Isolation of Human Antigen-Specific Antibodies from Memory B-Cells Nearly Two Years Post Vaccination

Isolation and production of therapeutic human monoclonal antibodies (mAbs) traditionally utilizes a handful of techniques including antibody engineering, phage display, hybridoma generation from transgenic mice or EBV immortalization of B-cells. Over the past decade a new approach has emerged that attempts to extract antigen-specific memory B-cells from the peripheral blood of individuals vaccinated or infected with the target. Initial attempts focused on culturing B-cells and inducing differentiation to plasmablasts for analysis of antibody-antigen specificity, but results were largely mixed due to difficult culture conditions and/or rarity of target cells. With advancing technology in cell sorting, single antigen-specific memory B-cells can be identified and sorted with fluorescently labeled antigens. This method has produced virus-specific mAbs from HIV-infected patients and tetanus-specific mAbs within weeks after Tdap immunization. Many other studies claim to have found antigen-specific mAbs months to years after immunization or clearance of an infection; however, these studies fail to provide direct evidence of antibody specificity by cloning and expressing the mAbs from B-cells. Here we report the efficient isolation of tetanus-specific mAbs from a subject Td-immunized almost two years prior to blood draw. Initially, the total B-cell population was isolated from peripheral blood mononuclear cells enriched by negative selection, then stained to identify tetanus-specific memory B-cells. These cells were individually sorted and PCR was performed to amplify heavy and light chain variable regions of the B-cell’s antibody mRNA. After sequencing, 15 of 42 samples produced both heavy and light chain antibody sequence and 11 mAbs were cloned and transiently expressed. ELISA analysis indicated 5 of the 11 mAbs bound the Hc protein fragment of tetanus toxin and 3 were specific for Hc. We plan to extend this initial success to additional targets and longer gaps between vaccination and B-cell isolation to identify functional therapeutic human antibodies

Human monoclonal antibodies to Plasmodium falciparum circumsporozoite protein for transient passive protection of malaria travelers to endemic areas

Plasmodium falciparum, is a protozoa that causes over 214 million cases of Malaria worldwide and the World Health Organization reported an estimated 438,000 deaths attributed to malaria in 2015. Current prevention strategies have reduced malaria cases but they are either costly, have poor efficacy or resistance has begun to develop. There is a global need for an effective pre-exposure prophylaxis treatment. The leading Malaria vaccine candidate is RTS,S which contains a monovalent Plasmodium falciparum circumsporozoite protein (CSP). The goal of this vaccine is to induce anti-CSP antibodies that would block sporozoite invasion of hepatocytes and thereby hinder parasite development into a blood-stage infection that causes malaria morbidity and mortality. Antibodies isolated from individuals who have received the RTS,S vaccine have been shown to prevent infection of hepatocytes, suggesting that CSP antibodies could be used prophylactically. However, phase III trial results of the vaccine have shown underwhelming efficacy in children. Growing resistance to transient protection strategies for travelers and low efficacy in vaccine trials suggest there is a need for a new treatment strategy. The generation of CSP specific human monoclonal antibodies (mAbs) would be useful as prevention especially for individuals that are temporarily exposed to Malaria in endemic regions such as travelers or military personnel. Isolation and production of therapeutic mAbs traditionally utilizes a handful of techniques including antibody engineering, phage display or hybridoma generation from transgenic mice. We have sorted antigen-specific memory B-cells from the peripheral blood of children naturally infected with malaria to isolate CSP-specific memory B-cells. These cells were individually sorted and PCR was performed to amplify antibody variable regions of the B-cell’s antibody mRNA. Samples that produced heavy and light chain antibody sequence were cloned and transiently expressed. We plan to characterize these mAbs for binding and neutralization of CSP to identify functional therapeutic mAbs

Analysis of sFlt Isoforms as Biomarkers for the Development of Preeclampsia

Preeclampsia is a multi-system disorder characterized by hypertension, edema and proteinuria affecting between 5-10% of pregnancies. A subset of cases progress to severe preeclampsia with exacerbated hypertension/proteinuria and evidence of nervous system, liver and/or kidney dysfunction, in addition to fetal growth restriction. Soluble fms-like tyrosine kinase-1 (sFlt) is minimally expressed in many tissues, including the placenta, and is a circulating antagonist to vascular endothelial growth factor. With progression of pregnancy, sFlt levels significantly rise, especially in women who develop preeclampsia. Diagnostic tests to predict preeclampsia in pregnant women are limited and current tests measure total sFlt in relationship to placental growth factor with varying sensitivity and specificity. We hypothesized that a pregnancy-specific splice variant of sFlt (sFlt1-14), almost exclusively expressed by the placenta, would serve as an improved serum biomarker for the development of preeclampsia. Monoclonal antibodies (mAbs) were developed that specifically bind the two predominant isoforms of sFlt (sFlt1 and sFlt1-14) by hybridoma generation from wild type mice immunized with c-terminal peptides of the two isoforms. Western blot, ELISA and affinity analysis indicated the mAbs were specific for sFlt1 or sFlt1-14 splice variants and recognized these proteins in biological fluids (amniotic fluid or serum). A quantitative capture ELISA was developed whereby total sFlt in biological fluid is captured by a unique human mAb and specific levels of sFlt1 or sFlt1-14 are detected by their respective mouse mAb, followed by anti-murine secondary antibody development. Using recombinant sFlt1 or sFlt1-14 as standards, these endogenous proteins were quantified in commercially available third trimester human pregnant sera. Future studies will measure these isoforms in sera prospectively collected from women with known outcomes of a healthy pregnancy or preeclampsia and the ability of absolute quantitation of the isoform(s) or a ratio of the two to predict the likely onset and severity of preeclampsia will be evaluated

