Immunization Of Rabbits Research Articles

Evaluation of antibody responses against the whole virions of goatpox and sheeppox viruses after subcutaneous immunization of rabbits

Antibody development is the integral process of generating and characterizing an antibody. It commences by inoculating the antigen of interest into laboratory animals, allowing the immune system develops large quantities of antibodies. This was aimed at developing antibodies against the virion of Goatpox and Sheeppox virus vaccines. The ability of Goatpox and Sheeppox vaccines was assessed. Regarding this study, the antibody titers against both Goatpox and Sheeppox viruses was increased in the same manner. The amount of IgG was determined to be 2.29 μg/μl and 2.18 μg/μl against virions of Goatpox virus and Sheeppox respectively. The purified IgG was analyzed by SDS-PAGE. Different bands of the purified antibodies were clearly visualized, and the molecular weight of IgG was estimated to be 67 kDa and 25 kDa. Additionally, antigen/antibody binding was confirmed by Western blot using GTPV A27 antigen. No significant differences in antibody titers were observed between the two groups (p < 0, 05).

Efficacy of an accelerated vaccination schedule against hepatitis E virus infection in pregnant rabbits.

An important goal of the Hepatitis E virus (HEV) vaccine is to prevent adverse pregnancy outcomes caused by different HEV genotypes during pregnancy, but studies directly evaluating maternal vaccination for HEV are lacking. Here we report maternal vaccination using HEV 239 vaccine in a pregnant rabbit model. Two dose of accelerated vaccination schedule (0, 7 days) induced high titers of anti-HEV protective antibodies in a short period of time in pregnant rabbits, which could protect the pregnant rabbits from HEV infection and adverse pregnancy outcomes. In addition, the immunized rabbits transfer maternal antibodies to pups through the placenta and breast milk, which protect neonates against HEV infection. Our results suggest that, besides vaccinating nonpregnant individuals, HEV 239 vaccine may also be discreetly considered for maternal vaccination.

Plasmodium knowlesi Duffy binding protein alpha region II (PkDBPαII) in clinical isolates from Peninsular Malaysia and Malaysian Borneo exhibit different immune responses in animal models

Plasmodium knowlesi utilizes the Duffy binding protein alpha (PkDBPα) to facilitate its invasion into human erythrocytes. PkDBPα region II (PkDBPαII) from Peninsular Malaysia and Malaysian Borneo has been shown to occur as distinct haplotypes, and the predominant haplotypes from these geographical areas demonstrated differences in binding activity to human erythrocytes in erythrocyte binding assays. This study aimed to determine the effects of genetic polymorphisms in PkDBPαII to immune responses in animal models. The recombinant PkDBPαII (~ 45kDa) of Peninsular Malaysia (PkDBPαII-H) and Malaysian Borneo (PkDBPαII-S) were expressed in a bacterial expression system, purified, and used in mice and rabbit immunization. The profile of cytokines IL-1ra, IL-2, IL-6, IL-10, TNF-α, and IFN-γ in immunized mice spleen was determined via ELISA. The titer and IgG subtype distribution of raised antibodies was characterized. Immunized rabbit sera were purified and used to perform an in vitro merozoite invasion inhibition assay. The PkDBPαII-immunized mice sera of both groups showed high antibody titer and a similar IgG subtype distribution pattern: IgG2b > IgG1 > IgG2a > IgG3. The PkDBPαII-H group was shown to have higher IL-1ra (P = 0.141) and IL-6 (P = 0.049) concentrations, with IL-6 levels significantly higher than that of the PkDBPαII-S group (P ≤ 0.05). Merozoite invasion inhibition assay using purified anti-PkDBPαII antibodies showed a significantly higher inhibition rate in the PkDBPαII-H group than the PkDBPαII-S group (P ≤ 0.05). Besides, anti-PkDBPαII-H antibodies were able to exhibit inhibition activity at a lower concentration than anti-PkDBPαII-S antibodies. PkDBPαII was shown to be immunogenic, and the PkDBPαII haplotype from Peninsular Malaysia exhibited higher responses in cytokines IL-1ra and IL-6, antibody IgM level, and merozoite invasion inhibition assay than the Malaysian Borneo haplotype. This suggests that polymorphisms in the PkDBPαII affect the level of immune responses in the host.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

