Anti-lipopolysaccharide Antibodies Research Articles

Synthesis of oligosaccharides from terminal B. pertussis LPS pentasaccharide and definition of the minimal epitope recognized by anti-pertussis antibodies.

Pertussis vaccines have been very effective in controlling whooping-cough epidemics but are ineffective in controlling circulation in older children and adults, thus facilitating the onset of future outbreaks. Antibodies against the lipopolysaccharide could reduce the carriage of the bacteria, its circulation, and transmission. The oligosaccharide fragments from the lipopolysaccharide may become a potential complement to existing vaccines in the form of protein glycoconjugates. An important step in the development of this type of vaccine is defining the minimal oligosaccharide epitope recognized by B. pertussis anti-lipopolysaccharide antibodies. This paper describes the complete synthesis of oligosaccharides containing two to five monosaccharide units corresponding to the pentasaccharide at the nonreducing end of the lipooligosaccharide and their recognition by mice and rabbit antibodies elicited against whole-cell B. pertussis. For the first time, we report that the terminal disaccharide, α-D-GlcNAcp-(1 → 4)-(2,3-di-NAc)-D-ManAp acid is the minimal structure recognized by antibodies induced by B. pertussis.

Detection of plasma anti-lipopolysaccharide (LPS) antibodies against enterohemorrhagic Escherichia coli (EHEC) in asymptomatic kindergarten teachers from Buenos Aires province

In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20–30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016–2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

In Silico and Electrochemical Studies for a ZnO-CuO-Based Immunosensor for Sensitive and Selective Detection of E. coli.

Escherichia coli is a harmful Gram-negative bacterium commonly found in the gut of warm-blooded organisms and affects millions of people annually worldwide. In this study, we have synthesized a ZnO–CuO nanocomposite (NC) by a co-precipitation method and characterized the as-synthesized NC using FTIR spectroscopy, XRD, Raman spectroscopy, and FESEM techniques. To fabricate the immunosensor, the ZnO–CuO NC composite was screen-printed on gold-plated electrodes followed by physisorption of the anti-LPS E. coli antibody. The biosensor was optimized for higher specificity and sensitivity. The immunosensor exhibited a high sensitivity (11.04 μA CFU mL–1) with a low detection limit of 2 CFU mL–1 with a redox couple. The improved performance of the immunosensor is attributed to the synergistic effect of the NC and the antilipopolysaccharide antibody against E. coli. The selectivity studies were also carried out with Staphylococcus aureus to assess the specificity of the immunosensor. Testing in milk samples was done by spiking the milk samples with different concentrations of E. coli to check the potential of this immunosensor. We further checked the affinity between ZnO–CuO NC with E. coli LPS and the anti-LPS antibody using molecular docking studies. Atomic charge computation and interaction analyses were performed to support our hypothesis. Our results discern that there is a strong correlation between molecular docking studies and electrochemical characterization. The interaction analysis further displays the strong affinity between the antibody–LPS complex when immobilized with a nanoparticle composite (ZnO–CuO).

Anti-lipopolysaccharide antibody administration mitigates ruminal lipopolysaccharide release and depression of ruminal pH during subacute ruminal acidosis challenge in Holstein bull cattle.

The effects of anti-lipopolysaccharide (LPS) antibody on rumen fermentation and LPS activity were investigated during subacute ruminal acidosis (SARA) challenge. Eleven Holstein cattle (164 ± 14 kg) were used in a 3 × 3 Latin square design. Cattle were fed a roughage diet on days −11 to −1 (pre-challenge) and day 2 (post-challenge), and a high-grain diet on days 0 and 1 (SARA challenge). For 14 days, 0-, 2-, or 4-g of anti-LPS antibody was administered once daily through a rumen fistula. Ruminal pH was measured continuously, and rumen fluid and blood samples were collected on days −1, 0, 1, and 2. Significantly lower ruminal LPS activity on day 1 was observed in the 2- and 4-g groups than those in the 0-g group. In addition, significantly higher 1-hr mean ruminal pH on SARA challenge period (days 0 and 1) was identified in the 4-g group than in the 0-g group. However, rumen fermentation measurements (total volatile fatty acid [VFA], VFA components, NH3-N and lactic acid) and peripheral blood metabolites (glucose, free fatty acid, beta-hydroxybutyrate, total cholesterol, blood urea nitrogen, aspartate aminotransferase and gamma-glutamyl transferase) were not different among the groups during the experimental periods. Therefore, anti-LPS antibody administration mitigates LPS release and pH depression without the depression of rumen fermentation and peripheral blood metabolites during SARA challenge in Holstein cattle.

