The O-glycan is essential for the induction of protective antibodies against lethal infection by flagella A-bearing Pseudomonas aeruginosa.

To address the problem of increased antimicrobial resistance, we developed a glycoconjugate vaccine comprised of O-polysaccharides (OPS) of the four most prevalent serotypes of Klebsiella pneumoniae (KP) linked to recombinant flagellin types A and B (rFlaA and rFlaB) of Pseudomonas aeruginosa (PA). Flagellin is the major subunit of the flagellar filament. Flagella A and B, essential virulence factors for PA, are glycosylated with different glycans. We previously reported that while both rFlaA and rFlaB were highly immunogenic, only the rFlaB antisera reduced PA motility and protected mice from lethal PA infection in a mouse model of thermal injury. Since recombinant flagellin is not glycosylated, we examined the possibility that the glycan on native FlaA (nFlaA) might be critical to functional immune responses. We compared the ability of nFlaA to that of native, deglycosylated FlaA (dnFlaA) to induce functionally active antisera. O glycan was removed from nFlaA with trifluoromethanesulfonic acid. Despite the similar high-titered anti-FlaA antibody levels elicited by nFlaA, rFlaA, and dnFlaA, only the nFlaA antisera inhibited PA motility and protected mice following lethal intraperitoneal bacterial challenge. Both the protective efficacy and carrier protein function of nFlaA were retained when conjugated to KP O1 OPS. We conclude that unlike the case with FlaB O glycan, the FlaA glycan is an important epitope for the induction of functionally active anti-FlaA antibodies.

Typhoidal salmonella disease in Mukuru informal settlement, Nairobi Kenya; carriage, diversity, and antimicrobial resistant genes.

INTRODUCTION: Multiple studies have shown that typhoid fever is endemic in developing countries characterized by poor hygiene. A unique way of Salmonella Typhi (S.Typhi) pathogenicity is establishing a persistent, usually asymptomatic carrier state in some infected individuals who excrete large numbers of bacteria in faeces. This study aimed to determine the isolation rate of S.Typhi from blood and stool samples among cases and asymptomatic individuals in the Mukuru informal settlement and identify antibiotic resistance patterns within the same population. MATERIALS AND METHODS: We recruited 1014 outpatient participants presenting with typhoid-like symptoms in selected health centres in Nairobi, Kenya. Bacterial isolation was done on Xylose Lysine Deoxycholate agar (XLD) and Mac Conkey agar (Oxoid), followed by standard biochemical tests. Identification was done using API20E, and S.Typhi was confirmed by serotyping using polyvalent antisera 0-9 and monovalent antisera d. The Kirby-Bauer disc diffusion method was used to test the antimicrobial susceptibility of S.Typhi isolates, while Multi-Drug Resistant (MDR) strains were characterized using conventional PCR. RESULTS: Of 1014 participants, 54 (5%) tested positive for S.Typhi. Thirty-eight (70%) of the S.Typhi isolated were from stool samples, while sixteen (30%) were from blood. Three (0.2%) of the isolates were from asymptomatic carriers. Of the 54 S.Typhi isolates, 20 (37%) were MDR. Resistance to ciprofloxacin and nalidixic acid was 43% and 52%, respectively. Resistance to amoxicillin-clavulanic acid (a beta-lactam inhibitor) was 2%. The BlaTEM-1 gene was present in 19/20 (95%) MDR isolates. CONCLUSION: MDR S.Typhi is prevalent in Mukuru Informal settlement. The sharp increase in nalidixic acid resistance is an indication of reduced susceptibility to fluoroquinolones, which are currently the recommended drugs for the treatment of typhoid fever. This study highlights the need for effective antimicrobial stewardship and routine surveillance of antimicrobial resistance (AMR) to inform policy on the prevention and control of MDR Typhoid disease.

Design of universal Ebola virus vaccine candidates via immunofocusing.