Therapeutic Monoclonal Antibodies to Prevent Tuberculosis Infection

Mycobacteria tuberculosis (Mtb) is a major cause of human morbidity and mortality. Transmission occurs through inhalation of aerosolized Mtb and the initial infection is believed to occur primarily in the alveolar macrophage, although Mtb can infect other cells residing in the lung including dendritic cells, pneumocytes and M cells. Several molecules derived from Mtb are involved in the attachment of the organism to host receptors (opsonic and non-opsonic), which have been reasonably well elucidated. However, a complete understanding of how Mtb attaches to the host and the relative importance of each mechanism on the outcome of infection remains elusive. We hypothesize that protection from infection is possible by blocking the critical initial surface interactions of the organism with the host cell using specific monoclonal antibodies (mAbs). To develop effective mAbs, the outermost layers of Mtb, the capsule and outer membrane, were isolated and characterized by protein gel and LC/MS/MS. Approximately 1000 different proteins were identified in the isolations, of which ~25% were unique to one of the two fractions. The capsule or outer membrane preparations were used as antigens to immunize CD1 mice for up to 12 weeks to generate antibodies via traditional hybridoma generation. Antibodies were screened, selected and characterized by their ability to bind whole cell Mtb by ELISA, demonstration of unique heavy chain variable region sequence and binding specificity by Western Blot. Of approximately 1500 screened hybridomas, 30 lead mAbs have been isolated with specificity to various targets. Preliminary results suggest several of the lead mAb candidates are able to prevent Mtb-induced macrophage cell death in vitro. Future studies will attempt to confirm efficacy in vivo after aerosolized infection in mice with mAb-coated Mtb or parenteral administration of mAb(s). Targets of functional mAbs will be determined and these antigens could serve as viable candidates for vaccine development

Pre-exposure prophylaxis with OspA-specific human monoclonal antibodies protects mice against tick transmission of Lyme disease spirochetes

Background. Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. Methods. Mice transgenic for human immunoglobulin genes were immunized with OspA protein of B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. Results. Nearly 100 unique OspA specific HuMabs were generated and four HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates based on borreliacidal activity. HuMab 319-44, 857-2 and 212-55 were borreliacidal against one or two Borrelia genospecies, whereas 221-7 was borreliacidal (IC50 \u3c 1nM) against B. burgdorferi, B. afzelii and B. garinii, the three main genospecies endemic in the US, Europe and Asia. All four HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi. Conclusions. Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and administration of these antibodies could be employed as pre-exposure prophylaxis for Lyme disease

Discovery and Development of Human Monoclonal Antibodies to Block RhD Alloimmunization During Pregnancy

Exposure of an Rh negative mother to red blood cells (RBCs) of an Rh positive fetus results in alloimmunization and development of anti-RhD antibodies. The anti-RhD antibodies cause hemolytic disease of the new born babies during subsequent pregnancies. Current prophylactic treatment involves polyclonal anti-RhD IgG purified from plasma of humans and is administered in approximately 20% of pregnancies. While the current prophylaxis is effective, it involves the use of human plasma and non-RhD specific antibodies, thus posing a risk of transmitting infections and undesired antibody reactions. Moreover, there is a serious scarcity of plasma donors to meet the requirement of anti-RhD antibodies. In this study we propose to discover and develop anti-RhD monoclonal human antibodies to replace the current polyclonal prophylaxis. We are using humanized BLT mice (fetal CD34+ stem cells, liver and thymus) reconstituted with RhD negative donor material and were immunized by using adenovirus containing RhD transgene. Serum samples were collected after 4-6 weeks of immunization. Our results show that the RhD immunized mice had considerably higher titer of IgG and IgA antibodies in the serum compared to the control, suggesting an immune response developed upon immunization. Splenocytes from antibody producing mice will be fused with a human fusion partner for the isolation of hybridomas producing human monoclonal antibodies. The immunoreactivity and functional activity of these antibodies will be discussed

Pressure Overload Induces Early Morphological Changes in the Heart

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes