Naive Serum Research Articles

A simple, field-applicable method to increase the infectivity of wild isolates of Plasmodium falciparum to mosquito vectors

BackgroundThe direct membrane feeding assay (DMFA), whereby gametocyte-infected blood is collected from human donors and from which mosquitoes feed through a membrane, is proving essential for assessing parameters influencing Plasmodium transmission potential in endemic countries. The success of DMFAs is closely tied to gametocyte density in the blood, with relatively high gametocytaemia ensuring optimal infection levels in mosquitoes. As transmission intensity declines with control efforts, the occurrence of asymptomatic individuals with low gametocyte densities, who can significantly contribute to the infectious reservoir, is increasing. This poses a limitation to studies relying on the experimental infection of large numbers of mosquitoes with natural isolates of Plasmodium. A simple, field-applicable method is presented for improving parasite infectivity by concentrating Plasmodium falciparum gametocytes.MethodsAnopheles gambiae received one of the following 5 blood treatments through DMFA: (i) whole blood (WB) samples from naturally-infected donors; (ii) donor blood whose plasma was replaced with the same volume of Plasmodium-naive AB + serum (1:1 control); (iii) plasma replaced with a volume of malaria-naïve AB + serum equivalent to half (1:1/2), or to a quarter (1:1/4), of the initial plasma volume; and (v) donor blood whose plasma was fully removed (RBC). The experiment was repeated 4 times using 4 distinct wild parasite isolates. Seven days post-infection, a total of 1,095 midguts were examined for oocyst presence.ResultsSubstituting plasma with reduced amounts (1:1/2 and 1:1/4) of Plasmodium-naive AB + serum led to a 31% and 17% increase of the mosquito infection rate and to a 85% and 308% increase in infection intensity compared to the 1:1 control, respectively. The full removal of plasma (RBC) reduced the infection rate by 58% and the intensity by 64% compared to the 1:1 control. Reducing serum volumes (1:1/2; 1:1/4 and RBC) had no impact on mosquito feeding rate and survival when compared to the 1:1 control.ConclusionsConcentrating gametocytic blood by replacing natural plasma by lower amount of naive serum can enhance the success of mosquito infection. In an area with low gametocyte density, this simple and practical method of parasite concentration can facilitate studies on human-to-mosquito transmission such as the evaluation of transmission-blocking interventions.

RDE Treatment Prevents Non-Specific Detection of SARS-CoV-2- and Influenza-Specific IgG Antibodies in Heat-Inactivated Serum Samples.

Assessing the levels of serum IgG antibodies is widely used to measure immunity to influenza and the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after natural infection or vaccination with specific vaccines, as well as to study immune responses to these viruses in animal models. For safety reasons, sometimes serum specimens collected from infected individuals are subjected to heat inactivation at 56 °C to reduce the risk of infecting personnel during serological studies. However, this procedure may affect the level of virus-specific antibodies, making the results of antibody immunoassays uninterpretable. Here, we evaluated the effect of the heat inactivation of human, ferret and hamster serum samples on the binding of IgG antibodies to the influenza and SARS-CoV-2 antigens. For this, serum samples of naive and immune hosts were analyzed in three variants: (i) untreated sera, (ii) heated at 56 °C for 1 h, and (iii) treated with receptor-destroying enzyme (RDE). The samples were studied through an in-house enzyme-linked immunosorbent assay (ELISA) using whole influenza virus or recombinant proteins corresponding to nucleocapsid (N) protein and the receptor-binding domain of SARS-CoV-2 Spike (RBD) as antigens. We demonstrated that the heat inactivation of the naive serum samples of various hosts can lead to false-positive results, while RDE treatment abolished the effect of the non-specific binding of IgG antibodies to the viral antigens. Furthermore, RDE also significantly decreased the level of virus-specific IgG antibodies in SARS-CoV-2 and influenza-immune sera of humans and animals, although it is unknown whether it actually removes true virus-specific IgG antibodies or only non-specifically binding artifacts. Nevertheless, we suggest that the RDE treatment of human and animal sera may be useful in preventing false-positive results in various immunoassays, while also neutralizing infectious virus, since the standard protocol for the use of RDE also includes heating the sample at 56 °C.