The Ebola virus causes hemorrhagic fever in humans and poses a significant threat to global public health. Although two viral vector vaccines have been approved to prevent Ebola virus disease, they are distributed in the limited ring vaccination setting and only indicated for prevention of infection from orthoebolavirus zairense (EBOV)-one of three orthoebolavirus species that have caused previous outbreaks. Ebola virus glycoprotein GP mediates viral infection and serves as the primary target of neutralizing antibodies. Here, we describe a universal Ebola virus vaccine approach using a structure-guided design of candidates with hyperglycosylation that aims to direct antibody responses away from variable regions and toward conserved epitopes of GP. We first determined the hyperglycosylation landscape on Ebola virus GP and used that to generate hyperglycosylated GP variants with two to four additional glycosylation sites to mask the highly variable glycan cap region. We then created vaccine candidates by displaying wild-type or hyperglycosylated GP variants on ferritin nanoparticles (Fer). Immunization with these antigens elicited potent neutralizing antisera against EBOV in mice. Importantly, we observed consistent cross-neutralizing activity against Bundibugyo virus and Sudan virus from hyperglycosylated GP-Fer with two or three additional glycans. In comparison, elicitation of cross-neutralizing antisera was rare in mice immunized with wild-type GP-Fer. These results demonstrate a potential strategy to develop universal Ebola virus vaccines that confer cross-protective immunity against existing and emerging filovirus species.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

Urine Immunofixation Electrophoresis for Diagnosis of Monoclonal Gammopathy: Evaluation of Methods for Urine Concentration.

BACKGROUND: Examination of urine by immunofixation electrophoresis (UIFE) is one of the tests recommended for screening and monitoring of monoclonal gammopathies, especially multiple myeloma. Unlike the serum free light chain measurement, a positive result on urine immunofixation is diagnostic for monoclonal immunoglobulin light chains. Urine is usually concentrated, generally by membrane filtration, prior to electrophoresis. METHODS: Alternative methods to membrane filtration for urine concentration were examined. Residual urine specimens submitted for urine protein electrophoresis were concentrated by precipitation of the proteins by ammonium sulfate salt precipitation, precipitation with ethanol and acetonitrile, and by desiccation. The concentrated specimens were subjected to immunofixation electrophoresis using antisera to free light chains (FLC). The results were compared with those from conventional immunofixation electrophoresis using specimens concentrated by membrane filtration. RESULTS: Ammonium sulfate, ethanol, and acetonitrile precipitation results were less than satisfactory. Concentration by desiccation provided results comparable, if not better than, those by membrane filtration and conventional UIFE. The cost of desiccation is minimal compared to more than $5.00/specimen cost of concentration by membrane filtration. The differences in the results with conventional UIFE and the method described here are likely due to (a) variability in the reactivity of different antisera to free monoclonal light chains, and (b) obscuration of monoclonal free light chains by co-migration with intact immunoglobulin monoclonal proteins. CONCLUSIONS: Concentrating urine by desiccation for immunofixation electrophoresis is technically simple, inexpensive, and provides results comparable to concentrating by membrane filtration. Using FLC provides a more sensitive assay than using conventional antisera.

Antibody landscape of C57BL/6 mice cured of B78 melanoma via a combined radiation and immunocytokine immunotherapy regimen.

Sera of immune mice that were previously cured of their melanoma through a combined radiation and immunocytokine immunotherapy regimen consisting of 12 Gy of external beam radiation and the intratumoral administration of an immunocytokine (anti-GD2 mAb coupled to IL-2) with long-term immunological memory showed strong antibody-binding against melanoma tumor cell lines via flow cytometric analysis. Using a high-density whole-proteome peptide array (of 6.090.593 unique peptides), we assessed potential protein-targets for antibodies found in immune sera. Sera from 6 of these cured mice were analyzed with this high-density, whole-proteome peptide array to determine specific antibody-binding sites and their linear peptide sequence. We identified thousands of peptides that were targeted by these 6 mice and exhibited strong antibody binding only by immune (after successful cure and rechallenge), not naïve (before tumor implantation) sera and developed a robust method to detect these differentially targeted peptides. Confirmatory studies were done to validate these results using 2 separate systems, a peptide ELISA and a smaller scale peptide array utilizing a slightly different technology. To the best of our knowledge, this is the first study of the full set of germline encoded linear peptide-based proteome epitopes that are recognized by immune sera from mice cured of cancer via radio-immunotherapy. We furthermore found that although the generation of B-cell repertoire in immune development is vastly variable, and numerous epitopes are identified uniquely by immune serum from each of these 6 immune mice evaluated, there are still several epitopes and proteins that are commonly recognized by at least half of the mice studied. This suggests that every mouse has a unique set of antibodies produced in response to the curative therapy, creating an individual "fingerprint." Additionally, certain epitopes and proteins stand out as more immunogenic, as they are recognized by multiple mice in the immune group.

Genotype analysis to clarify RhD variants in discrepant samples of Chilean population.

Introduction: The D antigen variants are classified as weak, partial, and extremely weak (DEL) and can be differentiated using molecular tests. In Chile, the laboratories of local blood centers do not identify variants of the D antigen, referring them for study to the Reference Laboratory of the Public Health Institute of Chile. So, our aim was to talk about the results of the molecular analysis of variants of the D antigen in samples that had different results in the serological classification. Methods: In the D antigen classification of the Rh system, 479 samples with serological discrepant results were sent for molecular analysis. The Rh phenotype was performed with monoclonal anti-C, anti-c, anti-E, and anti-e antisera by direct agglutination. To find the D antigen, researchers used direct agglutination with monoclonal antisera and indirect antiglobulin testing with the column (gel) agglutination method. Molecular analysis was performed with a polymerase chain reaction with sequence-specific primers (SSP-PCR) and sequencing. Results and discussion: The presence of D antigen variants was confirmed in 332 samples (69.3%), with an initial discrepancy in serological classification. In this group of discrepant samples, the frequency of weak RhD variants was 66% (219/332), that of extremely weak RhD was 28% (93/332), and that of partial RhD was 6% (20/332). The weak variants type 2 (27.4%), type 3 (8.4%), type 48 (8.4%), and type 1 (8.1%) were the next most prevalent variants after RHD*DEL43 (28%). The ccEe (R2r) phenotype was the most frequently detected (38.4%) and is present in 87% of the RHD*DEL43 samples. The E antigen is associated with the presence of this variant. Our analyses give the first description of D antigen variants in Chile. The most common variants are DEL type (RHD*DEL43) and weak (weak type 2), which are linked to the ccDEe (R2r) phenotype. These findings allow us to characterize the variants of the D antigen in Chile and, according to the obtained data, to design strategies for the management of donors, patients, and pregnant women.

Development, Histological, and Ultrastructural Evaluation of Sheep Hyperimmune Serum Therapy for COVID-19 / Desarrollo, Evaluación Histológica y Ultraestructural de la Terapia con Suero Hiperinmune de Oveja para COVID-19

SUMMARY: In response to the threat posed by new variants of SARS-CoV-2 and the urgent need for effective treatments in the absence of vaccines, the aim of this study was to develop a rapid and cost-effective hyperimmune serum (HS) derived from sheep and assess its efficacy. The utilization of a halal-certified, easily maintained in certain geographic regions, easy-to-handle animal such as sheep could provide a viable alternative to the expensive option of horses. Sheep were immunized with a whole inactivated SARS-CoV- 2 antigen to produce HS, which was evaluated for neutralizing potency using the PRNT50 assay. K18-hACE2 transgenic mice (n=35) were divided into three groups: control, SARS-CoV-2 exposure through inhalation, and SARS-CoV-2 exposed mice treated with HS. HS efficacy was assessed through serum proinflammatory cytokine levels, qRT-PCR analysis, histopathological examination of lungs and hearts, and transmission electron microscopy. Purified HS exhibited significant neutralizing activity (1/24,576). The SARS-CoV-2+HS group showed lower levels of TNF-α, IL-10, and IL-6 (P<0.01) and relatively lower levels of MCP-1 compared to the SARS-CoV-2 group. HS prevented death, reduced viral RNA levels in the lungs and hearts, protected against severe interstitial pneumonia, preserved lung tissue integrity, and prevented myocyte damage, while the SARS-CoV-2 group exhibited viral presence in the lungs. This study successfully developed a sheep-derived HS against the entire SARS-CoV-2 virus, resulting in a significant reduction in infection severity, inflammation, and systemic cytokine production. The findings hold promise for treating severe COVID-19 cases, including emerging viral variants, and immunocompromised patients.

En respuesta a la amenaza que suponen las nuevas variantes del SARS-CoV-2 y la urgente necesidad de tratamientos eficaces en ausencia de vacunas, el objetivo de este estudio fue desarrollar un suero hiperinmune (HS) rápido y rentable derivado de ovejas. y evaluar su eficacia. La utilización de un animal con certificación halal, de fácil mantenimiento en determinadas regiones geográficas y de fácil manejo, como las ovejas, podría proporcionar una alternativa viable a la costosa opción de los caballos. Las ovejas fueron inmunizadas con un antígeno de SARS-CoV-2 completamente inactivado para producir HS, cuya potencia neutralizante se evaluó mediante el ensayo PRNT50. Los ratones transgénicos K18-hACE2 (n = 35) se dividieron en tres grupos: control, exposición al SARS-CoV-2 mediante inhalación y ratones expuestos al SARS-CoV-2 tratados con HS. La eficacia de HS se evaluó mediante niveles de citoquinas proinflamatorias en suero, análisis qRT-PCR, examen histopatológico de pulmones y corazones y microscopía electrónica de transmisión. El HS purificado exhibió una actividad neutralizante significativa (1/24,576). El grupo SARS-CoV-2+HS mostró niveles más bajos de TNF-α, IL-10 e IL-6 (P<0,01) y niveles relativamente más bajos de MCP-1 en comparación con el grupo SARS-CoV-2. HS evitó la muerte, redujo los niveles de ARN viral en los pulmones y el corazón, protegió contra la neumonía intersticial grave, preservó la integridad del tejido pulmonar y evitó el daño de los miocitos, mientras que el grupo SARS-CoV-2 exhibió presencia viral en los pulmones. Este estudio desarrolló con éxito un HS derivado de ovejas contra todo el virus SARS-CoV-2, lo que resultó en una reducción significativa de la gravedad de la infección, la inflamación y la producción sistémica de citocinas. Los hallazgos son prometedores para el tratamiento de casos graves de COVID- 19, incluidas las variantes virales emergentes y los pacientes inmunocomprometidos.

Characterization of intrinsic and effective fitness changes caused by temporarily fixed mutations in the SARS-CoV-2 spike E484 epitope and identification of an epistatic precondition for the evolution of E484A in variant Omicron.

BACKGROUND: Intrinsic fitness costs are likely to have guided the selection of lineage-determining mutations during emergence of variants of SARS-CoV-2. Whereas changes in receptor affinity and antibody neutralization have been thoroughly mapped for individual mutations in spike, their influence on intrinsic replicative fitness remains understudied. METHODS: We analyzed mutations in immunodominant spike epitope E484 that became temporarily fixed over the pandemic. We engineered the resulting immune escape mutations E484K, -A, and -Q in recombinant SARS-CoV-2. We characterized viral replication, entry, and competitive fitness with and without immune serum from humans with defined exposure/vaccination history and hamsters monospecifically infected with the E484K variant. We additionally engineered a virus containing the Omicron signature mutations N501Y and Q498R that were predicted to epistatically enhance receptor binding. RESULTS: Multistep growth kinetics in Vero-, Calu-3, and NCI-H1299 were identical between viruses. Synchronized entry experiments based on cold absorption and temperature shift identified only an insignificant trend toward faster entry of the E484K variant. Competitive passage experiments revealed clear replicative fitness differences. In absence of immune serum, E484A and E484Q, but not E484K, were replaced by wildtype (WT) in competition assays. In presence of immune serum, all three mutants outcompeted WT. Decreased E484A fitness levels were over-compensated for by N501Y and Q498R, identifying a putative Omicron founder background that exceeds the intrinsic and effective fitness of WT and matches that of E484K. Critically, the E484A/Q498R/N501Y mutant and E484K have equal fitness also in presence of pre-Omicron vaccinee serum, whereas the fitness gain by E484K is lost in the presence of serum raised against the E484K variant in hamsters. CONCLUSIONS: The emergence of E484A and E484Q prior to widespread population immunity may have been limited by fitness costs. In populations already exposed to the early immune escape epitope E484K, the Omicron founder background may have provided a basis for alternative immune escape evolution via E484A. Studies of major antigenic epitope changes with and without their epistatic context help reconstruct the sequential adjustments of intrinsic fitness versus neutralization escape during the evolution of major SARS-CoV-2 variants in an increasingly immune human population.

A nanoparticle vaccine displaying the ookinete PSOP25 antigen elicits transmission-blocking antibody response against Plasmodium berghei.

