Serum anti-toxin B antibody correlates with protection from recurrent Clostridium difficile infection (CDI)

Background

Previous studies have demonstrated a correlation between Clostridium difficile anti-toxin A serum antibodies and protection against symptomatic disease and recurrence.

Methods

A neutralizing monoclonal antibody to C. difficile toxin A (CDA1) developed by MBL and Medarex, Inc. was studied in a phase II, randomized, double-blind, placebo-controlled trial in patients receiving standard of care treatment for C. difficile infection (CDI). Twenty-nine subjects received a single intravenous infusion of 10 mg/kg CDA1 and 17 subjects received placebo and were evaluated for recurrence of CDI during the 56-day study period. Serum antibodies against C. difficile toxin A and B were measured by ELISA and cytotoxicity assay at various time points before and after infusion.

Findings

CDI recurrence occurred in 5 of 29 (17%) in the CDA1 group and 3 of 17 (18%) (p = NS) in the placebo group with a trend toward delay in time to recurrence in the group treated with CDA1. The geometric mean concentration of antibody to an epitope of the receptor-binding domain of toxin B (0.300 and 1.20 μg/ml, respectively; p = 0.02) and geometric mean titer of neutralizing B antibody (8.00 and 100, respectively; p = 0.02) at study day 28 were lower for those subjects with recurrence compared to those who did not recur. In addition, a significantly greater proportion of subjects who recurred were infected with the epidemic BI/NAP1/027 strain compared with those that did not recur (88% vs. 22%; p = 0.002). Finally, in a multiple logistic regression analysis neutralizing anti-toxin B at day 14 (p < 0.001), anti-toxin A at day 28 (p < 0.001) and infection with the BI/NAP1/027 strain at enrollment (p = 0.002) were all predictive of CDI recurrence.

Interpretation

In this prospective study, lower concentrations of neutralizing anti-toxin B and anti-toxin A antibody and infection with the BI/NAP1/027 strain of C. difficile were significantly associated with recurrence of CDI.     

Active and passive immunization against Acinetobacter baumannii using an inactivated whole cell vaccine

The treatment of infections caused by Acinetobacter baumannii has become increasingly complicated due to the emergence of highly resistant strains. In the present study we demonstrate that immunization with an inactivated whole cell vaccine elicits a robust antibody response that provides protection against challenge with multiple A. baumannii strains in a murine model of disseminated sepsis. In addition, we show that passive immunization with serum raised against inactivated cells protects mice from subsequent infection. These results demonstrate that active and passive immunization using an inactivated whole cell vaccine may be an effective approach for preventing infection by A. baumannii.     

Functional and biological determinants affecting the duration of action and efficacy of anti-(+)-methamphetamine monoclonal antibodies in rats

These studies examined the in vivo pharmacokinetics and efficacy of five anti-methamphetamine monoclonal antibodies (mAbs, K_D values from 11 to 250 nM) in rats. While no substantive differences in mAb systemic clearance (t_1/2 = 6.1–6.9 days) were found, in vivo function was significantly reduced within 1–3 days for four of the five mAbs. Only mAb4G9 was capable of prolonged efficacy, as judged by prolonged high methamphetamine serum concentrations. MAb4G9 also maintained high amphetamine serum concentrations, along with reductions in methamphetamine and amphetamine brain concentrations, indicating neuroprotection. The combination of broad specificity for methamphetamine-like drugs, high affinity, and prolonged action in vivo suggests mAb4G9 is a potentially efficacious medication for treating human methamphetamine-related medical diseases.     

