Antibodies In Sera Of Mice Research Articles

Functional epitopes on hepatitis E virions and recombinant capsids are highly conformation-dependent

ABSTRACT Hepatitis E virus (HEV) is responsible for epidemic and sporadic acute hepatitis cases, especially in developing countries. Hepatitis E has become a vaccine-preventable disease in recent years with the development of a licensed vaccine. Most functional and neutralizing monoclonal antibodies (mAbs) are known to be highly sensitive to antigen conformation. In this study, a similar approach was used to characterize the conformational sensitivity of antibodies in human or mouse serum samples. Interestingly, comparative binding analysis using different antigen forms showed that the antibodies in the sera of naturally infected individuals, of human vaccinees and from mice immunized with the HEV p239 vaccine all exhibited a strong preference to particulate antigens over the monomeric form of the truncated capsid protein. The degree of discriminating the two test antigens is similar for serum samples to that for the well-characterized murine mAbs. A functional assay for assessing the inhibition of subviral particle cell entry by antibodies was used to determine the functional titers of anti-HEV antibodies in mouse sera. A good correlation was observed between the functional and binding titers in mouse sera determined using two different methods. This result supports the continued use of the enzyme-linked immunosorbent assay as the primary serological assay assuming that the coating antigen contains conformational and native-like epitopes, as in the case for HEV p239.

Abstract 1467: Bioengineering AAV6 vector-based vaccine for cancer treatment

Abstract Introduction: Adeno-associated virus (AAV) is a small virus of the parvovirus family and is widely used in clinical-stage gene therapy because of its low toxicity and limited side effects. There are a number of naturally occurring serotypes with different tissue tropism and further engineering of the viral capsid can increase the vectors suitability for use as a cancer vaccine. We recently showed that AAV serotype 6 (AAV6) successfully delivers a transgene to dendritic cells (DC) in vitro. In the present study we analyze several rationally designed AAV6 mutants to more efficiently target DC and further increase an antigen-specific immune response. Methods: AAV6 mutants encoding ovalbumin, tyrosinase, or GFP were developed and produced in the lab by standard procedure. Vaccination was performed as a single intramuscular (i.m.) injection in C57BL/6 mice. The immune response was measured against both ovalbumin (Ova) as a model antigen and tyrosinase (Tyr) as an endogenous antigen. The Ova-specific CD8 T-cells were identified in peripheral blood and splenocytes by binding epitope-specific tetramers. Activity of antigen-specific CD8 cells was estimated by IFNy production measured by ELISpot, and by in vivo and in vitro killing assays. A humoral response was analyzed by the presence of antigen-specific antibodies in mouse serum measured by ELISA or by Western Blotting. Vaccination with GFP was used to show direct antigen expression in DC. Briefly, 3 days after i.m. injection DC from drained lymph nodes were isolated by FACS (CD11c+/MHC II+) and the presence of GFP expression was analyzed by wide-field fluorescence microscopy. Results: Several significant observations were made: (i) optimized AAV6 directly targets DC in vivo as evidenced by the presence of GFP positive DC in mouse drained lymph nodes after i.m. injection with AAV6-GFP; (ii) AAV6 vectors induced a strong immune response not only against a strong immunogen such as Ova but also against a weak endogenous antigen such as Tyr. (iii) Transgene-specific cytotoxic CD8 T- cells showed superior killing capabilities against cancer cells in vitro and in cytotoxic assay in vivo. (iv) AAV6 based vaccine generates high transgene-specific antibodies titers detectable starting 3 weeks after injection. Our data allows us to choose the AAV6 mutant with superior capacity to induce an immune response against a transgene. As a future direction we will optimize the expression cassette to directly rout an antigen into the MHC class II processing and presentation pathway which is known to dramatically increase vaccine potency. The combination of optimized AAV capsid and expression cassette will be used to confirm an ability of our vaccine approach to reduce tumor growth and extend survival in a murine tumor model. In addition, our technology is highly compatible with, and complementary to current cancer treatments, including other immunotherapy approaches such as checkpoint (PD-1/PD-L1) inhibitors. Citation Format: Karina Krotova, Andrew Day, Edward Hinchcliffe, George Aslanidi. Bioengineering AAV6 vector-based vaccine for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1467.

Improved Antitumor Efficacy of Combined Vaccine Based on the Induced HUVECs and DC-CT26 Against Colorectal Carcinoma.

Angiogenesis is essential for the development, growth, and metastasis of solid tumors. Vaccination with viable human umbilical vein endothelial cells (HUVECs) has been used for antitumor angiogenesis. However, the limited immune response induced by HUVECs hinders their clinical application. In the present study, we found that HUVECs induced by a tumor microenvironment using the supernatant of murine CT26 colorectal cancer cells exerted a better antiangiogenic effect than HUVECs themselves. The inhibitory effect on tumor growth in the induced HUVEC group was significantly better than that of the HUVEC group, and the induced HUVEC group showed a strong inhibition in CD31-positive microvessel density in the tumor tissues. Moreover, the level of anti-induced HUVEC membrane protein antibody in mouse serum was profoundly higher in the induced HUVEC group than in the HUVEC group. Based on this, the antitumor effect of a vaccine with a combination of induced HUVECs and dendritic cell-loading CT26 antigen (DC-CT26) was evaluated. Notably, the microvessel density of tumor specimens was significantly lower in the combined vaccine group than in the control groups. Furthermore, the spleen index, the killing effect of cytotoxic T lymphocytes (CTLs), and the concentration of interferon-γ in the serum were enhanced in the combined vaccine group. Based on these results, the combined vaccine targeting both tumor angiogenesis and tumor cells may be an attractive and effective cancer immunotherapy strategy.

Recombinant lactococcus lactis secreting viral protein 1 of enterovirus 71 and its immunogenicity in mice.

To construct recombinant Lactococcus lactis (L. lactis) expressing viral protein 1 (VP1) of enterovirus 71 (EV71) and evaluate its immunogenicity to be used as an oral vaccine in BALB/c mice. Recombinant L. lactis competent in secreting VP1 (~ 30kDa) into the extracellular environment with the aid of the signal peptide Usp45 was produced. Enzyme-linked immunosorbent assay showed that significant VP1-specific antibody response including the production of both serum IgG and fecal IgA (p < 0.05) was elicited in BALB/c mice upon oral immunization with recombinant L. lactis. Moreover, in contrast to negative control, recombinant L. lactis induced adequate neutralizing antibodies in mouse sera (p < 0.05) as demonstrated in virus neutralization assay, whereas the presence of neutralizing antibodies in fecal samples was obvious but not significant (p > 0.05). Recombinant L. lactis expressing VP1 of EV71 has the potential to be used as an oral vaccine candidate. The findings may provide some preliminary evidences for further development of effective and needle-free EV71 vaccines.

Regulatory effect of humoral milieu on the viral DNA and surface antigen expression of hepatitis B virus (HBV) in vitro

While a few virions of hepatitis B virus (HBV) are sufficient to liver infection in vivo, even a large quantity of virion is unable to enter into hepatoma cells in vitro. To solve this paradox, we explored if humoral milieu such as serum or culture media, and its constituents, and pH would regulate the viral DNA and surface antigen expression of HBV in vitro. The viral DNA and surface antigen expression of HBV are significantly more stable in human serum than in mouse serum, and Dulbecco’s Modified eagle Medium (DMEM). In line, the increase of anti-HBcAg antibody in mouse serum, and DMEM implies that the virion was cleared in non-host humoral milieu. Further, lipid analysis revealed higher levels of lipids in human serum than those in mouse serum, and DMEM. Lipid removal analysis showed the decreased level of HBV DNA and surface antigen expression in human and mouse serum. In electrolytes analysis, human serum contained lower level of potassium than mouse serum. Last, on the viral DNA and surface antigen expression of HBV, the alkalinity of DMEM was more effective than neutral pH. Collectively, this study indicates that humoral milieu would regulate the viral DNA and surface antigen expression of HBV in vitro. This is the first report to regulate HBV growth/viability via simple manipulation of host humoral milieu.

Purification, characterization and immunogenicity assessment of glutaminase free L-asparaginase from Streptomyces brollosae NEAE-115

BackgroundL-asparaginase is a potential therapeutic enzyme widely used in the chemotherapy protocols of pediatric and adult patients with acute lymphoblastic leukemia. However, its use has been limited by a high rate of hypersensitivity in the long-term used. Hence, there is a continuing need to search for other L-asparaginase sources capable of producing an enzyme with less adverse effects.MethodsProduction of extracellular L-asparaginase by Streptomyces brollosae NEAE-115 was carried out using submerged fermentation. L-asparaginase was purified by ammonium sulphate precipitation and pure enzyme was reached using ion-exchange chromatography, followed by enzyme characterization. Anticancer activity towards Ehrlich Ascites Carcinoma (EAC) cells was investigated in female Swiss albino mice by determination of tumor size and the degree of tumor growth inhibition. The levels of anti-L-asparaginase IgG antibodies in mice sera were measured using ELISA method.ResultsThe purified L-asparaginase showed a total activity of 795.152 with specific activity of 76.671 U/mg protein and 7.835 − purification fold. The enzyme purity was confirmed by using SDS–PAGE separation which revealed only one distinctive band with a molecular weight of 67 KDa. The enzyme showed maximum activity at pH 8.5, optimum temperature of 37 °C, incubation time of 50 min and optimum substrate concentration of 7 mM. A Michaelis-Menten constant analysis showed a Km value of 2.139 × 10− 3 M with L-asparagine as substrate and Vmax of 152.6 UmL− 1 min− 1. The half-life time (T1/2) was 65.02 min at 50°С, while being 62.65 min at 60°С. Furthermore, mice treated with Streptomyces brollosae NEAE-115 L-asparaginase showed higher cytotoxic effect (79% tumor growth inhibition) when compared to commercial L-asparaginase group (67% tumor growth inhibition).ConclusionsThe study reveals the excellent property of this enzyme which makes it highly valuable for development of chemotherapeutic drug.

A virus-like particle vaccine protects mice against coxsackievirus A10 lethal infection

Coxsackievirus A10 (CVA10) has emerged worldwide as one of the main pathogens of hand, foot, and mouth disease (HFMD) in recent years. However, there is currently no commercial vaccine available to prevent CVA10 infection. Here we report the development of a recombinant virus-like particle (VLP) based candidate vaccine for CVA10. Co-expression of the capsid protein precursor P1 and the protease 3CD of CVA10 in Pichia pastoris resulted in cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3. These three subunit proteins co-assembled into CVA10 VLPs, which were visualized as spherical particles with a diameter of ∼30 nm under electron microscope. Immunization studies showed that CVA10 VLP could efficiently induce antigen-specific serum antibodies in mice. The anti-VLP sera were able to potently neutralize homologous and heterologous CVA10 strains. Importantly, passively transferred anti-VLP sera fully protected recipient neonatal mice from lethal CVA10 infection. In addition, neonatal mice born to the VLP-immunized dams were also completely protected from CVA10 lethal challenge. Collectively, these data show that CVA10 VLP represents a promising CVA10 vaccine candidate.

Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection.

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection.

Passive transfer autoimmunity in a mouse model of complex regional pain syndrome.

It has been proposed that complex regional pain syndrome (CRPS) is a posttraumatic autoimmune disease, and we previously observed that B cells are required for the full expression of CRPS-like changes in a mouse tibia fracture CRPS model. The current study used the mouse model to evaluate the progression of postfracture CRPS-like changes in wild-type (WT) and muMT fracture mice lacking B cells and antibodies. The pronociceptive effects of injecting WT fracture mouse serum antibodies into muMT fracture mice were also evaluated. Postfracture pain behaviors transitioned from being initially dependent on both innate and autoimmune inflammatory mechanisms at 3 weeks after fracture to being entirely mediated by antibody responses at 12 weeks after fracture and spontaneously resolving by 21 weeks after fracture. Furthermore, serum IgM antibodies from WT fracture mice had pronociceptive effects in the fracture limb when injected into muMT fracture mice. IgM antibody levels gradually increased in the fracture limb hind paw skin, sciatic nerve, and corresponding lumbar cord, peaking at 12 to 18 weeks after fracture and then declining. Immunohistochemistry localized postfracture IgM antibody binding to antigens in the fracture limb hind paw dermal cell nuclei. We postulate that fracture induces expression of neoantigens in the fracture limb skin, sciatic nerve, and cord, which trigger B cells to secret IgM antibodies that bind those antigens and initiate a pronociceptive antibody response. Autoimmunity plays a key role in the progression of nociceptive and vascular changes in the mouse fracture model and potentially contributes to the CRPS disease process.

Vi Capsular Polysaccharide Produced by Recombinant Salmonella enterica Serovar Paratyphi A Confers Immunoprotection against Infection by Salmonella enterica Serovar Typhi

Enteric fever is predominantly caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A, and accounts for an annual global incidence of 26.9 millions. In recent years, the rate of S. Paratyphi A infection has progressively increased. Currently licensed vaccines for typhoid fever, live Ty21a vaccine, Vi subunit vaccine, and Vi-conjugate vaccine, confer inadequate cross immunoprotection against enteric fever caused by S. Paratyphi A. Therefore, development of bivalent vaccines against enteric fever is urgently required. The immunogenic Vi capsular polysaccharide is characteristically produced in S. Typhi, but it is absent in S. Paratyphi A. We propose that engineering synthesis of Vi in S. Paratyphi A live-attenuated vaccine may expand its protection range to cover S. Typhi. In this study, we cloned the viaB locus, which contains 10 genes responsible for Vi biosynthesis, and integrated into the chromosome of S. Paratyphi A CMCC 50093. Two virulence loci, htrA and phoPQ, were subsequently deleted to achieve a Vi-producing attenuated vaccine candidate. Our data showed that, despite more than 200 passages, the viaB locus was stably maintained in the chromosome of S. Paratyphi A and produced the Vi polysaccharide. Nasal immunization of the vaccine candidate stimulated high levels of Vi-specific and S. Paratyphi A-specific antibodies in mice sera as well as total sIgA in intestinal contents, and showed significant protection against wild-type challenge of S. Paratyphi A or S. Typhi. Our study show that the Vi-producing attenuated S. Paratyphi A is a promising bivalent vaccine candidate for the prevention of enteric fever.

MEFA (multiepitope fusion antigen)-Novel Technology for Structural Vaccinology, Proof from Computational and Empirical Immunogenicity Characterization of an Enterotoxigenic Escherichia coli (ETEC) Adhesin MEFA.

Vaccine development often encounters the challenge of virulence heterogeneity. Enterotoxigenic Escherichia coli (ETEC) bacteria producing immunologically heterogeneous virulence factors are a leading cause of children’s diarrhea and travelers’ diarrhea. Currently, we do not have licensed vaccines against ETEC bacteria. While conventional methods continue to make progress but encounter challenge, new computational and structure-based approaches are explored to accelerate ETEC vaccine development. In this study, we applied a structural vaccinology concept to construct a structure-based multiepitope fusion antigen (MEFA) to carry representing epitopes of the seven most important ETEC adhesins [CFA/I, CFA/II (CS1–CS3), CFA/IV (CS4–CS6)], simulated antigenic structure of the CFA/I/II/IV MEFA with computational atomistic modeling and simulation, characterized immunogenicity in mouse immunization, and examined the potential of structure-informed vaccine design for ETEC vaccine development. A tag-less recombinant MEFA protein (CFA/I/II/IV MEFA) was effectively expressed and extracted. Molecular dynamics simulations indicated that this MEFA immunogen maintained a stable secondary structure and presented epitopes on the protein surface. Empirical data showed that mice immunized with the tagless CFA/I/II/IV MEFA developed strong antigen-specific antibody responses, and mouse serum antibodies significantly inhibited in vitro adherence of bacteria expressing these seven adhesins. These results revealed congruence of antigen immunogenicity between computational simulation and empirical mouse immunization and indicated this tag-less CFA/I/II/IV MEFA potentially an antigen for a broadly protective ETEC vaccine, suggesting a potential application of MEFA-based structural vaccinology for vaccine design against ETEC and likely other pathogens.

SYNTHETIC CONJUGATED ANALOGUES OF CAPSULE POLYSACCHARIDES OF PNEUMOCOCCUS - AN INSTRUMENT FOR DETECTION OF POST-VACCINATION ANTIBODIES

Evaluation ofthe ability of capsule polysaccharides (CP) of Streptococcus pneumoniae serotype 3 and 14 and their synthetic structure analogues, conjugated with bovine serum albumin (BSA), to detect antibodies in post-vaccination sera of mice. Materials andmethods. Oligosaccharides correspond- ing to one, one and a half and two repeating links of serotype 3 and 14 S. pneumoniae CP were synthe- sized, their conjugates with BSA were produced by squarate method as well. Ligand content-per BSA molecule was controlled by MALDI-TOF spectrometry. Immune sera were obtained after 2 intraperi- toneal administrations to mice of glucoconjugates adsorbed on aluminum hydroxide or 13-valent pneumococcal conjugated vaccine. Determination of levels of post-vaccination class G antibodies and their sub-isotypes was carried out in EIA. Immunization of mice with neoglucoconjugates resulted in formation of predominantly IgGl recognizing serotype 3 and 14 S. pneumoniae C. IgG1 in mice immunized with a 13-valent conjugated vaccine recognized serotype 3 S. pneumoniae CP, but detected serotype 14 S. pneumoniae CP weakly. All the conjugated synthetic oligosaccharides were characterized by a high ability to bind antibodies in blood of mice immunized with the polysaccharide conjugated vaccine. BSA-tetrasaccharide of serotype 3 S. pneumoniae and BSA-tetrasaccharide of serotype 14 S.pneumoniae were characterized by the highest ability to detect IgG1 against C. Synthetic oligosaccharides, conjugated with BSA protein-carrier, may be used to develop diagnostic test-systems for determination of antibodies in post-vaccination sera.

Inactivated coxsackievirus A10 experimental vaccines protect mice against lethal viral challenge

Coxsackievirus A10 (CVA10) has become one of the major causative agents of hand, foot and mouth disease (HFMD). It is now recognized that CVA10 should be targeted for vaccine development. We report here that β-propiolactone inactivated whole-virus based CVA10 vaccines can elicit protective immunity in mice. We prepared two inactivated CVA10 experimental vaccines derived from the prototype strain CVA10/Kowalik and from a clinical isolate CVA10/S0148b, respectively. Immunization with the experimental vaccines elicited CVA10-specific serum antibodies in mice. The antisera from vaccinated mice could potently neutralize in vitro infection with either homologous or heterologous CVA10 strains. Importantly, passive transfer of the anti-CVA10 sera protected recipient mice against CVA10/Kowalik or CVA10/S0148b infections. Moreover, active immunization with the inactivated vaccines also conferred protection against homologous and heterologous infections in mice. Collectively, our results demonstrate the proof-of-concept for inactivated whole-virus based CVA10 vaccines.

Retargeting FX-binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop.

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon: factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvβ3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development.

Yeast-produced recombinant virus-like particles of coxsackievirus A6 elicited protective antibodies in mice

Coxsackievirus A6 (CA6) has recently emerged as the predominant pathogen of hand, foot and mouth disease (HFMD), causing significant morbidity in children and adults. The increasing prevalence of CA6 infection and its associated disease burden underscore the need for effective CA6 vaccines. However, CA6 grows poorly in cultured cells, making it difficult to develop inactivated whole-virus or live attenuated vaccines. Here we report the development of a recombinant virus-like particle (VLP) based CA6 vaccine. CA6 VLPs were produced in Pichia pastoris yeast transformed with a vector encoding both P1 and 3CD proteins of CA6. Immunization with CA6 VLPs elicited CA6-specific serum antibodies in mice. Passive transfer of anti-VLP antisera protected recipient mice against lethal CA6 challenge. Collectively, these results demonstrate that CA6 VLPs represent a viable CA6 vaccine candidate which warrants further preclinical and clinical development.

Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis

We evaluated the potential of a liposomal antigen delivery system (ADS) containing Candida albicans cell wall surface proteins (CWSP) in mediating protection against systemic candidiasis. Treatment of bone-marrow-derived dendritic cells with CWSP-loaded dioctadecyldimethylammonium bromide:monoolein (DODAB:MO) liposomes enhanced and prolonged their activation comparatively to free antigen, indicating that liposome-entrapped CWSP were released more sustainable. Therefore, we immunized mice with CWSP either in a free form or loaded into two different DODAB:MO liposome formulations, respectively designated as ADS1 and ADS2, prior to intravenous C. albicans infection. Immunization with ADS1, but not with ADS2, conferred significant protection to infected mice, comparatively to immunization with CWSP or empty liposomes as control. ADS1-immunized mice presented significantly higher serum levels of C. albicans-specific antibodies that enhanced phagocytosis of this fungus. In these mice, a mixed cytokine production profile was observed encompassing IFN-γ, IL-4, IL-17A and IL-10. Nevertheless, only production of IL-4, IL-17 and IL-10 was higher than in controls. In this study we demonstrated that DODAB:MO liposomes enhance the immunogenicity of C. albicans antigens and host protection in a murine model of systemic candidiasis. Therefore, this liposomal adjuvant could be a promising candidate to assess in vaccination against this pathogenic fungus. Statement of SignificanceThis work describes the immunomodulation capacity of the previously validated antigen delivery system (ADS) composed by dioctadecyldimethylammonium bromide (DODAB) and monoolein (MO) lipids incorporating the cell wall surface proteins (CWSP) from C. albicans. Here, we not only present the ability of this system in facilitating antigen uptake by DCs in vitro, but also that this system induces higher levels of pro-inflammatory cytokines and opsonizing specific IgG antibodies in serum of mice immunized subcutaneously. We show that the ADS are efficient nanocarrier and modulate the immune response against intravenous C. albicans infection favoring mouse protection. In sum, we show that the incorporation of C. albicans antigens in DODAB:MO nanocarries are a promising vaccine strategy against C. albicans fungal infection.

Prevention of intestinal ischemia-reperfusion injury in humanized mice

Ischemia-reperfusion injury (I/R) is an inflammatory response to hypoxia that is dependent on antibodies. We have previously shown that the N2 peptide blocks binding of autoreactive antibody to ischemic tissue and reduces I/R injury in rodents. Whether these same antibodies exist and target the same antigen in humans is unknown. In this study, we create a novel "humanized" mice model of intestinal I/R and test the efficacy of N2. Humanized mice were generated by transplanting human lymphocytes into immunodeficient mice. Human T- and B-cell engraftment in the spleen and peritoneum was confirmed using flow cytometry, and circulating human antibody in mouse serum was measured with ELISA. Injury in the small intestine and lung after intestinal I/R was measured in the presence or absence of N2. Immunohistochemistry was used to assess for human antibody deposition in the small intestine. Humanized mice engrafted with functional CD20+Bcells generated high circulating serum levels of human antibody. N2 treatment significantly reduced intestinal injury severity scores after I/R (control: 28±1.5, N2: 9.1±3.4; P<.05). N2 also attenuated remote lung inflammation after I/R (control: 28±4, N2: 5.4±1.3; P<.05). Protection from I/R injury correlated with blockade of human antibody deposition on small intestine. N2 is an effective therapy for I/R injury in the presence of human immunity, supporting a conserved target of inflammatory attack in human reperfusion injury.

Chlamydomonas reinhardtii chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies.

An algae-based vaccine model against atherosclerosis was developed with positive findings in terms of antigen yield and immunogenicity in mouse. Several immunotherapies against atherosclerosis have been evaluated at the preclinical level thus far, with some of them currently under evaluation in clinical trials. In particular, the p210 epitope from ApoB100 is known to elicit atheroprotective responses. Considering that Chlamydomonas reinhardtii is an attractive host for the production and delivery of subunit vaccines, in this study a chimeric protein consisting of the B subunit of the cholera toxin and the p210 epitope from ApoB100 (CTB:p210) has been expressed in C. reinhardtii chloroplast as an attempt to establish an oral vaccine candidate against atherosclerosis. The Chlamydomonas-made CTB:p210 protein was successfully expressed at levels of up to 60µg per g of fresh weight biomass. The antigenic activity of the CTB and the p210 moiety was preserved in the CTB:p210 chimera. Moreover the algae-made CTB:p210 showed an immunogenic activity, when orally administered to BALB/c mice, as evidenced the presence of anti-p210 serum antibodies in mice treated with the algae-derived CTB:p210. The antibody response lasts for at least 80days after the last boost. This experimental model is proposed as a convenient tool in the development of low cost atherosclerosis vaccines of easy compliance and friendly delivery. Further studies will determine the therapeutic potential of this algae-made vaccine in atherosclerosis animal models.

Novel therapeutic compound tuftsin-phosphorylcholine attenuates collagen-induced arthritis.

Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin-PC (TPC), a novel helminth-based compound in collagen-induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 µg/0.1 ml) or orally (250 µg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme-linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (Treg ) and regulatory B (Breg ) cell phenotypes by fluorescence-activated cell sorter (FACS). TPC-treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P < 0.0001) in both subcutaneous and orally treated groups at day 31. Moreover, histology analysis demonstrated highly inflamed joints in control mice, whereas TPC-treated mice maintained normal joint structure. Furthermore, TPC decreased the titres of circulating collagen II antibodies in mice sera (P<0.0001), enhanced expression of IL-10 (P<0.0001) and inhibited production of tumour necrosis factor (TNF)-α, interleukin (IL)-17 and IL-1β (P<0.0001). TPC significantly expanded the CD4(+) CD25(+) forkhead box protein 3 (FoxP3(+) ) Treg cells and CD19(+) IL-10(+) CD5(high) CD1d(high) T cell immunoglobulin mucin-1 (TIM-1(+) ) Breg cell phenotypes (P<0.0001) in treated mice. Our data indicate that treatment with TPC attenuates CIA in mice demonstrated by low arthritic score and normal joints histology. TPC treatment reduced proinflammatory cytokines and increased anti-inflammatory cytokine expression, as well as expansion of Treg and Breg cells. Our results may lead to a new approach for a natural therapy for early rheumatoid arthritis onset.

Kinetics of Anti-Phlebotomus perniciosus Saliva Antibodies in Experimentally Bitten Mice and Rabbits.

BackgroundSand flies are hematophagous arthropods that act as vectors of Leishmania parasites. When hosts are bitten they develop cellular and humoral responses against sand fly saliva. A positive correlation has been observed between the number of bites and antibody levels indicating that anti-saliva antibody response can be used as marker of exposure to sand flies. Little is known about kinetics of antibodies against Phlebotomus perniciosus salivary gland homogenate (SGH) or recombinant salivary proteins (rSP). This work focused on the study of anti-P. perniciosus saliva antibodies in sera of mice and rabbits that were experimentally exposed to the bites of uninfected sand flies.Methodology/Principal FindingsAnti-saliva antibodies were evaluated by ELISA and Western blot. In addition, antibody levels against two P. perniciosus rSP, apyrase rSP01B and D7 related protein rSP04 were determined in mice sera. Anti-saliva antibody levels increased along the immunizations and correlated with the number of sand fly bites. Anti-SGH antibody levels were detected in sera of mice five weeks after exposure, and persisted for at least three months. Anti-apyrase rSP01B antibodies followed similar kinetic responses than anti-SGH antibodies while rSP04 showed a delayed response and exhibited a greater variability among sera of immunized mice. In rabbits, anti-saliva antibodies appeared after the second week of exposure and IgG antibodies persisted at high levels, even 7 months post-exposure.Conclusions/SignificanceOur results contributed to increase the knowledge on the type of immune response P. perniciosus saliva and individual proteins elicited highlighting the use of rSP01B as an epidemiological marker of exposure. Anti-saliva kinetics in sera of experimentally bitten rabbits were studied for the first time. Results with rabbit model provided useful information for a better understanding of the anti-saliva antibody levels found in wild leporids in the human leishmaniasis focus in the Madrid region, Spain.