BACKGROUND: Safe and effective vaccines are crucial for the control and eventual elimination of malaria. Novel approaches to optimize and improve vaccine efficacy are urgently required. Nanoparticle-based delivery platforms are considered potent and powerful tools for vaccine development. METHODS: In this study, we developed a transmission-blocking vaccine against malaria by conjugating the ookinete surface antigen PSOP25 to the Acinetobacter phage coat protein AP205, forming virus-like particles (VLPs) using the SpyTag/SpyCatcher adaptor system. The combination of AP205-2*SpyTag with PSOP25-SpyCatcher resulted in the formation of AP205-PSOP25 complexes (VLP-PSOP25). The antibody titers and avidity of serum from each immunization group were assessed by ELISA. Western blot and IFA were performed to confirm the specific reactivity of the elicit antisera to the native PSOP25 in Plasmodium berghei ookinetes. Both in vitro and in vivo assays were conducted to evaluate the transmission-blocking activity of VLP-PSOP25 vaccine. RESULTS: Immunization of mice with VLP-PSOP25 could induced higher levels of high-affinity antibodies than the recombinant PSOP25 (rPSOP25) alone or mixtures of untagged AP205 and rPSOP25 but was comparable to rPSOP25 formulated with alum. Additionally, the VLP-PSOP25 vaccine enhanced Th1-type immune response with remarkably increased levels of IgG2a subclass. The antiserum generated by VLP-PSOP25 specifically recognizes the native PSOP25 antigen in P. berghei ookinetes. Importantly, antisera generated by inoculation with the VLP-PSOP25 could inhibit ookinete development in vitro and reduce the prevalence of infected mosquitoes or oocyst intensity in direct mosquito feeding assays. CONCLUSIONS: Antisera elicited by immunization with the VLP-PSOP25 vaccine confer moderate transmission-reducing activity and transmission-blocking activity. Our results support the utilization of the AP205-SpyTag/SpyCatcher platform for next-generation TBVs development.

Multivariate Analysis as a Method to Evaluate Antigenic Relationships between Bovine Viral Diarrhea Virus 1b Isolates and Vaccine Strains.

The antigenicity of bovine viral diarrhea virus (BVDV) has been evaluated using virus-neutralizing titer data analyzed by principal component analysis (PCA) and has demonstrated numerous isolates to be antigenically divergent from US vaccine strains. The lack of BVDV-1b strains in currently licensed vaccines has raised concerns regarding the lack of protection against BVDV-1b field strains. The aim of this study was to evaluate the antigenic diversity of BVDV-1b strains and better understand the breadth of antigenic relatedness using BVDV-1b antisera and antisera from vaccine strains. Results from this analysis demonstrate the antigenic diversity observed among BVDV-1b isolates and genetic assignment into the BVDV-1b subgenotype is not representative of antigenic relatedness. This is demonstrated by BVDV-1b isolates (2280N, SNc, Illc, MSU, and 2337) observed to be as antigenically dissimilar as BVDV-2a isolates when using BVDV-1b antisera. Additionally, when BVDV-1a vaccine antisera was used for comparisons, a greater percentage of BVDV-1b isolates clustered with BVDV-1a vaccine strains as part of PC1, suggesting antigenic relatedness and potentially partial protection. Collectively, data from this study would suggest that while most BVDV-1b isolates are antigenically similar, there are antigenically dissimilar BVDV-1b isolates as determined by the lack of cross-reactivity, which may contribute to the lack of protection.

Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant, BA.2.86, has emerged and spread to numerous countries worldwide, raising alarm because its spike protein contains 34 additional mutations compared with its BA.2 predecessor1. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was no more resistant to human sera than the currently dominant XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from people who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. Although BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the 'inner face' of the RBD that is exposed only when this domain is in the 'up' position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.

Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.IMPORTANCEPathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

A novel Galleria mellonella experimental model for zoonotic pathogen Brucella.

Brucellosis is a major threat to public health and animal husbandry. Several in vivo vertebrate models, such as mice, guinea pigs, and nonhuman primates, have been used to study Brucella pathogenesis, bacteria-host interactions, and vaccine efficacy. However, these models have limitations whereas the invertebrate Galleria mellonella model is a cost-effective and ethical alternative. The aim of the present study was to examine the invertebrate G. mellonella as an in vivo infection model for Brucella. Infection assays were employed to validate the fitness of the larval model for Brucella infection and virulence evaluation. The protective efficacy of immune sera was evaluated by pre-incubated with a lethal dose of bacteria before infection. The consistency between the mouse model and the larval model was confirmed by assessing the protective efficacy of two Brucella vaccine strains. The results show that G. mellonella could be infected by Brucella strains, in a dose- and temperature-dependent way. Moreover, this larval model can effectively evaluate the virulence of Brucella strains in a manner consistent with that of mammalian infection models. Importantly, this model can assess the protective efficacy of vaccine immune sera within a day. Further investigation implied that haemolymph played a crucial role in the protective efficacy of immune sera. In conclusion, G. mellonella could serve as a quick, efficient, and reliable model for evaluating the virulence of Brucella strains and efficacy of immune sera in an ethical manner.