Alterations in immunodominance of Streptococcus mutans AgI/II: Lessons learned from immunomodulatory antibodies

Streptococcus mutans antigen I/II (AgI/II) has been widely studied as a candidate vaccine antigen against human dental caries. In this report we follow up on prior studies that indicated that anti-AgI/II immunomodulatory monoclonal antibodies (MAbs) exerted their effects by destabilizing the native protein structure and exposing cryptic epitopes. We show here that similar results can be obtained by immunizing mice with truncated polypeptides out of the context of an intra-molecular interaction that occurs within the full-length molecule and that appears to dampen the functional response against at least two important target epitopes. Putative T cell epitopes that influenced antibody specificity were identified immediately upstream of the alanine-rich repeat domain. Adherence inhibiting antibodies could be induced against two discrete domains of the protein, one corresponding to the central portion of the molecule and the other corresponding to the C-terminus.     

Malaria co-infection in children influences antibody response to schistosome antigens and inflammatory markers associated with morbidity

The epidemiological coexistence of schistosomiasis and malaria is frequently observed in developing countries. Co-infection with malaria in children could influence the development of acquired immunity associated with the resistance or the pathology of schistosomiasis. In the present study, performed during May to June 1996 in Senegal, the humoral immune response to Schistosoma haematobium 28 kDa glutathione S-transferase (Sh28GST) vaccinal antigen and to soluble egg antigens (SEA) has been evaluated in individuals infected by S. haematobium. Specific immunoglobulin G3 (IgG3) and IgE responses were significantly higher in co-infected children with Plasmodium falciparum compared with children infected with S. haematobium only. In addition, circulating levels of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and soluble tumor necrosis factor receptor II (sTNF-RII), 3 parameters associated with schistosomiasis morbidity, were significantly increased in co-infected children. Taken together, this study indicated that malaria co-infection can both influence the acquired specific immune response to schistosome antigens and unbalance the regulation of inflammatory factors closely involved in schistosomiasis pathology.     

Protection against Vibrio vulnificus infection by active and passive immunization with the C-terminal region of the RtxA1/MARTXVv protein

Vibrio vulnificus is a foodborne pathogen that is prevalent in coastal waters worldwide. Infection with V. vulnificus causes septicemia with fatality rates exceeding 50% even with aggressive antibiotic therapy. Several vaccine studies to prevent V. vulnificus infection have been performed but have had limited success. In this study, we identified the C-terminal region (amino acids 3491 to 4701) of the V. vulnificus multifunctional autoprocessing RTX (MARTX_Vv or RtxA1) protein, RtxA1-C, as a promising antigen that induces protective immune responses against V. vulnificus. Vaccination of mice with recombinant RtxA1-C protein with adjuvant elicited a robust antibody response and a dramatic reduction in blood bacterial load in mice infected intraperitoneally. Vaccination resulted in significant protection against lethal challenge with V. vulnificus. Furthermore, intraperitoneal passive immunization with serum raised against the recombinant RtxA1-C protein demonstrated marked efficacy in both prophylaxis and therapy. These results suggest that active and passive immunization against the C-terminal region of the RtxA1 protein may be an effective approach in the prevention and therapy of V. vulnificus infections.     

Corynebacterium pyruviciproducens promotes the production of ovalbumin specific antibody via stimulating dendritic cell differentiation and up-regulating Th2 biased immune response

Corynebacterium pyruviciproducens (C. pyruviciproducens), a newly discovered Corynebacterium, is gram-positive, non-flagellate, non-spore-forming lipophilic rod. No known pathogenic components of Corynebacteria have been found in this new bacterium, such as diphtheria toxin and tuberculostearic acid. In the present study, referring to Propionibacterium acnes (P. acnes), a well-known bacterial adjuvant, the stimulation of dendritic cells by C. pyruviciproducens was analyzed through detecting the levels of cytokine-secretion, ability of cell-proliferation and expression of membrane molecules. In addition, the effect of C. pyruviciproducens in promoting antibody production in vivo was detected. Compared with P. acnes, C. pyruviciproducens more strongly enhanced cytokine secretion including inflammatory factor IL-6 and Th1-associated molecule IL-12, and more effectively induced proliferation, activation or maturation of D2SC/1 (a murine dendritic cell line) and bone marrow-derived dendritic cells (BMDC). Vaccination studies in mice using ovalbumin (OVA) as a model antigen showed that C. pyruviciproducens effectively promoted antigen-specific humoral immune response by increasing OVA-specific antibody, Th2-biased response in spleen and high IL-4/IFN-γ ratio in serum.     

Evaluation of the adjuvant properties of Astragalus membranaceus and Scutellaria baicalensis GEORGI in the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models

Human vaccines are not available and current anti-toxoplasma treatment is disappointing. To investigate the possible adjuvant effect of aqueous extracts obtained from medicinal herbs of Astragalus membranaceus (Am) and Scutellaria baicalensis GEORGI (Sb) on the immune response to Toxoplasma gondii in the mouse models induced by ultraviolet (UV)-attenuated T. gondii, this paper studies the possible vaccination strategies to help combat infections with Toxoplasma and looking towards developing new vaccine and approaches. We used UV-attenuated T. gondii (UV-T.g) of RH strain as a vaccine and the extracts of Am (AmE) and Sb (SbE) as adjuvant. Mice were infected by intraperitoneal (i.p.) injection of 10² RH tachyzoites alone (infected controls), infected and treated with AmE (T.g + AmE) and SbE (T.g + SbE), respectively; and mice immunized i.p. with UV-T.g alone, UV-T.g co-administrated with AmE (UV-T.g + AmE) or SbE (UV-T.g + SbE), and then challenged with T.g, respectively. The animal survival time, parasite burden in peritoneal lavage fluids, liver histopathological analysis, and levels of serum antibodies among the groups were compared after either infection or challenge. The results showed that, compared to infected controls, infected mice treated with AmE or SbE, or vaccinated mice and then challenged, had significantly prolonged survival time, decreased parasite burden, improved liver histopathological score, and increased Th1-type cellular immune response; furthermore, vaccinated mice co-administrated with AmE or SbE had even longer survival, lower parasite burden, lower liver histopathological score, and higher Th1 response after challenge. Our data demonstrated that the protective immunity of UV-attenuated T. gondii could be markedly enhanced by AmE or SbE co-administration, which suggests that both AmE and SbE may have the potential to be used as effective vaccine adjuvant.     

Co-colonization by Haemophilus influenzae with Streptococcus pneumoniae enhances pneumococcal-specific antibody response in young children

Background

Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mcat) are common bacterial pathogens of respiratory infections and common commensal microbes in the human nasopharynx (NP). The effect of interactions among theses bacteria during co-colonization of the NP on the host immune response has not been evaluated. The objective of this study was to assess the impact of co-colonization by Hi or Mcat on the systemic antibody response to vaccine protein candidate antigens of Spn and similarly the impact of co-colonization by Spn and Mcat on antibody responses to Hi vaccine protein candidate antigens.

Methods

Serum samples were collected from healthy children at 6, 9, 15, 18, and 24 months of age when they were colonized with Spn, Hi, Mcat or their combinations. Quantitative ELISA was used to determine serum IgA and IgG against three Spn antigens and three Hi antigens, and as well as whole cells of non-typeable (NT) Spn and Hi.

Results

NP colonization by Spn increased serum IgA and IgG titers against Spn antigens PhtD, PcpA and PlyD and whole cells of NTSpn, and co-colonization of Hi or Mcat with Spn resulted in further increases of serum pneumococcal-specific antibody levels. NP colonization by Hi increased serum IgA and IgG titers against Hi antigens P6, Protein D and OMP26 and whole cells of NTHi, but co-colonization of Spn or Mcat with Hi did not result in further increase of serum NTHi-specific antibody levels.

Conclusion

Co-colonization of Hi or Mcat with Spn enhances serum antibody response to NTSpn whole cells and Spn vaccine candidate antigens PhtD, PcPA and PlyD1. Co-colonization appears to variably modulate pathogen species-specific host adaptive immune response.     

Epitope mapping of a common 57 kDa antigen of Leishmania species by monoclonal antibodies

BALB/c mice were immunized with freeze-thawed promastigote of Leishmania infantum. Five monoclonal antibodies (mAb) were selected, four IgM (designated as P1A9, P2G8, P5E3 and P6B3) and one IgG1 (P3D2). ELISA and Western blot analysis suggested that all monoclonal antibodies are specific to a band of 57 kDa antigen of L. infantum as well as other three Leishmania species (L. tropica, L. major and L. donovani). ELISA additivity tests revealed four epitopes on 57 kDa antigen as defined by four IgM monoclonal antibodies. Three distinct epitopes were recognized by P1A9, P2G8, and P6B3 antibodies and one epitope recognized by P5E3 antibody that shared with P2G8, and P6B3 epitopes. The 57 kDa protein was purified with affinity column and was shown to possess proteolytic activity. It seems that 57 kDa protein is the major surface Leishmania antigen (gp63) that has been used as subunit vaccine with appropriate adjuvant and induced protection against L. major infection in BALB/c mice.     

A recombinant bivalent fusion protein rVE confers active and passive protection against Yersinia enterocolitica infection in mice

In the present study, a bivalent chimeric protein rVE comprising immunologically active domains of Yersinia pestis LcrV and YopE was assessed for its prophylactic abilities against Yersinia enterocolitica O:8 infection in murine model. Mice immunized with rVE elicited significantly higher antibody titers with substantial contribution from the rV component (3:1 ratio). Robust and significant resistance to Y. enterocolitica infection with 100% survival (P < 0.001) was seen in rVE vaccinated mice when intra peritoneal (I.P.) challenged with 10⁸ CFU of Y. enterocolitica O:8 against the 75%, 60% and 75% survival seen in mice immunized with rV, rE, rV + rE, respectively. Macrophage monolayer supplemented with anti-rVE polysera illustrated efficient protection (89.41% survival) against challenge of Y. enterocolitica O:8. In contrast to sera from sham-immunized mice, immunization with anti-rVE polysera provided complete protection to BALB/c mice against I.P. challenge with 10⁸ CFU of Y. enterocolitica O:8 and developed no conspicuous signs of infection in necropsy. The histopathological analysis of microtome sections confirmed significantly reduced lesion size or no lesion in liver and intestine upon infection in anti-rVE immunized mice. The findings from this study demonstrated the fusion protein rVE as a potential candidate subunit vaccine and showed the functional role of antibodies in protection against Y. enterocolitica infections.     

A humanized monoclonal antibody targeting protein a promotes opsonophagocytosis of Staphylococcus aureus in human umbilical cord blood

Low and very-low-birth-weight (V/LBW) neonates are highly susceptible to bacterial sepsis and meningitis. Bacterial infections caused by Staphylococcus aureus can be particularly dangerous for neonates and can result in high mortality and long-term disabilities. Antibody-based strategies have been attempted to protect V/LBW neonates against staphylococcal disease. However, these efforts have so far been unsuccessful. Failures were attributed to the immaturity of the neonatal immune system but did not account for the anti-opsonic activity of Staphylococcal protein A (SpA). Here we show that monoclonal antibody 3F6, which blocks SpA activity, promotes complement-dependent cell-mediated phagocytosis of S. aureus in human umbilical cord blood. A substitution in the crystallizable fragment (Fc) region of 3F6 that enhances recruitment of complement component C1q further increases the phagocytic activity of cord blood. Our data demonstrate that the neonatal immune system possesses bactericidal activity that can be harnessed by antibodies that circumvent a key innate immune strategy of S. aureus.     

Vaccination with the Mycoplasma suis recombinant adhesion protein MSG1 elicits a strong immune response but fails to induce protection in pigs

Mycoplasma suis is the unculturable pathogen of porcine infectious anemia. The study was aimed to determine the immunogenicity and protective efficacy of MSG1, an immunodominant adhesin of M. suis as the first vaccine candidate against M. suis. The results demonstrated that recombinant MSG1 and Escherichia coli transformants expressing MSG1 (E. coli_MSG1) induced a strong humoral and cellular immunity against M. suis. The induced antibodies were found to be functionally active as confirmed by an in vitro adhesion inhibition assay. Both, IgG1 and IgG2 antibodies were induced, but E. coli_MSG1 immune response was characterized by a significantly higher IgG1 antibody production. Both vaccine candidates failed to protect against M. suis challenge. However, E. coli_MSG1 vaccination has a considerable effect on the severity of the disease as shown by higher post-challenge hemoglobin and hematocrit values in comparison to control groups. This indicated that a high IgG1 antibody titer is negatively connected with severity of M. suis-induced anemia. Furthermore, the induction of monospecific anti-MSG1 antibodies by both vaccine candidates clearly allows for the differentiation between infected and vaccinated animals (DIVA principle). Overall, the importance of MSG1 as potential vaccine candidate remains to be established. Future studies will evaluate the conditions (i.e. adjuvant, vaccination scheme, and application route) to optimize the effects of E. coli_MSG1 vaccines.     

Multi-serotype outer membrane vesicles of Shigellae confer passive protection to the neonatal mice against shigellosis

Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 Δstx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50 μg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3–4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future.     

Mucosal and systemic immunization with a novel attenuated pneumococcal vaccine candidate confer serotype independent protection against Streptococcus pneumoniae in mice

Despite the availability of effective vaccines, Streptococcus pneumoniae is still one of the major infectious diseases causing substantial morbidity and mortality in children under 5 years old. In this study, we demonstrate the protective efficacy of S. pneumoniae SPY1, a novel live attenuated vaccine strain against pneumococcal infection in murine models. This strain was characterized by defects in three important pneumococcal virulence factors including capsule, teichoic acids and pneumolysin. The lactate dehydrogenase assays and in vivo animal experiments demonstrated a significantly attenuated virulence and a reduced nasopharyngeal colonization for the SPY1 strain. We also show that mucosal and systemic immunization with the live SPY1 strain induced protective immune responses against pneumococci. Mucosal immunization with SPY1 offered better protection against colonization challenge with strains TIGR4 and serotype 19F than systemic SPY1 immunization. In invasive infection models, mucosal vaccination with the SPY1 strain conferred complete protection against D39 and clinical serotype 6B and 3 strains. Notably, intranasal vaccination with the SPY1 strain conferred superior protection against pneumococcal invasive disease compared with the commercial available vaccines. SPY1 strain was shown to elicit high levels of serotype-independent antibodies and a mixed cellular immune response. Besides, the SPY1 serum was able to passively protect mice against invasive challenge with D39 strain, indicating the protective effect of the antibody-mediated responses. Together, the SPY1 strain may be a promising live vaccine strain to protect pneumococcal infection.     

Role of antibodies in Sm-p80-mediated protection against Schistosoma mansoni challenge infection in murine and nonhuman primate models

Schistosomiasis is an important public health concern in more than 76 developing countries. Advent of an anti-schistosome vaccine would undoubtedly add to the existing control measures and may eventually help in the elimination of this disease. In the present study we have attempted to dissect the role(s) of antibodies in Sm-p80 mediated protection by intravenously transferring pooled sera from mice immunized with Sm-p80-pcDNA3 or purified IgG from baboons immunized with Sm-p80-pcDNA3, into naïve C57BL/6 mice, respectively, prior to challenge with cercariae. The passive transfer of antibodies from protected mice (homologous transfers) as well as transfer of total IgG from baboons (heterologous transfers), into naïve mice showed statistically significant reductions in worm burden and in the number of eggs in the tissues. Immunizations of antibody knockout mice (μMt−/−; B10.129S2 (B6)-Igh-6^tm1Cgn/J) with recombinant Sm-p80 in the presence of CpG-motif oligodeoxynucleotides as an adjuvant, resulted in substantial reduction of Sm-p80-mediated protection, compared to C57BL/6 (normal) control group of mice. Down regulation of cytokines that have important effects on B cell proliferation as well as the recovery of higher number of parasites in antibody knockout indicated a significant role(s) of antibodies in Sm-p80-mediated protection against Schistosoma mansoni in mice. In toto, these studies appear to suggest that antibodies play a significant role in Sm-p80 mediated protection.     

My continuing efforts toward the eradication of the vaccine-preventable diseases from Japan

When compared to the Western countries, unfortunately, the progress toward the eradication of vaccine-preventable diseases in Japan has been relatively slow and hampered by some problems inherent to Japan. Concerning this, I have been working on the vaccine-preventable diseases, that is, measles, influenza, Haemophilus influenzae, and Streptcoccus pneumoniae infections. My first work was to produce monoclonal antibodies against measles virus proteins, and by using these antibodies, I established the immunofluorescent measles virus detection system in tissue samples. I constructed the ‘Hokkaido measles-zero strategy’ to eliminate measles from Hokkaido prefecture, the northernmost island of Japan, within 5 years since 2001 with very kind efforts of pediatricians in Hokkaido and those who are engaged in political activities. Coworkers and I established the disease entity of influenza-associated encephalopathy. And finally, I worked as a member of some groups that accumulated the basic data on the occurrence of bacterial meningitis in Japan and urged the government to introduce the H. influenzae type b conjugate vaccine and the pediatric 7-valent pneumococcal conjugate vaccine in Japan. I would like to express many thanks to all of my colleagues for their contributions to these achievements.     

A genome-wide association study of host genetic determinants of the antibody response to Anthrax Vaccine Adsorbed

Several lines of evidence have supported a host genetic contribution to vaccine response, but genome-wide assessments for specific determinants have been sparse. Here we describe a genome-wide association study (GWAS) of protective antigen-specific antibody (AbPA) responses among 726 European-Americans who received Anthrax Vaccine Adsorbed (AVA) as part of a clinical trial. After quality control, 736,996 SNPs were tested for association with the AbPA response to 3 or 4 AVA vaccinations given over a 6-month period. No SNP achieved the threshold of genome-wide significance (p=5 × 10(-8)), but suggestive associations (p<1 × 10(-5)) were observed for SNPs in or near the class II region of the major histocompatibility complex (MHC), in the promoter region of SPSB1, and adjacent to MEX3C. Multivariable regression modeling suggested that much of the association signal within the MHC corresponded to previously identified HLA DR-DQ haplotypes involving component HLA-DRB1 alleles of *15:01, *01:01, or *01:02. We estimated the proportion of additive genetic variance explained by common SNP variation for the AbPA response after the 6 month vaccination. This analysis indicated a significant, albeit imprecisely estimated, contribution of variation tagged by common polymorphisms (p=0.032). Future studies will be required to replicate these findings in European Americans and to further elucidate the host genetic factors underlying variable immune response to AVA.     

Hepatocyte lesions and cellular immune response in yellow fever infection

The study of the in-situ cellular immune response is very important for the understanding of different liver infections. In the present study, 53 liver samples obtained by viscerotomy from patients who died during the course of jungle yellow fever were analyzed. The diagnosis was confirmed by serology, viral isolation and virus-specific immunohistochemistry. The specimens were analyzed by immunohistochemistry using specific antibodies for apoptosis, CD45RO, CD4, CD8, CD20, S100, CD57 and CD68. Quantitative analysis of the labeling pattern showed a clear predominance of the different phenotypes in the portal tract and midzone region of the acini. There was a predominance of T CD4+ lymphocytes, accompanied by the presence of T CD8+ lymphocytes, natural killer cells (CD57), macrophages and antigen-presenting cells (S100). The disproportion between the intensity of inflammation and the degree of hepatic injury was probably due to the intense apoptotic component, which classically does not induce an inflammatory response. The present study demonstrates that, despite the disproportion between injury and inflammation, the cellular immune response plays an important role in the pathogenesis of the hepatocytic injury observed in yellow fever, probably as a result of cytolytic actions through mechanisms involving MHC II and the activation of Fas receptors and granzymes/perforins.