Characterization of somatic and metabolic antigens of Cysticercus cellulosae and determination of immunodominant proteins

The present study shows the characterization of four somatic antigens (Whole Cyst Antigen (WCA), Cystic Fluid Antigen (CFA), Scolex Antigen (SA) and Membrane-Body Antigen (MBA)) and one metabolic antigen (Excretory-Secretory Antigen (ESA)) prepared from Cysticercus cellulosae originated from a naturally infected pig. Immunodominat proteins were determined using hyper immune rabbit sera. The Sodium Dodecyl SulphatePolyacrylamide Gel Electrophoresis (SDS-PAGE) profile of the antigens revealed different numbers and patterns of protein bands in the range of 11.80 to 176.74 kDa. The electrophoretic pattern of ESA also revealed a number of closely placed indistinct bands in the range of 20-34 kDa.. The proteins in the lower molecular weight range (&lt; 30 kDa) in the case of WCA, CFA, ESA, and the medium molecular weight range (30-60 kDa) in the case of SA and MBA were observed to be immunodominant in the Enzyme-Linked Immunoelectrotransfer Blot (EITB) assay. These immunodominant proteins could be further investigated as potential diagnostic antigens in the serodiagnosis of cysticercosis in pigs, and neurocysticercosis in human patients.

D-ribose-mediated glycation of fibrinogen: Role in the induction of adaptive immune response

A nonenzymatic reaction between reducing sugars and amino groups of proteins results in the formation of advanced glycation end products, which are linked to a number of chronic progressive diseases with macro- and microvascular complications. In this research, we sought to ascertain the immunological response to d-ibose-glycated fibrinogen. New Zealand White female rabbits were immunized with native and d-ribose-glycated (Rb-gly-Fb) fibrinogen and used for studying the immunological response. Serum from these rabbits analyzed using direct binding and competitive inhibition ELISA was found to contain a high titer of antibodies against Rb-gly-Fb; Rb-gly-Fb was much more immunogenic than its native form. The IgG against Rb-gly-Fb (Rb-gly-Fb-IgG) was highly specific against the immunogenic protein. Moreover, histopathology and immunofluorescence studies revealed the deposition of the Rb-gly-Fb-IgG immune complex in the glomerular basement membrane of the kidneys of immunized rabbits. Furthermore, immunization with Rb-gly-Fb increased the expression of genes encoding proinflammatory cytokines, tumour necrosis factor α, interleukin-6, interleukin-1β, and interferon-gamma, which is indicative of increased inflammation and the antigenic role of Rb-gly-Fb in provoking an immune response.

Intranasal immunization with recombinant Vaccinia virus encoding trimeric SARS-CoV-2 spike receptor-binding domain induces neutralizing antibody

Respiratory transmission of SARS-CoV-2 is considered to be the major dissemination route for COVID-19, therefore, mucosal immune responses have great importance in preventing SARS-CoV-2 from infection. In this study, we constructed a recombinant Vaccinia virus (VV) harboring trimeric receptor-binding domain (RBD) of SARS-CoV-2 spike protein (VV-tRBD), and evaluated the immune responses towards RBD following intranasal immunization against mice and rabbits. In BALB/c mice, intranasal immunization with VV-tRBD elicited robust humoral and cellular immune responses, with high-level of both neutralizing IgG and IgA in sera against SARS-CoV-2 psudoviruses, and a number of RBD-specific IFN-γ-secreting lymphocytes. Sera from immunized rabbits also exhibited neutralization effects. Notably, RBD-specific secretory IgA (sIgA) in both nasal washes and bronchoalveolar lavage fluids (BALs) were detectable and showed substantial neutralization activities. Collectively, a recombinant VV expressing trimeric RBD confers robust systemic immune response and mucosal neutralizing antibodies, thus warranting further exploration as a mucosal vaccine.