Immuno-affinity chromatography for purification of IgG from hyper-immune sera raised against 146S fraction of Foot and Mouth Disease Virus for diagnostic purposes.

Immunoaffinity chromatography (IAC) is a fundamental isolation and purification tool which is incorporated in a substantial range of therapeutic and diagnostic applications. This study has reappraised the usefulness of immunoaffinity chromatography for the purification of polyclonal antibodies. Protein A based IAC is a convenient and reliable method for purification of IgG, from hyperimmunesera (HIS) raised in experimental animals such as rabbits, guinea pigs and mice to be utilized in pharmaceutics and diagnostics. The 146S fraction of Foot and Mouth Disease virus (FMDV) TCID50=10 5.6 was cultured on a baby hamster kidney cell line 21 (BHK-21), concentrated using salt precipitation method using PEG 6000, purified by size exclusion chromatography (SEC) using Sepharose-30 at 254nm absorbance. Purification of 146S FMDV was analyzed using 12% SDS-PAGE which provided two bands of light and heavy chains. The alum-based vaccine, consisting of ≥10µg of 146S FMDV, was applied in 10 male rabbits and 10 male guinea pigs and two animals of each group were taken as a negative control. The titer of serum was calculated using virus neutralization test. A Protein-A kit (Thermo scientific- 44667, 0528.2) was used to purify HIS raised against 146S FMDV and validated using 12% SDS PAGE in reducing condition. The data demonstrate that protein-A affinity chromatography is an efficient tool for the purification of antibodies from hyper-immune sera raised against 146S FMDV and can be used for the production of diagnostic kits e.g. Enzyme linked immuno-sorbent assay (ELISA) and radioimmunoassay.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Introduction: Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (ß-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded ß-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Methods: Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Results: Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Discussion: Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

Potential of a bivalent vaccine for broad protection against enterovirus 71 and coxsackie virus 16 infections causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a highly contagious viral infection that is mainly caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16). As there are no specific therapeutics for HFMD, the development of a bivalent vaccine is required to cover a broad range of infections. In this study, the effectiveness of novel monovalent and bivalent vaccines targeting EV71 C4a and CVA16 was investigated for their ability to prevent viral infections in neonatal human scavenger receptor class B member 2 (hSCARB2) transgenic mice. As hSCARB2 serves as a key viral receptor for EV71, these transgenic mice are susceptible to EV71 strains and facilitate viral binding, internalization, and uncoating processes. Antisera prepared by vaccine immunization were transferred to 2-day-old hSCARB2 transgenic mice, which were then infected with EV71 C4a or CVA16 virus. The antisera generated by each monovalent or bivalent vaccine effectively protected against EV71 C4a and CVA16 infections. The examination of tissue damage and viral contents in various organs indicated that both monovalent and bivalent antisera reduced EV71 C4a viral load in the brainstem, and no significant tissue damage was observed. During CVA16 infection, the monovalent and bivalent antisera significantly reduced viral contents in both the brainstem and muscles. These results suggest that passive immunity by monovalent and bivalent antisera can effectively protect against EV71 C4a and CVA16 infections. Thus, the development of a bivalent vaccine that can provide broad protection against both CV and EV infections may be a promising strategy in preventing HFMD.

Screening and detection of multivalent human papillomavirus antibodies using a high-throughput liquid chip fluoroimmunoassay system.

