The Role of IgG Subclass in Antibody-Mediated Protection against Carbapenem-Resistant Klebsiella pneumoniae.

Monoclonal antibodies (MAbs) have the potential to assist in the battle against multidrug-resistant bacteria such as carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, the characteristics by which these antibodies (Abs) function, such as the role of antibody subclass, must be determined before such modalities can be carried from the bench to the bedside. We performed a subclass switch on anticapsular monoclonal murine IgG3 (mIgG3) hybridomas and identified and purified a murine IgG1 (mIgG1) hybridoma line through sib selection. We then compared the ability of the mIgG1 and mIgG3 antibodies to control CR-Kp sequence type 258 (ST258) infection both in vitro and in vivo We found by enzyme-limited immunosorbent assay (ELISA) and flow cytometry that mIgG3 has superior binding to the CR-Kp capsular polysaccharide (CPS) and superior agglutinating ability compared to mIgG1 The mIgG3 also, predictably, had better complement-mediated serum bactericidal activity than the mIgG1 and also promoted neutrophil-mediated killing at concentrations lower than that of the mIgG1 In contrast, the mIgG1 had marginally better activity in improving macrophage-mediated phagocytosis. Comparing their activities in a pulmonary infection model with wild-type as well as neutropenic mice, both antibodies reduced organ burden in a nonlethal challenge, regardless of neutrophil status, with mIgG1 having the highest overall burden reduction in both scenarios. However, at a lethal inoculum, both antibodies showed reduced efficacy in neutropenic mice, with mIgG3 retaining the most activity. These findings suggest the viability of monoclonal Ab adjunctive therapy in neutropenic patients that cannot mount their own immune response, while also providing some insight into the relative contributions of immune mediators in CR-Kp protection.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an urgent public health threat that causes life-threatening infections in immunocompromised hosts. Its resistance to nearly all antibiotics necessitates novel strategies to treat it, including the use of monoclonal antibodies. Monoclonal antibodies are emerging as important adjuncts to traditional pharmaceuticals, and studying how they protect against specific bacteria such as Klebsiella pneumoniae is crucial to their development as effective therapies. Antibody subclass is often overlooked but is a major factor in how an antibody interacts with other mediators of immunity. This paper is the first to examine how the subclass of anticapsular monoclonal antibodies can affect efficacy against CR-Kp Additionally, this work sheds light on the viability of monoclonal antibody therapy in neutropenic patients, who are most vulnerable to CR-Kp infection.

HIV Is Associated with Modified Humoral Immune Responses in the Setting of HIV/TB Coinfection.

Tuberculosis (TB) represents the largest cause of death in human immunodeficiency virus (HIV)-infected individuals in part due to HIV-related CD4+ T cell loss, rendering patients immunocompromised and susceptible to a loss of Mycobacterium tuberculosis control. However, in light of increasing data pointing to a role for humoral immunity in controlling M. tuberculosis infection, here, we aimed to define whether HIV infection also alters the humoral immune response in subjects with active and latent TB. We show that in the setting of active TB, HIV-positive individuals have significantly lower IgG responses to LAM and Ag85 than HIV-negative individuals. Furthermore, significant isotype/subclass-specific differences were frequently observed, with active TB, HIV-positive individuals demonstrating compromised antigen-specific IgM titers. HIV-infected individuals with active TB also exhibited a significant loss of influenza hemagglutinin- and tetanus toxoid-specific antibody titers at the isotype/subclass level, a symptom of broad humoral immune dysfunction likely precipitated by HIV infection. Finally, we illustrated that despite the influence of HIV infection, differences in M. tuberculosis-specific antibody profiles persist between latent and active TB disease. Taken together, these findings reveal significant HIV-associated disruptions of the humoral immune response in HIV/TB-coinfected individuals.IMPORTANCE TB is the leading cause of death from a single infectious agent globally, followed by HIV. Furthermore, TB represents the leading cause of death among people with HIV. HIV is known to cause severe defects in T cell immunity, rendering HIV/TB-coinfected individuals more susceptible to TB disease progression and complicating accurate TB disease diagnosis. Here, we demonstrate that HIV infection is additionally associated with severely compromised antibody responses, particularly in individuals with active TB. Moreover, despite the influence of HIV infection, antibody profiles still allow accurate classification of individuals with active versus latent TB. These findings reveal novel immunologic challenges associated with HIV/TB coinfection and additionally provide a basis with which to leverage the key antibody features identified to potentially combat TB globally via next-generation therapeutic or diagnostic design.