Effects of enzymolytic soybean meal on the growth performance, digestive enzyme activity, some serum indexes, carcase performance and meat quality of Rex rabbits

The purpose of this study was to investigate the effects of enzymolytic soybean meal (ESBM) on growth performance, digestive enzyme activity, serum biochemical parameters, immunity, carcase performance and meat quality of Rex rabbits. One hundred and twenty healthy weaned Rex rabbits were randomly divided into four groups (30 replicates per group and one rabbit per replicate). The control group was fed the basal diet and the ESBM treated groups (T1, T2 and T3) were fed 0.5%, 1% and 1.5% ESBM instead of the equivalent amount of soybean meal (SBM) in the basal diet, respectively. The study lasted 61 days, including a pre-feeding period of 5 days and a formal trial period of 56 days. Compared with the control group, the feed to weight ratio was significantly decreased in both T2 and T3 groups (p < 0.05). The α-amylase and trypsin activities of the caecum and serum albumin content were significantly increased in the T3 group (p < 0.05). The serum lysozyme activity was significantly increased in all treatment groups (p < 0.05). The full eviscerated slaughter ratio and half eviscerated slaughter ratio were significantly increased in the T1 group (p < 0.05). There were no significant differences in other indexes (p > 0.05). In conclusion, the replacement of SBM with ESBM (1.5%) in the diet can reduce the feed to weight ratio, increase the activity of some digestive enzymes, improve carcase performance, and enhance immunity of Rex rabbits. HIGHLIGHTS ESBM improved growth performance, gastrointestinal digestive enzyme activity and carcase performance of Rex rabbits. ESBM increased serum lysozyme activity and albumin content of Rex rabbits. ESBM could be used as a new kind of feed additive for Rex rabbits.

Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages

A vaccine targeting multiple stages of the Plasmodium falciparum parasite life cycle is desirable. The sporozoite surface Circumsporozoite Protein (CSP) is the target of leading anti-infective P. falciparum pre-erythrocytic vaccines. Pfs230, a sexual-stage P. falciparum surface protein, is currently in trials as the basis for a transmission-blocking vaccine, which inhibits parasite development in the mosquito vector. Here, recombinant full-length CSP and a Pfs230 fragment (Pfs230D1+) are co-displayed on immunogenic liposomes to induce immunity against both infection and transmission. Liposomes contain cobalt-porphyrin phospholipid (CoPoP), monophosphoryl lipid A and QS-21, and rapidly bind His-tagged CSP and Pfs230D1+ upon admixture to form bivalent particles that maintain reactivity with conformational monoclonal antibodies. Use of multicolor fluorophore-labeled antigens reveals liposome binding upon admixture, stability in serum and enhanced uptake in murine macrophages in vitro. Bivalent liposomes induce humoral and cellular responses against both CSP and Pfs230D1+. Vaccine-induced antibodies reduce parasite numbers in mosquito midguts in a standard membrane feeding assay. Mice immunized with liposome-displayed antigens or that passively receive antibodies from immunized rabbits have reduced parasite liver burden following challenge with transgenic sporozoites expressing P. falciparum CSP.