Anti-lipopolysaccharide antibody mitigates ruminal lipopolysaccharide release without acute-phase inflammation or liver transcriptomic responses in Holstein bulls.

Anti-lipopolysaccharide (LPS) antibody administration has the potential benefits of neutralizing and consequently controlling rumen-derived LPS during subacute ruminal acidosis. Four Holstein bulls were used in this crossover study with a 2-week wash-out period. Anti-LPS antibody (0 or 4 g) was administered once daily for 14 days. Significantly lower ruminal LPS and higher 1-h mean ruminal pH were identified in the 4 g group. However, blood metabolites, acute-phase proteins, cytokines, and hepatic transcriptomes were not different between the two groups. Therefore, anti-LPS antibody administration mitigated ruminal LPS release and pH depression without accompanying responses in acute-phase inflammation or hepatic transcriptomic expression.

Анти-ліпополісахаридні антитіла та осмотична стійкість еритроцитів у здорових осіб та пацієнтів з В-неходжкінською лімфомою з різними групами крові

Анти-ліпополісахаридні антитіла та осмотична стійкість еритроцитів у здорових осіб та пацієнтів з В-неходжкінською лімфомою з різними групами крові

Transcriptional profiling of Vibrio cholerae O1 following exposure to human anti- lipopolysaccharide monoclonal antibodies.

Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified >160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting.

Fluorescence immunoassay of E. coli using anti-lipopolysaccharide antibodies isolated from human serum

In this work, the gram-negative bacterium Escherichia coli strain BL21(DE3) (with lipopolysaccharide (LPS) in its outer membrane) and its modified ClearColi™ strain (lacking LPS) were used for the separation of anti-LPS antibodies from human serum by the following steps: (1) binding of the serum proteins to BL21(DE3); (2) dissociation of the bound proteins (including anti-LPS antibodies) from BL21(DE3) with acid; (3) filtering of the dissociated proteins using ClearColi to remove unwanted proteins; and (4) separation of the antibody fraction by protein-A column chromatography. The binding properties of the separated antibodies were analyzed by fluorescence-activated cell sorting to confirm their selective binding to LPS on the outer membrane of BL21(DE3), and by thermophoretic immunoassay to estimate their dissociation constant. The in vitro applicability of the separated anti-LPS antibodies was demonstrated through a fluorescence assay of BL21(DE3), after immobilizing the antibodies onto a modified microplate surface. The electrochemical detection of BL21(DE3) was also achieved after immobilizing the anti-LPS antibodies onto a gold electrode.

Spontaneous point mutations in the capsule synthesis locus leading to structural and functional changes of the capsule in serogroup A meningococcal populations

ABSTRACT Whole genome sequencing analysis of 100 Neisseria meningitidis serogroup A isolates has revealed that the csaABCD-ctrABCD-ctrEF capsule polysaccharide synthesis locus represents a spontaneous point mutation hotspot. Structural and functional properties of the capsule of 11 carriage and two disease isolates with non-synonymous point mutations or stop codons in capsule synthesis genes were analyzed for their capsular polysaccharide expression, recognition by antibodies and sensitivity to bactericidal killing. Eight of eleven carriage isolates presenting capsule locus mutations expressed no or reduced amounts of capsule. One isolate with a stop codon in the O-acetyltransferase gene expressed non-O-acetylated polysaccharide, and was not recognized by anti-capsule antibodies. Capsule and O-acetylation deficient mutants were resistant to complement deposition and killing mediated by anti-capsular antibodies, but not by anti-lipopolysaccharide antibodies. Two capsule polymerase mutants, one carriage and one case isolate, showed capsule over-expression and increased resistance against bactericidal activity of both capsule- and lipopolysaccharide-specific antibodies. Meningococci have developed multiple strategies for changing capsule expression and structure, which is relevant both for colonization and virulence. Here we show that point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading to variability in capsule expression.

