research, only a commercial vaccine based on a recombinant antigen is currently available. In this context, plants offer three decades of research and development on recombinant vaccine production to immunize hosts and as a delivery vehicle platform. Despite the experimental advances in plant-made vaccines to control several parasitosis and infectious diseases, no vaccine prototype has been developed against ticks. This review examines a panorama of ticks of veterinary importance, recombinant vaccine experimental developments, plant-made vaccine platforms, and perspectives on using this technology as well as the opportunities and limitations in the field of tick vaccine research.

Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia

Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect Leishmania in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(p < 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (p < 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia.

CT584 Is Not a Protective Vaccine Antigen against Respiratory Chlamydial Challenge in Mice.

Background:Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production.

A-270 Evaluation of a multiplex panel to determine antibody profile to monkeypox virus in vaccinated, exposed, and naïve individuals

Abstract Background Monkeypox Virus (MPXV) is a double-stranded DNA virus of the Orthopoxvirus genus that causes Mpox disease with symptoms such as swollen lymph nodes, chills, exhaustion, and blisters that appear on the face, inside the mouth, hands, feet etc. The genus orthopoxvirus (OPXV) has four species, namely, MPXV, smallpox-causing variola major virus (VARV), variola minor virus, and cowpox virus (CPXV), that cause disease in humans. In 1980, the World Health Assembly declared that smallpox had been eradicated due to a successful global vaccination program. This vaccine also provided cross-protection against other OPXV. Historically, the pox disease was rarely seen outside West and Central Africa. The first case of Mpox was identified in 1970 in a 9-year-old boy from the Democratic Republic of Congo (DRC), two years after the smallpox vaccination was discontinued. Since then, it has been reported in 11 African countries. In 2003, the first Mpox outbreak outside of Africa was in the USA and was linked to contact with infected pet prairie dogs. The recent 2022-2023 Mpox outbreak has generated renewed interest in the scientific community to understand the level of OPXV-specific immunity in the various geographic regions, among different age groups, and especially in the risk groups and its impact on future outbreaks by using integrated serosurveillance. To meet the need of the hour, we developed a novel multiplex assay for the detection of human IgG antibodies using Luminex® xMAP® technology for the detection of the human IgG profiles. Methods We have evaluated 11 different recombinant antigens from VARV, Vaccinia virus (VACV), and MPXV in a multiplex assay for detecting human IgG antibodies to OPXV using Luminex® xMAP® technology. We have covalently coupled magnetic fluorescent microspheres to three recombinant proteins from VARV, three from VACV, and five from MPXV to interrogate IgG responses. This 15-plex assay also includes four internal controls that assure the assay and reagent performance. In this preliminary evaluation, we have tested 84 de-identified serum and plasma from healthy subjects. We evaluated 24 serum samples of MPXV-exposed individuals procured commercially. We performed principal component analysis to determine which of these 11 antigens contributed the maximum differential and a result analysis algorithm was developed to predict these individuals' positive and negative antibody outcomes. Results The healthy subjects that were assumed to have been vaccinated showed positive response to A35R of MPXV; A36R of VARV, A33R of VACV, the homologous proteins in the three species and were distinct from unvaccinated younger individuals. There was some reactivity with B6R of MPXV protein. In MPXV-exposed subjects, we observed strong positive antibody responses to A35R of MPXV, A36R of VARV, A33R of VACV, and B6R of MPXV proteins in 19 out of 24 individuals. Fifteen individuals showed a moderate positive response to the A27L protein of VACV, and 8 out of 24 showed a weak positive response to the A29 protein of MPXV. Conclusions This panel is a useful tool to understand the level of OPXV-specific immunity by measuring IgG profiles for successful serosurveillance programs for public health.