The recombinant Erns and truncated E2-based indirect enzyme-linked immunosorbent assays to distinguishably test specific antibodies against classical swine fever virus and bovine viral diarrhea virus

BackgroundClassical swine fever (CSF) virus is the causative agent of an economically important, highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV). BVDV infection in pigs can mimic CSF clinical signs, which cause difficulty in differentiation. Serological test for detection of virus specific antibodies is a valuable tool for diagnosis and surveillance of CSFV and BVDV infections in animals. The aim of this study was to develop the CSFV Erns and BVDV tE2 -based ELISAs to distinguishably test specific antibodies against CSFV and BVDV.MethodsThe CSFV Erns and truncated E2 (tE2, residues 690–865) of BVDV were expressed in E. coli and purified by Ni–NTA affinity chromatography, respectively. Employing Erns or tE2 protein as diagnostic antigen, indirect ELISAs were developed to distinguishably test specific antibodies against CSFV and BVDV. The specificity and sensitivity of ELISAs were evaluated using a panel of virus specific sera of pigs, immunized rabbits and immunized mice. A total 150 clinical serum samples from farm pigs were measured by the developed ELISAs and compared with virus neutralizing test (VNT).ResultsIndirect ELISA was established based on recombinant CSFV Erns or BVDV tE2 protein, respectively. No serological cross-reaction between antibodies against CSFV and BVDV was observed in sera of immunized rabbits, immunized mice or farm pigs by detections of the Erns and tE2 -based ELISAs. Compared to VNT, the CSFV Erns -based ELISA displayed a high sensitivity (93.3%), specificity (92.0%) and agreement rate (92.7%), and the sensitivity, specificity and agreement rate of BVDV tE2 -based ELISA was 92.3%, 95.2% and 94.7%, respectively.ConclusionThe newly developed ELISAs are highly specific and sensitive and would be valuable tools for serological diagnosis for CSFV and BVDV infections.

Factors of cell immunity under exposure to the Eimeria spp

One of the actual problems in farming rabbits is a decrease of their resistance, that is often conditioned by the availability of eimeriosis pathogens, which is significantly widespread both in foreign countries and in Ukraine. A lot of researchers studied the particular qualities of the development of prothymeriosis immunity, the role of lymphocyte populations, and the antigenic composition at various stages of development of eimeria. The aim of the research was to study the influence of the association of pathogens Eimeria sрp. with different levels of invasion intensity on indicators of cellular immunity of rabbits. For the experiments, analogue groups of male rabbits of the Californian breed aged 3-5 months were selected. During scatological research, we have found that rabbits with eimeriosis had different levels of invasion intensity (II), according to which the animals were divided into three groups: I - low level of invasion intensity (II = 1838.89 ± 1114.68 oocysts in 1 g of feces), II - medium level (II = 39787.50 ± 13422.34 oocysts in 1 g of feces) and group III - high level (II = 88578.57 ± 17776.32 oocysts in 1 g of feces). In the blood of all rabbits with eimeriosis (association of the pathogens Eimeria magna, E. media, E. perforans, E. stiedae) there were leukocytosis, absolute lymphocytosis against the background of relative segmented neutropenia regardless of the intensity of invasion. At the same time, in the blood of animals of all experimental groups, in comparison with the control, the number of eosinophils was significantly (p &lt;0.001) higher in absolute and percentage (p &lt;0.05) values. The same pattern has been found for basophiles. The absolute number of T- and B-lymphocytes, T-helpers and T-active lymphocytes was significantly higher compared to a low percentage of O-lymphocytes. So, if sick rabbits have pathogens of Eimeria spp., the defense mechanisms of innate and adaptive immunity with the participation of blood cells are activated. With an increase in the intensity of eimeriosis invasion in the blood of sick animals, the number of leukocytes, eosinophils and basophils increases against the background of a decrease in the percentage of segmented neutrophils. In the blood of rabbits with eimeriosis, a higher percentage of B-lymphocytes was noted, which had a weak positive correlation (r=0.28) with the level of invasion intensity, and a smaller number of O-lymphocytes. Key words: eimeriosis, leukogram, T-lymphocyte, B-lymphocyte, О-lymphocyte, T-helper, T-suppressor, T-active lymphocyte.

Dietary Moringa oleifera leaf powder improves jejunal permeability and digestive function by modulating the microbiota composition and mucosal immunity in heat stressed rabbits.