Design and production of conjugate vaccines against S. Paratyphi A using an O-linked glycosylation system in vivo

Enteric fever, mainly caused by Salmonella enterica serovar Paratyphi A, remains a common and serious infectious disease worldwide. As yet, there are no licensed vaccines against S. Paratyphi A. Biosynthesis of conjugate vaccines has become a promising approach against bacterial infection. However, the popular biosynthetic strategy using N-linked glycosylation systems does not recognize the specialized O-polysaccharide structure of S. Paratyphi A. Here, we describe an O-linked glycosylation approach, the only currently available glycosylation system suitable for an S. Paratyphi A conjugate vaccine. We successfully generated a recombinant S. Paratyphi A strain with a longer O-polysaccharide chain and transformed the O-linked glycosylation system into the strain. Thus, we avoided the need for construction of an O-polysaccharide expression vector. In vivo assays indicated that this conjugate vaccine could evoke IgG1 antibody to O-antigen of S. Paratyphi A strain CMCC 50973 and elicit bactericidal activity against S. Paratyphi A strain CMCC 50973 and five other epidemic strains. Furthermore, we replaced the peptides after the glycosylation site (Ser) with an antigenic peptide (P2). The results showed that the anti-lipopolysaccharide antibody titer, bactericidal activity of serum, and protective effect during animal challenge could be improved, indicating a potential strategy for further vaccine design. Our system provides an easier and more economical method for the production of S. Paratyphi A conjugate vaccines. Modification of the glycosylation site sequon provides a potential approach for the development of next-generation “precise conjugate vaccines.”

Vibrio cholerae O1 secretes an extracellular matrix in response to antibody-mediated agglutination.

Vibrio cholerae O1 is one of two serogroups responsible for epidemic cholera, a severe watery diarrhea that occurs after the bacterium colonizes the human small intestine and secretes a potent ADP-ribosylating toxin. Immunity to cholera is associated with intestinal anti-lipopolysaccharide (LPS) antibodies, which are known to inhibit V. cholerae motility and promote bacterial cell-cell crosslinking and aggregation. Here we report that V. cholerae O1 classical and El Tor biotypes produce an extracellular matrix (ECM) when forcibly immobilized and agglutinated by ZAC-3 IgG, an intestinally-derived monoclonal antibody (MAb) against the core/lipid A region of LPS. ECM secretion, as demonstrated by crystal violet staining and scanning electron microscopy, occurred within 30 minutes of antibody exposure and peaked by 3 hours. Non-motile mutants of V. cholerae did not secrete ECM following ZAC-3 IgG exposure, even though they were susceptible to agglutination. The ECM was enriched in O-specific polysaccharide (OSP) but not Vibrio polysaccharide (VPS). Finally, we demonstrate that ECM production by V. cholerae in response to ZAC-3 IgG was associated with bacterial resistant to a secondary complement-mediated attack. In summary, we propose that V. cholerae O1, upon encountering anti-LPS antibodies in the intestinal lumen, secretes an ECM (or O-antigen capsule) possibly as a strategy to shield itself from additional host immune factors and to exit an otherwise inhospitable host environment.

Review article: novel methods for the treatment of non-alcoholic steatohepatitis - targeting the gut immune system to decrease the systemic inflammatory response without immune suppression.

SummaryBackgroundThe systemic immune system plays a role in inflammation and fibrogenesis associated with non‐alcoholic steatohepatitis (NASH) and has become a potential target for drug development. In particular, the gut immune system has been suggested as a means for generating signals that can target the systemic immune system.AimTo describe seven novel methods being developed for the treatment of NASH that target the gut immune system for alleviation of the systemic inflammatory response, including oral administration of fatty‐liver‐derived proteins, anti‐CD3 antibodies, tumour necrosis factor fusion protein, anti‐lipopolysaccharide antibodies, glucosylceramide, delayed‐release mercaptopurine, and soy‐derived extracts.MethodsA search for these methods for oral immunotherapy for NASH was conducted.ResultsOral administration of these compounds provides an opportunity for immune modulation without immune suppression, with the advantage of being independent of a single molecular/inflammatory pathway. These modes of oral immune therapy demonstrate superior safety profiles, such that the patient is not exposed to general immune suppression. Moreover, these approaches target the whole spectrum of the disease and may serve as adjuvants to other therapies, such that they provide a platform for treatment of concomitant disorders in patients with NASH, including diabetes and hyperlipidaemia. Most of the compounds reviewed are currently in phase II trials, and it is anticipated that the acquisition of more clinical data in the next few years will enable the use of this new class of drugs for the treatment of NASH.ConclusionOral immunotherapy may provide a novel platform for the treatment of NASH.

New Pharmacologic Agents That Target Inflammation and Fibrosis in Nonalcoholic Steatohepatitis-Related Kidney Disease.

Epidemiologic data show an association between the prevalence and severity of nonalcoholic fatty liver disease and the incidence and stage of chronic kidney disease (CKD); furthermore, nonalcoholic steatohepatitis (NASH)-related cirrhosis has a higher risk of renal failure, a greater necessity for simultaneous liver-kidney transplantation, and a poorer renal outcome than cirrhosis of other etiologies even after simultaneous liver-kidney transplantation. These data suggest that NASH and CKD share common proinflammatory and profibrotic mechanisms of progression, which are targeted incompletely by current treatments. We reviewed therapeutic approaches to late preclinical/early clinical stage of development in NASH and/or CKD, focusing on anti-inflammatory and antifibrotic treatments, which could slow the progression of both disease conditions. Renin inhibitors and angiotensin-converting enzyme-2 activators are new renin-angiotensin axis modulators that showed incremental advantages over angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers in preclinical models. Novel, potent, and selective agonists of peroxisome proliferator-activated receptors and of farnesoid X receptor, designed to overcome limitations of older compounds, showed promising results in clinical trials. Epigenetics, heat stress response, and common effectors of redox regulation also were subjected to intensive research, and the gut was targeted by several approaches, including synbiotics, antilipopolysaccharide antibodies, Toll-like receptor-4 antagonists, incretin mimetics, and fibroblast growth factor 19 analogs. Promising anti-inflammatory therapies include inhibitors of NOD-like receptor family, pyrin domain containing 3 inflammasome, of nuclear factor-κB, and of vascular adhesion protein-1, chemokine antagonists, and solithromycin, and approaches targeting common profibrogenic pathways operating in the liver and the kidney include galectin-3 antagonists, and inhibitors of rho-associated protein kinase and of epidermal growth factor activation. The evidence, merits, and limitations of each approach for the treatment of NASH and CKD are discussed.

Bactericidal Immunity to Salmonella in Africans and Mechanisms Causing Its Failure in HIV Infection.

BackgroundNontyphoidal strains of Salmonella are a leading cause of death among HIV-infected Africans. Antibody-induced complement-mediated killing protects healthy Africans against Salmonella, but increased levels of anti-lipopolysaccharide (LPS) antibodies in some HIV-infected African adults block this killing. The objective was to understand how these high levels of anti-LPS antibodies interfere with the killing of Salmonella.Methodology/Principal FindingsSera and affinity-purified antibodies from African HIV-infected adults that failed to kill invasive S. Typhimurium D23580 were compared to sera from HIV-uninfected and HIV-infected subjects with bactericidal activity. The failure of sera from certain HIV-infected subjects to kill Salmonella was found to be due to an inherent inhibitory effect of anti-LPS antibodies. This inhibition was concentration-dependent and strongly associated with IgA and IgG2 anti-LPS antibodies (p<0.0001 for both). IgG anti-LPS antibodies, from sera of HIV-infected individuals that inhibit killing at high concentration, induced killing when diluted. Conversely, IgG, from sera of HIV-uninfected adults that induce killing, inhibited killing when concentrated. IgM anti-LPS antibodies from all subjects also induced Salmonella killing. Finally, the inhibitory effect of high concentrations of anti-LPS antibodies is seen with IgM as well as IgG and IgA. No correlation was found between affinity or avidity, or complement deposition or consumption, and inhibition of killing.Conclusion/SignificanceIgG and IgM classes of anti-S. Typhimurium LPS antibodies from HIV-infected and HIV-uninfected individuals are bactericidal, while at very high concentrations, anti-LPS antibodies of all classes inhibit in vitro killing of Salmonella. This could be due to a variety of mechanisms relating to the poor ability of IgA and IgG2 to activate complement, and deposition of complement at sites where it cannot insert in the bacterial membrane. Vaccine trials are required to understand the significance of lack of in vitro killing by anti-LPS antibodies from a minority of HIV-infected individuals with impaired immune homeostasis.

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.

Effects of Central Injection of Anti-LPS Antibody and Blockade of TLR4 on GnRH/LH Secretion during Immunological Stress in Anestrous Ewes

The present study was designed to examine the effect of intracerebroventricular (icv) administration of antilipopolysaccharide (LPS) antibody and blockade of Toll-like receptor 4 (TLR4) during immune stress induced by intravenous (iv) LPS injection on the gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion in anestrous ewes. Injection of anti-LPS antibody and TLR4 blockade significantly (P < 0.01) reduced the LPS dependent lowering amount of GnRH mRNA in the median eminence (ME). Moreover, blockade of TLR4 caused restoration of LH-β transcription in the anterior pituitary decreased by the immune stress. However, there was no effect of this treatment on reduced LH release. The results of our study showed that the blockade of TLR4 receptor in the hypothalamus is not sufficient to unblock the release of LH suppressed by the immune/inflammatory challenges. This suggests that during inflammation the LH secretion could be inhibited directly at the pituitary level by peripheral factors such as proinflammatory cytokines and circulating endotoxin as well.