The effect of heat inactivation of ferrets serum samples on the detection of SARS-CoV-2-specific IgG antibodies in elisa

BACKGROUND: Determination of serum antibody levels to the novel coronavirus SARS-CoV-2 is a necessary tool for assessing humoral immunity in COVID-19 patients or individuals vaccinated with specific vaccines, as well as for studying the immune responses to the viral antigens in animal models. Serum specimens of infected humans and animals are considered potentially infectious material, and therefore heat inactivation of samples at 56 C for 1 hour is recommended to reduce the risk of infection of personnel during serological studies. However, this procedure may affect the detection of virus-specific IgG and IgM antibodies, making interpretation of the results difficult.
 AIM: The goal is to evaluate the effect of heat inactivation of ferret serum samples on the binding of IgG antibodies to the SARS-CoV-2 antigens.
 MATERIALS AND METHODS: Serum samples of SARS-CoV-2 naive and immune ferrets were analyzed in three variants: (1) native sera, (2) serum samples than were heated at 56C for 1 hour, and (3) serum samples that were treated with receptor-degrading enzyme (RDE). The samples were studied in enzyme-linked immunosorbent assay (ELISA) using recombinant RBD protein (receptor-binding domain of SARS-CoV-2 S-protein) as a substrate, followed by determination of specific antibody levels before and after treatments.
 RESULTS: It has been shown that heat inactivation of the ferrets naive serum samples can lead to false-positive results, while RDE treatment can neutralize the effect of non-specific binding of IgG antibodies to the RBD domain of the SARS-CoV-2 S protein.
 CONCLUSIONS: Structural rearrangement of SARS-CoV-2-specific IgG antibodies and the formation of immunoglobulin complexes during heat inactivation of serum samples can affect the avidity of the antigen-antibody complex and lead to false-positive results when performing enzyme immunoassay. One of the possible methods to reduce the risk of artifacts is the treatment of blood sera with RDE, which eliminates the effect of heat inactivation.

Investigations on the effects of anti-Leishmania major serum on the progression of Leishmania infantum infection in vivo and in vitro - implications of heterologous exposure to Leishmania spp.

Leishmaniasis is a vector-borne parasitic disease caused by protozoa of the genus Leishmania. Twenty different species are known to cause disease in humans with varying degrees of pathology. These diseases are transmitted throughout the geographic range of phlebotomine sandflies, found between the latitudes 50°N and 40°S. This study explores antibody dependent enhancement (ADE) as the cause of disease exacerbation in heterologous exposure of L. major primed mice to L. infantum challenge. BALB/c mice received serum from L. major infected or naive mice. All mice were challenged with L. infantum and tissue parasite burdens were recorded. Animals that received anti-L. major serum exhibited significantly higher parasite burdens. Surprisingly, these parasite burdens were higher than those of mice infected with L. major and challenged with L. infantum. In vitro phagocytosis assays were carried out to measure parasite uptake in the presence of naive vs. anti-L. major serum. J774A.1 murine monocytes were cultured with either L. major or L. infantum in the presence of anti-L. major serum, naive serum, or no serum. Significantly higher rates of L. major uptake by J774A.1 cells occurred in the presence of anti-L. major serum, but no measurable increase of L. infantum phagocytosis was seen. Our results suggest that increased disease severity observed in vivo in mice previously exposed to L. major and challenged with L infantum is not a result of extrinsic ADE. We speculate that intrinsic ADE, due to biased memory T cell responses caused by Fcγ signaling, could account for disease exacerbation seen in the animal model.