The Profile of Immunoglobulin A and Immunoglobulin G Subclasses in Sprague Dawley Rats Experimentally Infected with Brucella abortus Biotype 1.

This study measured total serum immunoglobulin A (IgA), immunoglobulin G (IgG)1, IgG2a response against whole cell antigen (WCA), outer membrane protein (OMP), periplasmic protein (PP), cytoplasmic protein (CP), and crude Brucella protein (CBP) of Brucella abortus in experimental brucellosis induced with B. abortus biotype 1 in Sprague Dawley (SD) rats during a 17-week infection period. Six- to 8-week-old SD rats (n = 44) were experimentally infected with 1 × 109 colony forming unit of B. abortus biotype 1 through the intraperitoneal route. Serial serum samples were collected from the rat at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days after inoculation. The sera were tested by enzyme linked immunosorbent assay. We have noticed a very low level and short persistence of IgA antibody in our experiment. The low level and short persistence of IgA antibody suggest that this antibody isotype might not be protective against brucellosis in rats. Both Th1 and Th2 specific immune responses were recorded in our study with the production of IgG1 and IgG2a antibody isotopes, respectively. We noticed significant dominant IgG2a antibody responses over IgG1 responses throughout the experiment (p < 0.001) against WCA and OMP. The mixed Th1 and Th2 dominant immune responses mediated by IgG2a and IgG1 antibody isotypes were observed against CP, PP, and CBP. Data of our study suggest that IgG2a dominant responses in the early stages of disease play the main role in conferring protection against brucellosis and with the progress of disease IgG1 dominant responses were elicited.

Proteome array of antibody responses to Chlamydia trachomatis infection in nonhuman primates.

AIMS: Nonhuman primates have been used to investigate pathogenic mechanisms and evaluate immune responses following Chlamydia trachomatis inoculation. This study aimed to systemically profile antibody responses to C. trachomatis infection in nonhuman primates. MATERIALS AND METHODS: Sera were obtained from 4 pig-tailed and 8 long-tailed macaques which were intravaginally or ocularly infected with live C. trachomatis organisms, and analyzed by C. trachomatis proteome array of antigens. KEY FINDINGS: The sera from 12 macaques recognized total 172 C. trachomatis antigens. While 84 antigens were recognized by pig-tailed macaques intravaginally infected with serovar D strain, 125 antigens were recognized by long-tailed macaques ocularly infected with serovar A, and 37 antigens were recognized by both. Ocular inoculation with virulent A2497 strain induced antibodies to more antigens. Among the antigens uniquely recognized by A2497 strain infected macaques, outer membrane complex B antigen (OmcB) induced robust antibody response. Although macaques infected by less virulent A/HAR-13 strain failed to develop antibodies to OmcB, reinfection by A2497 strain induced high levels of antibodies to OmcB. SIGNIFICANCE: Proteome array has revealed a correlation of chlamydial infection invasiveness with chlamydial antigen immunogenicity, and identified antibody responses to OmcB potentially as biomarkers for invasive infection with C. trachomatis.

Commensal Microbes Affect Host Humoral Immunity to Bordetella pertussis Infection.

As important players in the host defense system, commensal microbes and the microbiota influence multiple aspects of host physiology. Bordetella pertussis infection is highly contagious among humans. However, the roles of the microbiota in B. pertussis pathogenesis are poorly understood. Here, we show that antibiotic-mediated depletion of the microbiota results in increased susceptibility to B. pertussis infection during the early stage. The increased susceptibility was associated with a marked impairment of the systemic IgG, IgG2a, and IgG1 antibody responses to B. pertussis infection after antibiotic treatment. Furthermore, the microbiota impacted the short-lived plasma cell responses as well as the recall responses of memory B cells to B. pertussis infection. Finally, we found that the dysbiosis caused by antibiotic treatment affects CD4+ T cell generation and PD-1 expression on CD4+ T cells and thereby perturbs plasma cell differentiation. Our results have revealed the importance of commensal microbes in modulating host immune responses to B. pertussis infection and support the possibility of controlling the severity of B. pertussis infection in humans by manipulating the microbiota.

PNAG-specific equine IgG1 mediates significantly greater opsonization and killing of Prescottella equi (formerly Rhodococcus equi) than does IgG4/7.

Prescottella equi (formerly Rhodococcus equi) is a facultative intracellular bacterial pathogen that causes severe pneumonia in foals 1-6 months of age, whereas adult horses are highly resistant to infection. We have shown that vaccinating pregnant mares against the conserved surface polysaccharide capsule, ß-1 → 6-linked poly-N-acetyl glucosamine (PNAG), elicits opsonic killing antibody that transfers via colostrum to foals and protects them against experimental infection with virulent. R. equi. We hypothesized that equine IgG1 might be more important than IgG4/7 for mediating protection against R. equi infection in foals. To test this hypothesis, we compared complement component 1 (C1) deposition and polymorphonuclear cell-mediated opsonophagocytic killing (OPK) mediated by IgG1 or IgG4/7 enriched from either PNAG hyperimmune plasma (HIP) or standard plasma. Subclasses IgG1 and IgG4/7 from PNAG HIP and standard plasma were precipitated onto a diethylaminoethyl ion exchange column, then further enriched using a protein G Sepharose column. We determined C1 deposition by enzyme-linked immunosorbent assay (ELISA) and estimated OPK by quantitative microbiologic culture. Anti-PNAG IgG1 deposited significantly (P < 0.05) more C1 onto PNAG than did IgG4/7 from PNAG HIP or subclasses IgG1 and IgG4/7 from standard plasma. In addition, IgG1 from PNAG HIP mediated significantly (P < 0.05) greater OPK than IgG4/7 from PNAG HIP or IgG1 and IgG4/7 from standard plasma. Our findings indicate that anti-PNAG IgG1 is a correlate of protection against R. equi in foals, which has important implications for understanding the immunopathogenesis of R. equi pneumonia, and as a tool for assessing vaccine efficacy and effectiveness when challenge is not feasible.

Antibodies prevalence against Haemophilus influenzae type b in Jeddah population, Saudi Arabia. II. Antibodies subclasses.

Our previous reported that Jeddah, Saudi Arabia population have a comparable and protective herd immunity represented by IgG, IgA and IgM against Haemophilus influenzae type b (Hib), by using indirect ELISA test was used to evaluate Haemophilus influenzae type b (Hib) anti-polyribosyl-ribitol phosphate (PRP) total antibodies (IgM, IgG, IgA) in 1,003 sera samples. Current report was evaluated the IgG and IgA subclasses responsible about this protection using same methodology. IgG, IgA, and their subclasses are responsible about this circulating protection. Our study will consider the first report evaluate the levels of IgA subclasses and its relation to ages, as well as the relations between anti-Hib antibody subclass and age. The current results demonstrated that the highest levels concentrated in IgG1 and IgG2, while IgG3 and IgG4 showed the lowest levels. So, their concentrations were arranged in IgG1 > IgG2 > IgG3 > IgG4. The results indicated that the age and gender have no effect on both IgG or IgA subclasses in healthy immunized individuals enrolled. While, IgA1 concentrations were significantly higher than IgA2 in all age categories regardless of gender. It seem that the IgG1, IgG2 and IgA1 subclasses were the main constituent of Jeddah herd immunity against Hib. Finally, to the best of our knowledge, there were no previous reports that focusing on the levels of IgA subclasses and its relation to ages, so our study considers the first worldwide.

Immunoglobulin G subclass switching impacts sensitivity of an immunoassay targeting Francisella tularensis lipopolysaccharide.

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

LT adjuvant modulates epitope specificity and improves the efficacy of murine antibodies elicited by sublingual vaccination with the N-terminal domain of Streptococcus mutans P1.

In this study, we evaluated the immunogenicity, protective efficacy and peptide-based immune signatures of antibodies raised in mice after sublingual immunization with a recombinant form of the P1 (aka AgI/II, PAc) adhesin (P139-512) of Streptococcus mutans, a major etiological agent of dental caries. Sublingual administration of P139-512 in combination with the mucosal adjuvant LTK4R (a derivative of heat-labile LT toxin) induced strong and long-lasting systemic and mucosal immune responses. Incorporation of the adjuvant resulted in an enhancement of the anti-adhesive and anti-colonization activity against S. mutans as evaluated both under in vitro and in vivo conditions. Incorporation of the adjuvant to the vaccine formulation also changed the epitope specificity of the induced antibodies as determined by immunological signatures of sera collected from vaccinated mice. Use of a peptide microarray library led to the identification of peptide targets recognized by antibodies in serum samples with enhanced anti-adhesive effects. Altogether, the results presented herein showed that the sublingual administration of a P1-based subunit vaccine represents a promising approach for the prevention of dental caries caused by S. mutans. In addition, the present study disclosed the role of adjuvants on the epitope specificity and functionality of antibodies raised by subunit vaccines.

Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells.

We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs) generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT) or lipopolysaccharide (LPS). Using a novel proteomics approach, we were able to identify sialidase as another major antigen targeted by the antibody response to Vibrio cholerae infection. Antitoxin MAbs targeted both the A and B subunits, and most were also potent neutralizers of enterotoxigenic Escherichia coli heat-labile toxin. LPS-specific MAbs uniformly targeted the O-specific polysaccharide, with no detectable responses to either the core or the lipid moiety of LPS. Interestingly, the LPS-specific antibodies varied widely in serotype specificity and functional characteristics. One participant infected with the Ogawa serotype produced highly mutated LPS-specific antibodies that preferentially bound the previously circulating Inaba serotype. This demonstrates durable memory against a polysaccharide antigen presented at the mucosal surface and provides a mechanism for the long-term, partial heterotypic immunity seen following cholera. IMPORTANCE: Cholera is a diarrheal disease that results in significant mortality. While oral cholera vaccines are beneficial, they do not achieve equivalent protection compared to infection with Vibrio cholerae Although antibodies likely mediate protection, the mechanisms of immunity following cholera are poorly understood, and a detailed understanding of antibody responses to cholera is of significance for human health. In this study, we characterized the human response to cholera at the single-plasmablast, monoclonal antibody level. Although this approach has not been widely applied to the study of human bacterial infection, we were able to uncover the basis of cross-reactivity between different V. cholerae serotypes and the likely impact of prior enterotoxigenic Escherichia coli exposure on the response to cholera, as well as identify novel antigenic targets. In addition to improving our understanding of the repertoire and function of the antibody response to cholera in humans, this study has implications for future cholera vaccination efforts.

Unveiling Unexpected Immune Activities Induced by Your Pneumococcal Vaccine.

In modern-day vaccine design, a good pneumococcal capsular polysaccharide vaccine is measured by its ability to induce opsonic antibodies. These antibodies label bacteria for phagocytosis by neutrophils and thereby overcome the capsule's barrier function. Doyle and Pirofski have raised a serious challenge to the current paradigm by describing anti-capsular antibodies that are highly protective but nonopsonic [C.R. Doyle and L. Pirofski, mBio 7(1):e02260-15, 2016, doi:10.1128/mBio.02260-15]. In fact, some functions are not related to neutrophils or phagocytosis at all. An increased awareness of these activities is critical not only for accurate comparisons of vaccine candidates but also for improvements in vaccination outcomes in settings of neutropenia. When vaccine developers select a single gatekeeper assay (e.g., an opsonophagocytic assay for bacteria or a neutralization assay for viruses), promising vaccine candidates may be missed. Doyle and Pirofski stress that multiple functions, not just one, should be investigated to enhance discovery of antibody mechanisms and to best assess vaccine-induced correlates of immune protection.

VapA-specific IgG and IgG subclasses responses after natural infection and experimental challenge of foals with Rhodococcus equi.

Rhodococcus equi is a common cause of pneumonia in young foals worldwide and has considerable economic effects on the global equine industry. Despite ongoing efforts, no vaccine is currently available to prevent rhodococaal pneumonia. This is due, in part, to an incomplete understanding of the protective immune response to this bacterium. While antibodies to VapA, a lipoprotein produced by virulent R. equi, are useful in differentiating antibody production in response to pathogenic versus non-pathogenic strains, the significance of the humoral response of foals to this lipoprotein remains poorly defined. The objectives of this study were to evaluate changes in VapA-specific IgG and IgG subclasses after exposure and infection of neonatal foals. Experimental foals included those challenged with R. equi at 1 (n=18), 2 (n=4) and 3 (n=6) weeks of age. Confirmed naturally infected (n=7) and not infected (n=3) foals were also included. All foals were bled 24h after birth and weekly thereafter for a period of 8 weeks. Antibody changes over time were evaluated. Following birth, VapA-specific IgGs significantly (p<0.05) decreased over time in all foals as a result of normal decay of passively transferred antibodies. Both VapA-specific IgGa and IgG(T) significantly increased (p<0.05) after experimental challenge, however, the rise in IgG(T) occurred earlier. Only a significant (p<0.05) increase in VapA-specific IgG(T) over time was seen after natural infection. Whether VapA-specific IgG(T) can be used to differentiate rhodococcal from other pneumonias requires further investigation under field conditions.

Antibody orientation at bacterial surfaces is related to invasive infection.

Several of the most significant bacterial pathogens in humans, including Streptococcus pyogenes, express surface proteins that bind IgG antibodies via their fragment crystallizable (Fc) region, and the dogma is that this protects the bacteria against phagocytic killing in blood. However, analysis of samples from a patient with invasive S. pyogenes infection revealed dramatic differences in the presence and orientation of IgG antibodies at the surface of bacteria from different sites. In the throat, IgG was mostly bound to the bacterial surface via Fc, whereas in the blood IgG was mostly bound via fragment antigen-binding (Fab). In infected and necrotic tissue, the Fc-binding proteins were removed from the bacterial surface. Further investigation showed that efficient bacterial IgGFc-binding occurs only in IgG-poor environments, such as saliva. As a consequence, the bacteria are protected against phagocytic killing, whereas in blood plasma where the concentration of IgG is high, the antibodies preferentially bind via Fab, facilitating opsonization and bacterial killing. IgG-poor environments represent the natural habitat for IgGFc-binding bacteria, and IgGFc-binding proteins may have evolved to execute their function in such environments. The lack of protection in plasma also helps to explain why cases of severe invasive infections with IgGFc-binding bacteria are so rare compared with superficial and uncomplicated infections.

A vaccine candidate for post-weaning diarrhea in swine constructed with a live attenuated Salmonella delivering Escherichia coli K88ab, K88ac, FedA, and FedF fimbrial antigens and its immune responses in a murine model.

In order to construct a novel vaccine candidate for preventing post-weaning diarrhea in swine, the individual genes for Escherichia coli K88ab, K88ac, FedA, and FedF fimbriae were inserted into a secretion plasmid pBP244 containing asd, lepB, secA, and secB. These were transformed into Salmonella Typhimurium Δlon ΔcpxR Δasd. Secretion of the individual recombinant fimbrial antigens was confirmed by immunoblot analysis. Groups 1 and 2 mice received a single oral dose of the vaccine mixture and S. Typhimurium carrying pBP244 only as a control, respectively. In groups 3 and 4, mice were primed and boosted with the vaccine mixture and S. Typhimurium carrying pBP244 only as a control, respectively. In general, all immunized mice had significantly increased serum immunoglobulin (Ig)G (P < 0.05) and intestinal secretory IgA against the individual fimbrial antigens compared with those mice in the control group. In the IgG2a and IgG1 titer assay, only IgG2a titer was increased in group 1, while both IgG2a and IgG1 titers were increased in group 3. Furthermore, the vaccine strains were not detected in the excreted feces of any immunized mice. Thus, the vaccine candidate can be highly immunogenic and be safe to the environment.

Immunogenicity of a combined schedule of 7-valent pneumococcal conjugate vaccine followed by a 23-valent polysaccharide vaccine in adult recipients of heart or lung transplants.

A combined schedule of 7-valent pneumococcal conjugate vaccine (PCV7) followed by 23-valent pneumococcal polysaccharide vaccine (PPV23) was evaluated retrospectively in 26 adult recipients of heart or lung transplants. PCV7 was immunogenic in these patients but there appeared to be no benefit from the additional PPV23 dose.

Cyclooxygenase-1 orchestrates germinal center formation and antibody class-switch via regulation of IL-17.

The cyclooxygenase (COX) enzymes are known modulators of innate immune cell function; however, their contributions to adaptive immunity are relatively unknown. We investigated the roles of COX-1 and COX-2 in the humoral immune response to infection with the Lyme disease pathogen Borrelia burgdorferi. We report that in vitro, murine B cells constitutively expressed COX-1 and up-regulated expression of both COX-1 and COX-2 as well as their products PGE(2), PGF(2alpha), and thromboxane B(2) and their receptors following stimulation with B. burgdorferi or anti-CD40. In vitro inhibition of COX-1 and/or COX-2 in murine B cells resulted in decreased eicosanoid production and altered Ab production. Importantly, infection of mice lacking COX-1, but not COX-2, activity resulted in a defect in Ig class-switching and a lack of Borrelia-specific IgG production. This defect correlated with decreased germinal center formation and IL-6 and IL-17 production, and it could be partially recovered by restoration of IL-6, but fully recovered by IL-17. Furthermore, sera from COX-1 inhibitor-treated mice were dramatically less effective in killing B. burgdorferi, but borreliacidal activity was restored in COX-1 inhibitor-treated mice administered IL-17. We conclude that IL-17 plays a role in Ab production and Ig class-switching in response to infection and that COX-1 is a critical, previously unrecognized regulator of this response.