Heat stress (HS) has detrimental effects on intestinal health by altering digestive and immune responses in animals. Dietary Moringa oleifera leaf powder (MOLP) has been implicated in ameliorating the impact of HS, but its effects in terms of intestinal function improvement under HS remain poorly characterized. Therefore, the current study investigated the impact of HS and MOLP supplementation on tight junction barriers, intestinal microbiota (jejunal digesta), and differentially expressed genes (DEGs) in jejunal mucosa of heat-stressed rabbits by using the next-generation sequencing techniques. A total of 21 male New Zealand White rabbits (32weeks old mean body weight of 3318 ± 171g) were divided into three groups (n = 7/group) as control (CON, 25°C), heat stress (HS, 35°C for 7h daily), and HS with MOLP supplementation (HSM, 35°C for 7h daily) gavage at 200mg/kg body weight per day for 4weeks. The results indicated that MOLP supplementation increased mRNA expression of tight junction proteins and glutathione transferase activity, while the malonaldehyde concentration was decreased in the jejunal mucosa compared to HS group (P < 0.05). Furthermore, MOLP decreased the concentrations of lipopolysaccharide, pro-inflammatory cytokines, and myeloperoxidase compared with HS group (P < 0.05). Intestinal microbiota analysis revealed that at phyla level, the relative abundance of Bacteroidetes was higher in HSM group compared to CON and HS groups. MOLP supplementation also resulted in higher abundance of putatively health-associated genera such as Christensenellaceae R-7 gut group, Ruminococcaceae NK4A214 group, Ruminococcus 2, Lachnospiraceae NK4A136 group, and Lachnospiraceae unclassified along with higher butyrate levels in HSM group as compared to HS group. The analysis of DEGs revealed that MOLP reversed inflammatory response by downregulation of genes, such as TNFRSF13C, LBP, and COX2 in enriched KEGG pathway of NF-kβ pathway. MOLP supplementation also significantly upregulated the expression of genes in protein digestion and absorption pathway, including PRSS2, LOC100349163, CPA1, CPB1, SLC9A3, SLC1A1, and SLC7A9 in HSM group. Three genes of fibrillar collagens, i.e., COL3A1, COL5A3, and COL12A1 in protein digestion were also down-regulated in HSM group. In conclusion, MOLP supplementation could improve jejunal permeability and digestive function, positively modulate microbiota composition and mucosal immunity in heat-stressed rabbits.

Quantitative proteomics analysis reveals core and variable tick salivary proteins at the tick-vertebrate host interface.

Few studies have examined tick proteomes, how they adapt to their environment, and their roles in the parasite-host interactions that drive tick infestation and pathogen transmission. Here we used a proteomics approach to screen for biologically and immunologically relevant proteins acting at the tick-host interface during tick feeding and, as proof of principle, measured host antibody responses to some of the discovered candidates. We used a label-free quantitative proteomic workflow to study salivary proteomes of (i) wild Ixodes ricinus ticks fed on different hosts, (ii) wild or laboratory ticks fed on the same host, and (iii) adult ticks cofed with nymphs. Our results reveal high and stable expression of several protease inhibitors and other tick-specific proteins under different feeding conditions. Most pathways functionally enriched in sialoproteomes were related to proteolysis, endopeptidase, and amine-binding activities. The generated catalogue of tick salivary proteins enabled the selection of six candidate secreted immunogenic peptides for rabbit immunizations, three of which induced strong and durable antigen-specific antibody responses in rabbits. Furthermore, rabbits exposed to ticks mounted immune responses against the candidate peptides/proteins, confirming their expression at the tick-vertebrate interface. Our approach provides insights into tick adaptation strategies to different feeding conditions and promising candidates for developing antitick vaccines or markers of exposure of vertebrate hosts to tick bites.

Evaluation of New Polyclonal Antibody Developed for Serological Diagnostics of Tomato Mosaic Virus.

Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to the vector-less transmission of ToMV. Resistant breeding generally has low effectiveness. The most practical approach is the use of a rapid diagnostic assay of the virus’ presence before the symptoms occur in plants, followed by the eradication of virus-infected plants. Such approaches also include serological detection methods (ELISA and Western immunoblotting), where antibodies need to be developed for an immunochemical reaction. The development and characterization of polyclonal antibodies for the detection of ToMV with appropriate parameters (sensitivity, specificity, and cross-reactivity) were the subjects of this study. A new polyclonal antibody, AB-1, was developed in immunized rabbits using the modified oligopeptides with antigenic potential (sequences are revealed) derived from the coat protein of ToMV SL-1. the developed polyclonal antibody. AB-1, showed higher sensitivity when compared with commercially available analogs. It also detected ToMV in infected pepper and eggplant plants, and detected another two tobamoviruses (TMV and PMMoV) and ToMV in soil rhizosphere samples and root residues, even two years after the cultivation of the infected tomato plant.

Abstract 323: Generation and application of a novel monoclonal antibody to UBQLN4 to characterize the dsDNA damage response

Abstract Ubiquilin-4 (UBQLN4) is a member of the ubiquilin protein family whose primary function is to maintain protein homeostasis in cells. Recent research indicates that UBQLN4 contributes to genomic stability through interactions with ubiquitylated MRE11. In a dysregulated state, UBQLN4 can shift double-strand break repair toward the homologous recombination repair (HRR) pathway or canonical non-homologous end joining (c-NHEJ) pathway. The latter pathway is associated with an overexpression of UBQLN4 and can be suggestive of a sensitivity to PARP1 inhibitors. As with any protein with diagnostic or therapeutic implications, having trustworthy antibodies is required to generate actionable and repeatable data. To help standardize the validation of these antibodies, the International Working Group for Antibody Validation was created in correlation with a 2016 Nature publication by Uhlen et al. In this publication, a series of “validation pillars” were proposed to give guidance on validating antibodies for scientific use. In response to that publication, we have institutionally adapted that language and many of those principles to supplement our existing validation scheme of antibodies produced on site, including a novel monoclonal antibody against UBQLN4. Four UBQLN4 peptides or full-length UBQLN4 were synthesized and purified internally and used for rabbit immunization, B-cell sorting, and screening for activity via ELISA. Variable regions were sequenced to avoid clonal redundancy, and a subset of these clones were selected to be characterized via a strip-style western blot. Clones were also assessed by immunohistochemistry (IHC) using tissue microarrays with multiple tissue and cell types, and specificity was determined via reciprocal IPs and western blot. Over 200 clones were sequenced and screened by ELISA. These results allowed us to narrow the set down to 153 clones that were screened by strip-style western blot. 74 of these were assessed by IHC, 9 clones were screened by reciprocal IPs, and finally, 6 clones by western blot. Specificity was assessed throughout the process by the utilizing independent antibodies, complementary assays, a UBQLN4 knockout cell line, and UBQLN4-overexpressing cells. Over the course of narrowing this clone set from 200 clones to a single antibody, we were able to meet the pillar requirements outlined in our validation scheme, empowering us to identify a superior antibody useable in several assays, including WB, IP, IHC, and ICC. Going forward, to further detail the use of this target's role in DNA repair, multiplex immunofluorescent studies will be performed in conjunction with Co-IP to identify additional protein binding partners unique to the DNA repair process. Citation Format: Brian D. McWilliams, Mike Spencer. Generation and application of a novel monoclonal antibody to UBQLN4 to characterize the dsDNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 323.

Characterization of rabbit polyclonal antibody against camel recombinant nanobodies.

Nanobodies (Nbs) are recombinant single-domain fragments derived from camelids’ heavy-chain antibodies (HCAbs). Nanobodies are increasingly used in numerous biotechnological and medical applications because of their high stability, solubility, and yield. However, one major obstacle prohibiting Nb expansion is the affordability of specific detector antibodies for their final revelation. In this work, the production of a specific anti-Nb antibody as a general detector for camel antibodies, conventional cIgG, and HCAb, and their derived Nbs was sought. Thus, a T7 promoter plasmid was constructed and used to highly express six different Nbs that were used in a successful rabbit immunization. Affinity-purified rabbit anti-Nb rIgG was able to detect immobilized or antigen-bound Nbs via enzyme-linked immunosorbent assay, and its performance was comparable to that of a commercial anti-6× His antibody. Its capacities in dosing impure Nbs, detecting Nbs displayed on M13 phages, and revealing denatured Nbs in immune blotting were all proven. As expected, and because of shared epitopes, rabbit anti-Nb cross-reacted with cIgG, HCAbs, and 6× His-tagged proteins, and the percentage of each fraction within anti-Nb rIgG was determined. Anti-Nb is a promising tool for the checkpoints throughout the recombinant Nb technology.

Membrane location of cardiolipin antigen in Treponema pallidum: further study on the origin of nontreponemal antibodies.

Aim: The present study examined the membrane location of cardiolipin antigen in treponemes. Materials & methods: The authors used different methods to disrupt the outer membrane of treponemes, detected the location of the cardiolipin antigen and analyzed the immune response in rabbits immunized with various antigens. Results: All organisms were labeled with nontreponemal antibodies on immunoelectron and fluorescence microscopy, except the citrate buffer-treated group, which is a method leading to relatively complete removal. Except for citrate buffer-treated spirochetes, all treponemes produced low-titer, nontreponemal antibodies in immunized rabbits. Conclusion: These findings indicated that the cardiolipin antigen was localized in the outer membrane of spirochetes. This study provided further evidence of the origin of nontreponemal antibodies during Treponema pallidum infection.

Immunization of Rabbits with a Quadrivalent Shigella Bioconjugate Vaccine Induces Functional Antibodies Reactive with Shigella Isolates from Kenya.

ABSTRACTDiarrheal diseases are a leading cause of global morbidity and mortality, disproportionately affecting children in resource-limited settings. Although improvements in hygiene and access to clean water are helpful, vaccines are considered essential due to the low infectious dose of Shigella species and increasing antibiotic resistance. Building on achievements with conjugate vaccines, a safe and immunogenic novel bioconjugate vaccine linking Shigella O-antigen to Pseudomonas aeruginosa exoprotein A has been developed to induce immunity against Shigella flexneri 2a, 3a, and 6 and S. sonnei. This study evaluated the breadth of reactivity and functionality of pooled serum from rabbits immunized with monovalent and quadrivalent Shigella bioconjugates formulated with or without an adjuvant against Shigella serotypes isolated in Kenya. Rabbit sera were assayed by colony blot for reactivity with 67 isolates of Shigella serotypes targeted by the vaccine, S. flexneri (2a, 3a, and 6) and S. sonnei, and 42 isolates of Shigella serotypes not targeted by the vaccine, S. flexneri (1b, 2b, 4a, and 4b), S. boydii, and S. dysenteriae. Shigella isolates testing positive in the colony blot assay were then used to assess functional activity using a bactericidal assay. Of the 41 Shigella isolates targeted by the vaccine, 22 were reactive with the adjuvanted quadrivalent and the respective monovalent rabbit sera. The S. flexneri 2a and 3a monovalent rabbit serum cross-reacted with S. flexneri 3a, 2b, and 2a, respectively. Immunization with the adjuvanted quadrivalent vaccine also induced cross-reactivity with isolates of S. flexneri 2b, 4a, and 4b. Collectively, these results suggest that the Shigella quadrivalent vaccine may be more broadly protective than designed, offering a promising solution to Shigella infections.IMPORTANCE Diarrheal diseases are the third leading cause of death globally, disproportionally affecting low- to middle-income countries like Kenya, with Shigella species being the leading cause of bacterial diarrhea, especially in children. The low infectious dose and high antibiotic resistance levels complicate treatment, leading to long-term sequelae that necessitate control measures such as vaccines to reduce morbidity and mortality rates, especially among children under 5 years of age. A quadrivalent bioconjugate Shigella vaccine was recently developed to safely and effectively induce immunity against four important Shigella spp. This study demonstrates the breadth of reactivity and functionality of the parenterally administered bioconjugate vaccine by evaluating the ability of rabbit sera to bind and kill Shigella isolates recently collected in Kenya. These results suggest that the Shigella quadrivalent vaccine may be more broadly protective than designed and may offer a promising solution to the morbidity and mortality associated with Shigella infections.