Immunoassays are commonly used in disease diagnosis and vaccine evaluation but can be costly and time-consuming when confronted with multivalent targets, such as antisera containing antibodies to human papillomavirus (HPV), because of their limited ability to discriminate between multiple analytes in a single reaction well. This study describes the development of a high-throughput liquid chip system that combines immunoassay techniques and magnetic beads to allow the simultaneous screening and quantitative detection of antibodies to four types of HPV using the Luminex fluoroimmunoassay system. Groups of beads embedded with fluorescent dyes at various ratios were coated with optimized HPV capture antigens and demonstrated excellent dose-dependent response to four monoclonal antibodies used as reference standards. This assay is sensitive, accurate, repeatable, and simple to perform, enabling multiplex antibody detection with a high degree of orthogonality. The performance of the Luminex system was compared with conventional immunoassays for quantitative detection of quadrivalent HPV antibodies in antisera of mice immunized with five lots of HPV vaccines, verifying the accuracy and detection efficiency of the assay. This strategy is a promising approach to characterizing antibodies present in polyclonal antisera and has promising applications in research, clinical, and industrial settings, for example, streamlining vaccine efficacy trials and vaccine lot inspection and release procedures.

The CombE-IDMS Assay as an Alternate Potency Method for Adjuvanted Quadrivalent Influenza Vaccines.

The potency of all currently licensed inactivated influenza viral vaccines is assayed by the single radial immunodiffusion (SRID) method. SRID relies upon antisera and reference antigen reagents which are produced, standardized, and distributed in the mass quantities needed for vaccine manufacturers only after a significant amount of time has elapsed from the seasonal strain recommendations issued by the WHO; this time delay is exacerbated under conditions of an emerging pandemic. Previously, the limited trypsin digestion isotope dilution mass spectrometry (LTD-IDMS) method, which does not require antisera or reference antigens, demonstrated comparable quantitation of immunologically active hemagglutinin, the primary viral antigen, to SRID in stressed vaccine materials. Here, we demonstrate a streamlined improvement to the LTD-IDMS method by eliminating the need for its precipitation and washing steps, saving time and labor in the sample preparation process while paving the way for plate-based high-throughput analysis. This is accomplished using dissimilar proteases in the pretreatment (a combination of chymotrypsin and elastase) and analytical (trypsin) digestion steps so that any pretreatment digests will not cause interference while monitoring analytical tryptic digests by IDMS. The combination of enzymes (CombE)-IDMS method is tested alongside LTD-IDMS and SRID for the first time on MF59 adjuvanted seasonal cell-based quadrivalent influenza vaccines (aQIVc) under stressed conditions of heating, oxidation, lowered and elevated pH, and freeze-thaw. Overall, a correlation in the degradation trend is observed between CombE-IDMS and SRID in the four strains of the quadrivalent formulation, highlighting the method's stability indicating capability as a rapid alternate potency assay in a highly complex formulation of aQIVc.

Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of the medically most important Naja spp. African snakes.

BACKGROUND: Envenomations by African snakes represent a high burden in the sub-Sahara region. The design and fabrication of polyspecific antivenoms with a broader effectiveness, specially tailored for its use in sub-Saharan Africa, require a better understanding of the immunological features of different Naja spp. venoms of highest medical impact in Africa; and to select the most appropriate antigen combinations to generate antivenoms of wider neutralizing scope. METHODOLOGY/PRINCIPAL FINDINGS: Rabbit-derived monospecific antisera were raised against the venoms of five spitting cobras and six non-spitting cobras. The effects of immunization in the animal model were assessed, as well as the development of antibody titers, as proved by immunochemical assays and neutralization of lethal, phospholipase A2 and dermonecrotic activities. By the end of the immunization schedule, the immunized rabbits showed normal values of all hematological parameters, and no muscle tissue damage was evidenced, although alterations in aspartate aminotransferase (AST) and alkaline phosphatase (ALP) suggested a degree of hepatic damage caused mainly by spitting cobra venoms. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within the spitting and no-spitting cobras, yet some antisera showed more extensive cross-reactivity than others. The antisera with the widest coverage were those of anti-Naja ashei and anti-N. nigricollis for the spitting cobras, and anti-N. haje and anti-N. senegalensis for the non-spitting cobras. CONCLUSIONS/SIGNIFICANCE: The methods and study design followed provide a rationale for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against cobra venoms in sub-Saharan Africa. Results suggest that venoms from N. ashei, N. nigricollis within the spitting cobras, and N. haje and N. senegalensis within the non-spitting cobras, generate antisera with a broader cross-reactivity. These experimental results should be translated to larger animal models used in antivenom elaboration to assess whether these predictions are reproduced.