Bovine immune response to papillomatous digital dermatitis (PDD)-associated spirochetes is skewed in isolate reactivity and subclass elicitation.

Papillomatous digital dermatitis (PDD) is a growing cause of lameness of dairy cattle worldwide. Farms with PDD-afflicted cows experience economic loss due to treatment costs, decreased milk production, lower reproductive efficiency and premature culling. Cows exhibit both humoral and cellular immune responses to PDD-associated spirochetes. This study was undertaken to further characterize the bovine humoral response to PDD-associated spirochetes. Forty-seven sera samples collected from cattle (Field cattle) on three different dairy operations in Iowa were analyzed. In addition, sera were obtained from six young steers (Test cattle) that received a mixed inoculum of four previously isolated Treponema phagedenis-like spirochetes (1A, 3A, 4A and 5B) on two separate occasions. Relative levels of total IgG, IgG1, IgG2 and IgM reactive to each individual spirochete were determined. Field cattle had a higher mean antibody response to 5B compared to the other isolates and T. phagedenis. Test cattle reacted most strongly with 4A following initial exposure, shifting to a greater reactivity with 5B and a reactivity profile similar to field cattle following secondary exposure. No measurable IgM was detected. IgG1 was produced predominately in all cattle. Low to moderate levels of total IgG reactivity to T. phagedenis occurred with sera from all cattle.

Comparison of serum humoral responses induced by oral immunization with the hepatitis B virus core antigen and the cholera toxin B subunit.

The hepatitis B virus core (HBc) virus-like particle (VLP) is known as one of the most immunogenic antigens and carrier vehicles in different immunization strategies. Recent findings are suggesting the potential of the HBc VLPs as an oral immunogen. Here, we focus on the induction of serum humoral responses by oral administration of HBc VLPs in preparations substantially free of lipopolysaccharide and immunomodulating encapsidated RNA. The full-length HBc antigen was used, because the C-terminal arginine-rich tail may contribute to the immunogenicity of the antigen as the region is involved in cell surface heparan sulfate binding and internalization of the protein. Serum antibody levels and isotypes were determined following oral administration of the HBc VLPs with the perspective of using the HBc VLP as an immunostimulatory and carrier molecule for epitopes of blood-borne diseases in oral immunization vaccination strategies. Following oral administration of the HBc VLP preparations to mice, a strong serum humoral response was induced with mainly immunoglobulin G2a (IgG2a) antibodies, pointing toward a Th1 response which is essential in the control of intracellular pathogens. Intraperitoneal immunization with the HBc VLP induced a stronger, mixed Th1/Th2 response. Finally, a comparison was made with the induced serum humoral response following oral administration of the recombinant cholera toxin B pentamer, a commonly used oral immunogen. These immunizations, in contrast, induced predominantly antibodies of the IgG1 isotype, indicative of a Th2 response. These data suggest that the HBc VLP can be an interesting carrier molecule in oral vaccine development.

Generation and characterization of hybridoma antibodies for immunotherapy of tularemia.

Tularemia is caused by the Gram-negative facultative intracellular bacterium Francisella tularensis, which has been classified as a category A select agent-a likely bioweapon. The high virulence of F. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. We have characterized 14 anti-Francisella hybridoma antibodies derived from mice infected with F. tularensis live vaccine strain (LVS) for potential use as immunotherapy of tularemia. All 14 antibodies cross-reacted with virulent F. tularensis type A clinical isolates, 8 bound to a purified preparation of LVS LPS, and 6 bound to five protein antigens, identified by proteome microarray analysis. An IgG2a antibody, reactive with the LPS preparation, conferred full protection when administered either systemically or intranasally to BALB/c mice post challenge with a lethal dose of intranasal LVS; three other antibodies prolonged survival. These anti-Francisella hybridoma antibodies could be converted to chimeric versions with mouse V regions and human C regions to serve as components of a recombinant polyclonal antibody for clinical testing as immunotherapy of tularemia. The current study is the first to employ proteome microarrays to identify the target antigens of anti-Francisella monoclonal antibodies and the first to demonstrate the systemic and intranasal efficacy of monoclonal antibodies for post-exposure treatment of respiratory tularemia.