Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has developed substantial antigenic variability. As the majority of the population now has pre-existing immunity due to infection or vaccination, the use of experimentally generated animal immune sera can be valuable for measuring antigenic differences between virus variants. Here, we immunized Syrian hamsters by two successive infections with one of nine SARS-CoV-2 variants. Their sera were titrated against 16 SARS-CoV-2 variants, and the resulting titers were visualized using antigenic cartography. The antigenic map shows a condensed cluster containing all pre-Omicron variants (D614G, Alpha, Delta, Beta, Mu, and an engineered B.1+E484K variant) and considerably more diversity among a selected panel of Omicron subvariants (BA.1, BA.2, BA.4/BA.5, the BA.5 descendants BF.7 and BQ.1.18, the BA.2.75 descendant BN.1.3.1, the BA.2-derived recombinants XBB.2 and EG.5.1, and the BA.2.86 descendant JN.1). Some Omicron subvariants were as antigenically distinct from each other as the wildtype is from the Omicron BA.1 variant. Compared to titers measured in human sera, titers in hamster sera are of higher magnitude, show less fold change, and result in a more compact antigenic map topology. The results highlight the potential of sera from hamsters for the continued antigenic characterization of SARS-CoV-2.

Flagellum-mediated motility is essential to Pseudomonas aeruginosa (P. aeruginosa) virulence. Antibody against flagellin reduces motility and inhibits the spread of the bacteria from the infection site. The standard soft-agar assay to demonstrate anti-flagella motility inhibition requires long incubation times, is difficult to interpret, and requires large amounts of antibody. We have developed a time-lapse video microscopy method to analyze anti-flagellin P. aeruginosa motility inhibition that has several advantages over the soft agar assay. Antisera from mice immunized with flagellin type A or B were incubated with Green Fluorescent Protein (GFP)-expressing P. aeruginosa strain PAO1 (FlaB+) and GFP-expressing P. aeruginosa strain PAK (FlaA+). We analyzed the motion of the bacteria in video taken in ten second time intervals. An easily measurable decrease in bacterial locomotion was observed microscopically within minutes after the addition of small volumes of flagellin antiserum. From data analysis, we were able to quantify the efficacy of anti-flagellin antibodies in the test serum that decreased P. aeruginosa motility. This new video microscopy method to assess functional activity of anti-flagellin antibodies required less serum, less time, and had more robust and reproducible endpoints than the standard soft agar motility inhibition assay.

BACKGROUND: Snakebite envenomation inflicts a high burden of mortality and morbidity in sub-Saharan Africa. Antivenoms are the mainstay in the therapy of envenomation, and there is an urgent need to develop antivenoms of broad neutralizing efficacy for this region. The venoms used as immunogens to manufacture snake antivenoms are normally selected considering their medical importance and availability. Additionally, their ability to induce antibody responses with high neutralizing capability should be considered, an issue that involves the immunization scheme and the animal species being immunized.
RESULTS: Using the lethality neutralization assay in mice, we compared the intrageneric neutralization scope of antisera generated by immunization of horses with monospecific, bispecific/monogeneric, and polyspecific/monogeneric immunogens formulated with venoms of Bitis spp., Echis spp., Dendroaspis spp., spitting Naja spp. or non-spitting Naja spp. It was found that the antisera raised by all the immunogens were able to neutralize the homologous venoms and, with a single exception, the heterologous congeneric venoms (considering spitting and non-spitting Naja separately). In general, the polyspecific antisera of Bitis spp, Echis spp, and Dendroaspis spp gave the best neutralization profile against venoms of these genera. For spitting Naja venoms, there were no significant differences in the neutralizing ability between monospecific, bispecific and polyspecific antisera. A similar result was obtained in the case of non-spitting Naja venoms, except that polyspecific antiserum was more effective against the venoms of N. melanoleuca and N. nivea as compared to the monospecific antiserum.
CONCLUSIONS: The use of polyspecific immunogens is the best alternative to produce monogeneric antivenoms with wide neutralizing coverage against venoms of sub-Saharan African snakes of the Bitis, Echis, Naja (non-spitting) and Dendroaspis genera. On the other hand, a monospecific immunogen composed of venom of Naja nigricollis is suitable to produce a monogeneric antivenom with wide neutralizing coverage against venoms of spitting Naja spp. These findings can be used in the design of antivenoms of wide neutralizing scope for sub-Saharan Africa.

BACKGROUND: 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.
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.
CONCLUSIONS: 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.

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.

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.

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.

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.
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.
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.

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.

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.
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.
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.
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.