Scalable Process for High-Yield Production of PfCyRPA Using Insect Cells for Inclusion in a Malaria Virosome-Based Vaccine Candidate.

Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) has been identified as a promising blood-stage candidate antigen to include in a broadly cross-reactive malaria vaccine. In the last couple of decades, substantial effort has been committed to the development of scalable cost-effective, robust, and high-yield PfCyRPA production processes. Despite insect cells being a suitable expression system due to their track record for protein production (including vaccine antigens), these are yet to be explored to produce this antigen. In this study, different insect cell lines, culture conditions (baculovirus infection strategy, supplementation schemes, culture temperature modulation), and purification strategies (affinity tags) were explored aiming to develop a scalable, high-yield, and high-quality PfCyRPA for inclusion in a virosome-based malaria vaccine candidate. Supplements with antioxidants improved PfCyRPA volumetric titers by 50% when added at the time of infection. In addition, from three different affinity tags (6x-His, 4x-His, and C-tag) evaluated, the 4x-His affinity tag was the one leading to the highest PfCyRPA purification recovery yields (61%) and production yield (26 mg/L vs. 21 mg/L and 13 mg/L for 6x-His and C-tag, respectively). Noteworthy, PfCyRPA expressed using High Five cells did not show differences in protein quality or stability when compared to its human HEK293 cell counterpart. When formulated in a lipid-based virosome nanoparticle, immunized rabbits developed functional anti-PfCyRPA antibodies that impeded the multiplication of P. falciparum in vitro. This work demonstrates the potential of using IC-BEVS as a qualified platform to produce functional recombinant PfCyRPA protein with the added benefit of being a non-human expression system with short bioprocessing times and high expression levels.

Ubiquitin of Entamoeba histolytica induces antibody response in patients with invasive amoebiasis.

Entamoeba histolytica causes amoebic liver abscess (ALA) in humans. The injury of target cells by E. histolytica includes processes controlled by the ubiquitin Ehub. Previously, we found immunodominance of Ehub glycan moieties using immunized rabbits. In this work, we analysed dominance of antibodies to the glycoprotein Ehub in the sera from 52 patients with ALA. Controls were sera from 20 healthy people living in endemic areas with a high seroprevalence of antibodies to amoebas, and 20 patients with alcoholic hepatitis (AH) to rule out the cross-reaction of Ehub with autoantibodies induced by liver damage. Antigens were trophozoite extract, glycoprotein Ehub and the recombinant protein E. histolytica recombinant ubiquitin (rEhub). The sera from healthy volunteers and patients with AH do not have antibodies to glycoprotein Ehub. Surprisingly, only the antibodies from patients with ALA recognized the glycoprotein Ehub, and some sera gave a faint reaction with the recombinant protein, especially because evolutionarily, the ubiquitin is conserved between species. This is the first report demonstrating that antibodies to ubiquitin Ehub are induced exclusively in patients with invasive amoebiasis, and the antibody response is mainly to the glycoprotein, indicating glycans are immunodominant. Inhibitors of the Ehub glycans could be potential treatment for amoebiasis by selectively damaging trophozoites.