Serum from patients with hepatitis E virus-related acute liver failure induces human liver cell apoptosis

The pathogenesis of acute liver failure has not been fully elucidated. The present study investigated the effects of the serum from patients with hepatitis E virus (HEV)-related acute liver failure on human liver cell survival and apoptosis, and evaluated the protective effects of anti-lipopolysaccharide(LPS) antibody recognizing core polysaccharide against acute liver failure serum-induced apoptosis. Serum was collected from patients with HEV-related acute liver failure. The levels of endotoxin (LPS) in the serum were measured using a quantitative tachypleus amebocyte lysate endotoxin detection kit with a chromogenic endpoint. Serum with a mean concentration of LPS was incubated with L02 human liver cells and the rate of apoptosis was detected by flow cytometry. The apoptotic rate was also evaluated in liver cells incubated with antibody and the HEV-related acute liver failure serum. The results indicated that the concentration of LPS in the serum of patients with HEV-related acute liver failure was 0.26±0.02 EU/ml, which was significantly higher than that of the control group (P<0.05). The rate of apoptosis in the human liver cells induced by acute liver failure serum was 5.83±0.42%, which was significantly increased compared with that in the cells treated with the serum of healthy individuals (P<0.05). The apoptotic rate of the cells incubated with antibody and the acute liver failure serum was 5.53±0.51%, which was lower than that of the cells incubated with acute liver failure serum alone (P>0.05). These results indicate that the serum of patients with HEV-related acute liver failure induces the apoptosis of human liver cells. LPS may be directly involved in the apoptosis of human liver cells. Moreover, the presence of the antibody did not significantly reduce the level of apoptosis of liver cells exposed to HEV-related acute liver failure serum.

Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

Recent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection with Salmonella enterica serovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity against S. Typhimurium in a standard in vitro assay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) from S. Typhimurium but not from Escherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing of S. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing of S. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against an S. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

Genomic correlates of variability in immune response to an oral cholera vaccine

Cholera is endemic to many countries. Recent major outbreaks of cholera have prompted World Health Organization to recommend oral cholera vaccination as a public-health strategy. Variation in percentage of seroconversion upon cholera vaccination has been recorded across populations. Vaccine-induced responses are influenced by host genetic differences. We have investigated association between single-nucleotide polymorphic (SNP) loci in and around 296 immunologically relevant genes and total anti-lipopolysaccharide (LPS) antibody response to a killed whole-cell vaccine, comprising LPS from multiple strains of Vibrio cholerae. Titers derived from standard vibriocidal assays were also analyzed to gain further insights on validated SNP associations. Vaccination was administered to 1000 individuals drawn from India. Data on two independent random subsets, each comprising ∼500 vaccinees, were used for discovery of genomic associations and validation, respectively. Significant associations of four SNPs and haplotypes in three genes (MARCO, TNFAIP3 and CXCL12) with AR were discovered and validated, of which two in TNFAIP3 and CXCL12 were also significantly associated with immunity (fourfold increase in vibriocidal titers). CXCL12 is a neutrophil and lymphocyte chemoattractant that is upregulated in response to V. cholerae infection. LPS in the vaccine possibly provides signals that mimic those of the live bacterium. TNFAIP3 promotes intestinal epithelial barrier integrity and provides tight junction protein regulation; possible requirements for adequate response to the vaccine. LPS is a potent activator of innate immune responses and a ligand of MARCO. Variants in this gene have been found to be associated with LPS response, but not with high vibriocidal titer level.

Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes

Vibrio cholerae is considered one of the major health threats in developing countries. Lack of efficient vaccine, short incubating time of the disease, and bacterium ability to survive in aquatic environment have made cholera one of the most epidemic diseases yet known. The lipopolysaccharide is one of the bacterium key antigens used to classify V. cholerae into 206 serogroups. V. cholerae serogroup O1 is a causative agent of all cholera pandemics. Research has shown that anti-lipopolysaccharide (LPS) antibodies could provide protective immunity in cholera cases. In this research, we used N-terminal fragments of the camel's heavy-chain antibodies called VHH or nanobodies and produced a phagemid library. The obtained library was panned against V. cholerae O1 LPS, and four monoclonal nanobodies were isolated. Isolated nanobodies were tested in LPS ELISA and bacterial ELISA. The nanobody with the highest affinity toward the bacterium was used in an in vivo challenge and successfully neutralized the bacterium infection. The isolated nanobody showed high thermostability and proteolytic resistance in characterization tests.