Efficient antitumor effects of a novel oncolytic adenovirus fully composed of species B adenovirus serotype 35

Oncolytic adenoviruses (OAds) are among the most promising oncolytic viruses. Almost all oncolytic adenoviruses are composed of human adenovirus serotype 5 (Ad5) (OAd5). However, expression of the primary infection receptor for Ad5, coxsackievirus-adenovirus receptor (CAR), often declines on malignant tumor cells, resulting in inefficient infection in CAR-negative tumor cells. In addition, at least 80% of adults have neutralizing antibodies against Ad5. In this study, we developed a novel OAd fully composed of OAd35. OAd35 recognizes CD46, which is ubiquitously expressed on almost all human cells and is often upregulated on malignant tumor cells, as an infection receptor. Moreover, 20% or fewer adults have neutralizing antibodies against Ad35. OAd35 mediated efficient cell lysis activities at levels similar to OAd5 in CAR-positive tumor cells, while OAd35 showed higher levels of cell lysis activities than OAd5 in CAR-negative tumor cells. Anti-Ad5 serum significantly inhibited in vitro tumor cell lysis activities of OAd5, whereas OAd35 exhibited comparable levels of in vitro tumor cell lysis activities in the presence of anti-Ad5 and naive serum. OAd35 significantly suppressed growth of the subcutaneous CAR-positive and CAR-negative tumors following intratumoral administration. These results indicated that OAd35 is a promising alternative oncolytic virus for OAd5.

In situ Vaccine Plus Checkpoint Blockade Induces Memory Humoral Response.

In a syngeneic murine melanoma (MEL) model, we recently reported an in situ vaccination response to combined radiation (RT) and intra-tumoral (IT) injection of anti-GD2 hu14. 18-IL2 immunocytokine (IC). This combined treatment resulted in 71% complete and durable regression of 5-week tumors, a tumor-specific memory T cell response, and augmented response to systemic anti-CTLA-4 antibody checkpoint blockade. While the ability of radiation to diversify anti-tumor T cell response has been reported, we hypothesize that mice rendered disease-free (DF) by a RT-based ISV might also exhibit a heightened B cell response. C57BL/6 mice were engrafted with 2 × 106 GD2+ B78 MEL and treated at a target tumor size of ~200 mm3 with 12 Gy RT, IT-IC on day (D)6-D10, and anti-CTLA-4 on D3, 6, and 9. Serum was collected via facial vein before tumor injection, before treatment, during treatment, after becoming DF, and following rejection of subcutaneous 2 × 106 B78 MEL re-challenge on D90. Flow cytometry demonstrated the presence of tumor-specific IgG in sera from mice rendered DF and rejecting re-challenge with B78 MEL at D90 after starting treatment. Consistent with an adaptive endogenous anti-tumor humoral memory response, these anti-tumor antibodies bound to B78 cells and parental B16 cells (GD2-), but not to the unrelated syngeneic Panc02 or Panc02 GD2+ cell lines. We evaluated the kinetics of this response and observed that tumor-specific IgG was consistently detected by D22 after initiation of treatment, corresponding to a time of rapid tumor regression. The amount of tumor-specific antibody binding to tumor cells (as measured by flow MFI) did not correlate with host animal prognosis. Incubation of B16 MEL cells in DF serum, vs. naïve serum, prior to IV injection, did not delay engraftment of B16 metastases and showed similar overall survival rates. B cell depletion using anti-CD20 or anti-CD19 and anti-B220 did not impact the efficacy of ISV treatment. Thus, treatment with RT + IC + anti-CTLA-4 results in adaptive anti-tumor humoral memory response. This endogenous tumor-specific antibody response does not appear to have therapeutic efficacy but may serve as a biomarker for an anti-tumor T cell response.

Antibodies against the DNABII protein integration host factor (IHF) inhibit sinus implant biofilms.

Chronic rhinosinusitis is a common, costly condition often treated with endoscopic sinus surgery and intraoperative placement of intranasal sinus implant materials. Whereas these materials aid in postoperative healing, they also support bacterial biofilm formation and thus contribute to negative outcomes. This study examined pretreatment of sinus implant materials with antibody against an essential bacterial biofilm structural component, the DNABII family of DNA-binding proteins, as a strategy to prevent biofilm formation. Sinus implant materials were equilibrated in immunoglobulin G (IgG)-enriched antiserum against the DNABII protein integration host factor (IHF), individually or in combination with amoxicillin-clavulanate prior to inoculation with nontypeable Haemophilus influenzae (NTHI), a predominant pathogen of chronic rhinosinusitis. After 16 hours, the bacterial burden was quantitated and compared to pretreatment with saline, IgG-enriched naive serum, or amoxicillin-clavulanate alone. NTHI readily formed biofilms on all three materials in vitro. However, pretreatment of each material with IgG-enriched anti-IHF resulted in a significant decrease in bacterial burden compared to controls (P ≤ 0.05). Moreover, a significant and synergistic outcome was achieved with a cocktail of anti-IHF plus amoxicillin-clavulanate (P ≤ 0.05) with complete inhibition of NTHI biofilm formation on all three materials. Biofilm formation was well supported in vitro on three sinus implant materials that vary in composition and resorption characteristics; however, pretreatment of each with DNABII protein targeted antibodies in combination with a previously ineffective antibiotic was highly effective to prevent the formation NTHI biofilms. These data demonstrate the potential for clinical utility of pretreatment of sinus implant and additional surgical materials with anti-DNABII antibodies. NA Laryngoscope, 130:1364-1371, 2020.

Pre-Existing Maternal Antibodies Cause Rapid Prenatal Rejection of Allotransplants in the Mouse Model of In Utero Hematopoietic Cell Transplantation.

In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative nonimmunosuppressive alternative to postnatal hematopoietic stem cell transplantation for the treatment of congenital hemoglobinopathies. Anti-HLA donor-specific Abs (DSA) are associated with a high incidence of graft rejection following postnatal hematopoietic stem cell transplantation. We determine if DSA present in the mother can similarly cause graft rejection in the fetus following IUHCT. Ten million C57BL/6 (B6, H2kb) bone marrow cells were transplanted in utero into gestational day 14 BALB/c (H2kd) fetuses. The pregnant BALB/c dams carrying these fetuses either had been previously sensitized to B6 Ag or were injected on gestational days 13-15 with serum from B6-sensitized BALB/c females. Maternal-fetal Ab transmission, Ab opsonization of donor cells, chimerism, and frequency of macrochimeric engraftment (chimerism >1%) were assessed by flow cytometry. Maternal IgG was transmitted to the fetus and rapidly opsonized donor cells following IUHCT. Donor cell rejection was observed as early as 4 h after IUHCT in B6-sensitized dams and 24 h after IUHCT in dams injected with B6-sensitized serum. Efficient opsonization was strongly correlated with decreased chimerism. No IUHCT recipients born to B6-sensitized dams or dams injected with B6-sensitized serum demonstrated macrochimeric engraftment at birth compared with 100% of IUHCT recipients born to naive dams or dams injected with naive serum (p < 0.001). In summary, maternal donor-specific IgG causes rapid, complete graft rejection in the fetus following IUHCT. When a third-party donor must be used for clinical IUHCT, the maternal serum should be screened for DSA to optimize the chance for successful engraftment.

Endotoxin neutralization by an O-antigen specific monoclonal antibody: A potential novel therapeutic approach against Klebsiella pneumoniae ST258

ABSTRACTKlebsiella pneumoniae ST258 is a globally distributed multi-drug resistant pathogen responsible for severe invasive infections. In this study, the different virulence potential of K. pneumoniae ST258 isolates in endotoxin susceptible versus resistant animal models was shown. Furthermore, ST258 clinical isolates were found highly sensitive to the bactericidal effect of naive animal and human serum. These observations imply that LPS, released from the rapidly lysed bacteria, may contribute to the high mortality associated with ST258 bacteremia cases.A humanized version (mAb A1102) of a previously described murine mAb specific for the conserved LPS O-antigen, was tested for endotoxin neutralization. A1102 was able to neutralize TLR-4 activation by ST258-derived LPS in vitro with an efficacy exceeding that of polymyxin B by 3 orders of magnitude. Passive immunization with A1102 afforded a significant level of protection in a galactosamine-sensitized mouse model of endotoxemia, induced by ST258-derived LPS, or upon challenge with live bacteria. Efficacy was retained using an aglycosylated IgG, as well as upon complement depletion, suggesting that Fc-independent endotoxin neutralization may be the main protective mechanism in this model, in spite of the complement-dependent bactericidal and opsonic activities additionally observed for A1102 in vitro. Furthermore, rabbits that are naturally highly susceptible to endotoxin, were also significantly protected by low doses of A1102 when challenged with an ST258 strain.Given this unique mode of action and the high protective efficacy of this mAb, passive immunization, as prophylactic or adjunct therapeutic approach for the treatment of infections caused by ST258 isolates should be considered.

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.

A pig-to-mouse coronary artery transplantation model for investigating the pathogenicity of anti-pig antibody.

Rejection of Gal-free (GTKO) donor pig cardiac xenografts is strongly associated with vascular non-Gal antibody binding, endothelial cell (EC) injury, and activation and microvascular thrombosis. We adopted a pig-to-SCID/beige small animal transplant model to compare the pathogenicity of baboon and human anti-pig antibody. Wild-type (GT(+) ) or GTKO porcine coronary arteries (PCAs) were transplanted into the infrarenal aorta of SCID/beige mice. Three days after transplant, recipients were infused with anti-pig antibody (anti-SLA class I, an isotype control, naive or sensitized baboon serum, or naive human serum). PCAs were recovered 24h after antibody infusion and examined using histology, immunohistochemistry, and insitu hybridization. Dose-dependent intragraft thrombosis occurred after infusion of anti-SLA I antibody (but not isotype control) in GT(+) and GTKO PCA recipients. Naive baboon serum induced thrombosis in GT(+) grafts. Thrombosis was significantly reduced by pre-treating naive baboon serum with Gal polymer and not observed when this serum was infused to GTKO PCA recipients. Naive human serum caused dose-dependent intragraft thrombosis of GTKO PCAs. In all cases, thrombosis involved graft-specific vascular antibody and complement deposition, macrophage adherence, EC delamination, and subendothelial thrombus formation. This study provides the first direct invivo comparison of the pathogenicity of naive human and baboon serum. The results suggest that human preformed non-Gal antibody may have increased pathogenicity compared to baboon. This model, which showed a rejected graft histopathology similar to antibody-mediated rejection in cardiac xenotransplantation, may be useful to assess the pathogenicity of individual protein or carbohydrate specific non-Gal reactive antibodies.

3300 A phase 1b/2 study of ribociclib (LEE011; CDK4/6 inhibitor) in combination with binimetinib (MEK162; MEK inhibitor) in patients with NRAS-mutant melanoma

s S663 and refractory MM patients a significant humoral immune response against proteins related with these three conditions. Materials and Methods: We studied repository serum with clinical data included in MGUS, naive and refractory MM. We performed a systematic multivariate analysis of proteins overexpressed in monoclonal gammopathy as representative of a potential start to create preventive vaccines for MM and in refractory cancer patients who progress despite treatment. We found four potential oncogenic drivers such as HIF-1 alpha, RAD51, Sox2 and Mcl-1. Afterwards we performed an antigen–specific indirect ELISA for both IgG and IgA using recombinant proteins to interrogate human MGUS naive and refractory MM serum and demonstrated if they were immunogenic. Results: Using indirect ELISA both antigen specific IgG and IgA using recombinant proteins with the four proteins we only found a statistical difference for anti-Mcl-1 specific IgG in MGUS (p = 0.01), naive MM (p = 0.005) and relapsed MM (p = 0.05). For anti-Mcl-1 IgA we found in MGUS (p=0001), naive MM (p = 0.002) and relapsed MM (p = 0.01). Here, we showed that even in refractory MM the antigenspecific humoral immune response was detected and it was clear that patients with the highest immune responses had better clinical outcomes in terms of OS. Discussion: MM is a challenging hematological malignancy and immune therapy is a good possibility for treatment either to prevent recurrence or in combination to improve the outcomes of the current standard of care treatment. We showed that one out of the four proteins had biological and clinical relevance in MGUS, naive and relapsed MM is immunogenic both for IgG and IgA. We will move forward to prepare a peptide vaccine and test animal immunogenicity for Mcl-1. Also it could be a good approach in combination with standard of care treatment in progressive or active disease to improve clinical outcomes. No conflict of interest. Proffered Paper Session (Monday, 28 September) Melanoma and Skin Cancer

P57 Hydrogen sulfide treatment reduces activation of ischemia–reperfusion injury-induced tissue inflammatory processes and resultant recipient adaptive immune response to donor tissue during allogeneic renal transplantation

Background Organ procurement is inherently associated with ischemia–reperfusion injury (IRI), resulting from loss and subsequent restoration of blood flow and is detrimental to short- and long-term graft function and survival. Treatment of donor organs with endogenously produced gasotransmitters such as hydrogen sulfide (H 2 S) is a novel method of mitigating IRI during transplantation, as previously shown in a model of murine syngeneic renal transplantation (RTx) [1] . The protective effects of H 2 S against IRI during allogeneic RTx, in which genetically dissimilar grafts are targeted by the recipient immune system causing organ rejection, have not yet been studied. Since H 2 S is known to exhibit anti-inflammatory actions during tissue IRI, we postulated that H 2 S treatment during allogeneic RTx could reduce graft inflammation and modulate the resultant recipient immune response against donor tissue. Methods Following bilateral native nephrectomy, recipient Lewis rats underwent RTx with left kidneys obtained from Brown Norway donor rats that were flushed, at the time of procurement, with either cold (4°C) University of Wisconsin standard preservation solution (UW group) or cold UW + H 2 S donor molecule (150 μ M NaHS) (H 2 S group) and stored for 6 h at 4°C in the same solution. Sham animals were also followed. Renal grafts were obtained at post-operative day 2 and RNA was isolated from renal homogenates for gene microarray analysis (Affymetrix Rat Gene 1.0 ST Microarray). Additionally, serum was collected post-operatively at day 1 to 2 and at time of sacrifice to determine the relative abundance of allo-reactive serum antibodies. TIB-120 cells (ATCC), a rat B-cell line heterozygous for Brown Norway and Lewis alleles, were incubated with serum from transplant recipients in each treatment group, stained for bound rat IgG/M antibodies and analyzed via flow cytometry. Results Upon microarray analysis both treatment groups exhibited significant enrichment in genes involved in inflammatory and innate immune responses as well as cytokine signaling and antigen processing at day 2 compared to Sham (p 2 S treated grafts had markedly reduced enrichment scores in each of these pro-inflammatory processes compared to UW. Upon allo-antibody binding analysis both UW and H 2 S treatment groups exhibited similar trends, with more cells unbound by allo-antibodies at day 1 to 2 and few unbound cells at time of sacrifice. However, while cells incubated with UW serum at day 1 to 2 exhibited markedly more allo-antibody binding (27% unbound cells) compared to cells incubated with naive Sham serum (37% unbound cells), cells incubated with H 2 S serum at day 1 to 2 exhibited similar levels of allo-antibody binding (40% cells unbound) as those incubated with Sham serum. These findings indicate that H 2 S treated animals experienced less acute graft inflammation, leading to a delayed induction of adaptive immune response to donor tissue compared to UW alone. Conclusions These findings are the first to report that H 2 S treatment may modulate the recipient immune response to donor tissue during allogeneic RTx. This phenomenon could represent an important underlying mechanism of H 2 S-mediated protection against renal injury early in the transplant curriculum, ultimately prolonging the survival and function renal allografts.

Optimal Germinal Center B Cell Activation and T-Dependent Antibody Responses Require Expression of the Mouse Complement Receptor Cr1

Follicular dendritic cells (FDCs) and complement receptor (Cr)1 and complement receptor (Cr)2 are important for the generation of humoral immunity. Cr1/2 expression on B cells and FDCs was shown to provide a secondary signal for B cell activation, to facilitate transport of Ag in immune follicles, and to enhance retention of immune complexes by FDCs. We show in this study that murine B cells predominantly express the Cr2 product from the Cr2 gene, whereas FDCs almost exclusively express the Cr1 isoform generated from the Cr2 gene. To define the specific role of Cr1, we created an animal that maintains normal cell-restricted expression of Cr2 but does not express Cr1. Cr1-deficient (Cr1KO) mice develop normal B1 and B2 immature and mature B cell subsets and have normal levels of naive serum Abs but altered levels of natural Abs. Immunization of the Cr1KO animal demonstrates deficient Ab responses to T-dependent, but not T-independent, Ags. Germinal centers from the immunized Cr1KO animal possess a deficiency in activated B cells, similar to that seen for animals lacking both Cr1 and Cr2 or C3. Finally, animals lacking only Cr1 respond similarly to wild-type animals to infections with Streptococcus pneumoniae, a pathogen to which animals lacking C3 or both Cr1 and Cr2 are particularly sensitive. Altogether, these data suggest that the production of Cr1, primarily by FDCs, is critical in the generation of appropriately activated B cells of the germinal center and the generation of mature Ab responses.

Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement

Adenovirus type 5 (Ad5) specifically binds coagulation factor X (FX), and FX is normally essential for intravenously injected Ad5 vectors to transduce the liver. We demonstrate that the ability of FX to enhance liver transduction by Ad5 vectors is due to an unexpected ability of FX to protect Ad5 from attack by the classical complement pathway. In vitro, naive mouse serum neutralized Ad5 when FX was blocked from binding Ad5. This neutralization was mediated by natural IgM and the classical complement pathway. In vivo, FX was essential for Ad5 vectors to transduce the livers of wild-type mice, but FX was not required for liver transduction in mice that lack antibodies, C1q or C4. We conclude that Ad5 recruits FX as a defense against complement and that the sensitivity of Ad5 to inactivation by complement must be taken into account when designing vectors for systemic gene therapy.

Humoral Immunity to AAV-6, 8, and 9 in Normal and Dystrophic Dogs

Adeno-associated virus (AAV)-6, 8, and 9 are promising gene-delivery vectors for testing novel Duchenne muscular dystrophy gene therapy in the canine model. Humoral immunity greatly influences in vivo AAV transduction. However, neutralizing antibodies to AAV-6, 8, and 9 have not been systemically examined in normal and dystrophic dogs. To gain information on the seroprevalence of antibodies to AAV-6, 8, and 9, we measured neutralizing antibody titers using an in vitro transduction inhibition assay. We examined 72 naive serum samples and 26 serum samples obtained from dogs that had received AAV gene transfer. Our data demonstrated that AAV-6 neutralizing antibody was the most prevalent antibody in dogs irrespective of age, gender, disease status (dystrophic or not), and prior parvovirus vaccination history. Surprisingly, high-level anti-AAV-6 antibody was detected at birth in newborn puppies. Further, a robust antibody response was induced in affected, but not normal newborn dogs following systemic AAV gene transfer. Taken together, our data have provided an important baseline on the seroprevalence of AAV-6, 8, and 9 neutralizing antibodies in normal and Duchenne muscular dystrophy dogs. These results will help guide translational AAV gene-therapy studies in dog models of muscular dystrophy.

Carbohydrate Mimetic Peptides Augment Carbohydrate-Reactive Immune Responses in the Absence of Immune Pathology

Among the most challenging of clinical targets for cancer immunotherapy are Tumor Associated Carbohydrate Antigens (TACAs). To augment immune responses to TACA we are developing carbohydrate mimetic peptides (CMPs) that are sufficiently potent to activate broad-spectrum anti-tumor reactivity. However, the activation of immune responses against terminal mono- and disaccharide constituents of TACA raises concerns regarding the balance between “tumor destruction” and “tissue damage”, as mono- and disaccharides are also expressed on normal tissue. To support the development of CMPs for clinical trial testing, we demonstrate in preclinical safety assessment studies in mice that vaccination with CMPs can enhance responses to TACAs without mediating tissue damage to normal cells expressing TACA. BALB/c mice were immunized with CMPs that mimic TACAs reactive with Griffonia simplicifolia lectin 1 (GS-I), and tissue reactivity of serum antibodies were compared with the tissue staining profile of GS-I. Tissues from CMP immunized mice were analyzed using hematoxylin and eosin stain, and Luxol-fast blue staining for myelination. Western blots of membranes from murine mammary 4T1 cells, syngeneic with BALB/c mice, were also compared using GS-I, immunized serum antibodies, and naive serum antibodies. CMP immunization enhanced glycan reactivities with no evidence of pathological autoimmunity in any immunized mice demonstrating that tissue damage is not an inevitable consequence of TACA reactive responses.

Antiviral Antibodies Target Adenovirus to Phagolysosomes and Amplify the Innate Immune Response

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

JVRS-100 adjuvanted universal influenza A vaccine based on a multiple antigenic peptide of M2e protects from lethal challenge (132.6)

Abstract Background: The conventional vaccine strategy for control of influenza A is vulnerable to antigenic drift and the emergence of unmatched epidemic strains that cause primary vaccine failure. A vaccine strategy that targets an influenza antigen, which is less susceptible to antigenic variation, would be a major improvement. Methods: Balb/c mice were vaccinated three times with the M2e peptide in the context of a multiple antigenic peptide (MAP) complex and combination with the cationic lipid DNA complex adjuvant (JVRS-100). Antibody titers were monitored over the time course of vaccination and the mice received a lethal challenge with H1N1 (PR/8/34) or H3N2 (HKx31) two weeks after the final vaccination. Results: Vaccination of mice with JVRS-100-MAP4-M2e compared to MAP4-M2e resulted in increased survival following lethal challenge with 6x LD50 PR/8/34 H1N1 (100% vs. 30%, P=0.002) or 10x LD50 HKx31 H3N2 (80% vs. 20%). Recipients of 200µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (6x LD50 PR/8/34) resulted in survival of 60% (P=0.014) compared with control mice which received 200µl naive serum (0% survival). Furthermore, recipients of 300µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (2x LD50 PR/8/34) resulted in survival of 100% (P=0.0026) compared with control mice which received 200µl naive serum (0% survival). Conclusion: These studies demonstrate that a simple, synthetic M2e vaccine in a MAP configuration with a strong adjuvant may result in a viable vaccine candidate for universal flu vaccination and the protective immunity is primarily due to an antibody response.

Preliminary investigation into the killing effect of kingfish (Seriola lalandi) serum and mucus against the monogenean parasites Benedenia seriolae and Zeuxapta seriolae

The survival of two economically important monogenean parasites, Benedenia seriolae and Zeuxapta seriolae, exposed to either naive or infected yellowtail kingfish Seriola lalandi serum and mucus was observed over an 8-h period. Infection status of the host had no effect on survival of monogeneans in either serum or mucus therefore data were grouped according to each parasite. Serum exposure at the highest concentration of 1:5 did not have any effect on Z. seriolae, however, 50% killing activity against B. seriolae was observed within 3 h at concentrations greater than 1:100. This killing activity was completely abolished following heat treatment and inactivation of the serum. Prior incubation of parasites with heat-treated serum for 2 h in order to potentially coat the parasites with antibodies did not enhance the activity of fresh serum against either species. Addition of 5 mM EDTA, but not 5 mM EGTA-Mg, inhibited the killing ability of fresh serum against B. seriolae, suggesting that previously observed activity against this parasite was most likely mediated via the alternative complement pathway. Cutaneous mucus was not found to have any effect on B. seriolae or Z. seriolae survival. Overall these results suggest that the blood-feeding gill parasite Z. seriolae is considerably more resistant to host immune responses compared with skin- and mucus-dwelling B. seriolae.