Antibody detection and kinetics of antibody production during early stages of immunization with hepatitis B virus vaccine

Antibodies to influenza virus and human immunodeficiency virus are detectable in B cells during the early stages of the immune response, prior to their occurrence in plasma. To investigate similar phenomena in a model of immunization against hepatitis B virus (HBV) infection, medical students in Ghana were screened for HBV markers, HBV surface (HBs) antigen (HBsAg), and HBV core antibodies (anti-HBc). Consenting volunteers, 24 of whom were seronegative (susceptible) and 2 of whom were positive for anti-HBc (prior infection), were vaccinated on day 0, day 40, and 6 months. Two sets of 10 blood samples, sequentially collected at intervals of 2 days following each immunization on days 0 and 40, were processed into B-cell lysates and plasma. Solid-phase HBsAg coated on microtiter plates for enzyme immunoassay or nitrocellulose membranes for dot blot assay was used to detect anti-HBs activity by an indirect antiglobulin assay. A commercially procured sandwich immunoassay was used, along with an enzyme-linked immunosorbent assay and a dot blot assay, for the detection of anti-HBs in B-cell lysates and plasma. Following the first injection of vaccine, a single sample of B-cell lysate collected between 5 and 21 days revealed anti-HBs in 18/21 subjects with no plasma antibodies detectable by sandwich immunoassay. After the booster dose was injected on day 40, a single sample of B-cell lysate collected between 44 and 49 days showed anti-HBs in 16/19 subjects, and this was accompanied by plasma antibodies in 8 subjects. In contrast, between 8 and 13 days, both subjects with prior HBV infection showed anti-HBs in B-cell lysates and plasma. Thus, primary immunization with the HBV vaccine appears to transiently elicit low-affinity anti-HBs in B-cell lysates into plasma.     

DNA immunization followed by a single boost with cells: a protein-free immunization protocol for production of monoclonal antibodies against the native form of membrane proteins

Recent advancements in antibody-based therapies require the development of an efficient method for generation of monoclonal antibodies (MAbs) against the native form of membrane proteins. We examined DNA immunization followed by a single boost with cells as a protein-free immunization protocol for production of MAbs. Mice immunized with plasmid cDNAs encoding human CD30 or Ret tyrosine kinase were given a single boost with cells expressing the corresponding antigen prior to cell fusion. A total of nine cell fusion experiments revealed that the cell boost is necessary for efficient generation of hybridomas and the DNA-cell boost method gave good yields of specific MAbs (5–59 MAbs from one mouse). All IgG isotypes except IgG3 were generated, although IgG2a was the dominant isotype. All the MAbs reacted with native antigens expressed on cells in a fluorescence-activated cell sorter (FACS) analysis as well as with recombinant CD30 or Ret protein genetically fused with human Fc in an enzyme-linked immunosorbent assay (ELISA). The affinities of the anti-CD30 MAbs to CD30-Fc protein ranged from 0.9 to 12.4 nM K_ds, which were comparable to existing MAbs to these proteins, which range from 3.0 to 13.0 nM. Western blot analysis and topographical epitope mapping experiments based on the mutual competition of pairs of the anti-CD30 MAbs revealed that about 40% of the epitopes were linear epitopes and that each epitope was topographically classified into one of six groups. The large number of MAbs that react with high affinities to a variety of epitopes on the native form of antigens indicates that the method presented in this paper could be generally useful for generating MAbs to other membrane proteins.     

Antibody production by transferred lymphoid cells: Inhibition by competition of antigens

A/Jax and C57BL mice were immunized with either human red blood cells or with both human cells and (A/Jax×C57BL)F₁ hybrid spleen cells, and sensitized spleen cells were transferred to isogeneic or hybrid hosts. Anti-human haemagglutinin titres were lower in hosts receiving C57BL rather than A/Jax cells, were lower in hybrid than isogeneic hosts, and were lower in animals receiving doubly- than in those receiving singly-sensitized cells.

The results can be best interpreted on the basis of competition of antigens, rather than by postulating allergic death of sensitized lymphoid cells exposed to an overwhelming dose of antigen.

Competition of antigen was found to occur in A/Jax and C57BL mice actively immunized with both human red cells and hybrid spleen cells, when antibody titres were compared with those of singly-immunized controls. Anti-human haemagglutinin titres in actively immunized hybrid mice were intermediate between those of the parental strains, suggesting that differences in titres between the parental strains were due to physiological differences in the antibody-forming system, rather than to the more poorly reacting strain sharing antigenic determinants with and therefore being partially tolerant to human red cells.

     

Antibody production by injection of living cells expressing non self antigens as cell surface type II transmembrane fusion protein

Multi-well assays based on the Boyden chamber have enabled highly parallel studies of chemotaxis-the directional migration of cells in response to molecular gradients-while direct-viewing approaches have allowed more detailed questions to be asked at low throughput. Boyden-based plates provide a count of cells that pass through a membrane, but no information about cell appearance. In contrast, direct-viewing devices enable the observation of cells during chemotaxis, which allows measurement of many parameters including area, shape, and location. Here we show automated chemotaxis and cell morphology assays in a 96-unit direct-viewing plate. Using only 12000 primary human neutrophils per datum, we measured dose-dependent stimulation and inhibition of chemotaxis and quantified the effects of inhibitors on cell area and elongation. With 60 parallel conditions we demonstrated 5-fold increase in throughput compared to previously reported direct-viewing approaches.     

Antibody production by `cloned' cell populations

A method for the instrasplenic cloning of lymph node cells, based on the injection into X-irradiated recipients of cells from phytohaemagglutinin treated donors was developed. Clones were tested for their capacity to form anti-Shigella antibodies, and to produce a graft versus host immune response. It was found that each of the randomly chosen clones produced antibodies to Shigella. When immunized `cloned' cell populations were injected into secondary recipients, and the latter were again immunized with Shigella, an increased response was obtained. Clones produced by bone marrow cells in spleens of X-irradiated animals, similarly tested for the immunological competence did not produce antibodies to Shigella. Cells of lymphoid clones of parental strain origin produced a graft versus host response in newborn F₁ hybrids. Bone marrow derived clones did not. Thus, each cloned cell population appears to be a priori pluripotential with respect to antibody formation. The immunological findings, analysed in relation to the population sizes of the tested clones, seem to indicate that each immune competent cell may, in fact, be pluripotential.     

Lymphokine production by T cells generated during infection with Trichinella spiralis

Lymphokine production by mesenteric lymph node cells (MLNC) taken from mice during infection with the intestinal nematode parasite Trichinella spiralis was investigated. Upon stimulation in vitro with a protective crude antigen preparation of the infective L1 larvae, MLNC proliferated in a specific manner, and were observed to release the T-cell lymphokines IL-2, IL-3 and interferon. IL-2 and IL-3 release by MLNC was greatest when taken during the early intestinal phases of infection. Interferon was also released by cells taken from infected mice, with highest levels observed shortly after expulsion of the parasite from the gut. MLNC taken during the early intestinal phases of infection were also able to respond and proliferate to an exogenous source of IL-2, suggesting clonal expansion of T cells within the node. Lymphokine release from T. spiralis specific T-cell lines was also examined.     

Antibody responsiveness during immunization and challenge of genetically modified antibody responder mice with murine hepatitis virus 3

The aim of this study was to evaluate some immunological patterns involved in natural and acquired resistance against MHV3 using the original model of genetically modified lines of mice selected for high (HIII) and low (LIII) antibody responsiveness. As previously shown, a lower pre-existing anti-MHV antibody level was found in susceptible HIII mice as compared to resistant LIII mice. Mortality rates of the F1 (H x L) hybrids and F2 and backcross segregants reflected co-dominance of both characters and the survivors had higher preexisting anti-MHV antibody titers. The present data show that both lines had the potential to synthesize antibodies and that the resistance acquired by the susceptible HIII mice paralleled the antibody synthesis. Nevertheless, higher antibody titers were necessary to confer resistance in HIII mice than in LIII ones. When compared to uvMHV3, a single immunization with a related infectious MHV strain induced a higher antibody synthesis and led the HIII mice to resist the MHV3 challenge. A direct correlation between the antibody level and resistance to infection was always observed in HIII mice. Although mounting a Th2 response as indicated by IgG1 responses, they were also able to readily synthesize large amounts of IgG2a antibodies after immunization or during infection, reflecting a Th1 response. The transfer of anti-MHV antibodies to susceptible HIII mice was capable of conferring resistance to MHV3, providing the antibodies were present before virus infection and in large amounts. The resistance and the survival time of these animals increased with the level of antibody administered. If these direct and clear data suggest that HIII mice can acquire resistance through antibodies, the basis of the resistance of the resistant LIII mice may rely on mechanisms less dependent on antibodies.     

Antibody response in rabbits to immunization with Mycobacterium leprae.

Mycobacterium leprae purified from liver tissue of an infected armadillo (the A/10 preparation) was tested for antigenic composition by immunization of rabbits and characterization of the antibody response by crossed immunoelectrophoresis. The rabbit antisera detected seven distinct components in the M. leprae preparation. This number is far lower than in similar experiments with other mycobacteria. The M. leprae sonic extract gave far fewer lines after polyacrylamide gel electrophoresis and staining with Coomassie brillant blue than sonic extracts prepared from BCG, M. smegmatis, and M. phlei adjusted to the same protein concentration based on the Folin assay. The seven components detected in M. leprae cross-reacted extensively with M. avium, BCG, M. lepraemurium, M. smegmatis, and Nocardia asteroides. The seven components are involved in immune reactions in leprosy; antibodies against all of them were demonstrated in sera from patients with lepromatous leprosy, but the specificity of the antibodies varied from patient to patient. The reason for the demonstration of so few antigenic components and some of the implications of these findings for the use of armadillo-grown M. leprae to develop specific skin test reagents and in other aspects of leprosy research are discussed.     

Kinetics of virus production from single cells

The production of virus by infected cells is an essential process for the spread and persistence of viral diseases, the effectiveness of live-viral vaccines, and the manufacture of viruses for diverse applications. Yet despite its importance, methods to precisely measure virus production from cells are lacking. Most methods test infected-cell populations, masking how individual cells behave. Here we measured the kinetics of virus production from single cells. We combined simple steps of liquid-phase infection, serial dilution, centrifugation, and harvesting, without specialized equipment, to track the production of virus particles from BHK cells infected with vesicular stomatitis virus. Remarkably, cell-to-cell differences in latent times to virus release were within a factor of two, while production rates and virus yields spanned over 300-fold, highlighting an extreme diversity in virus production for cells from the same population. These findings have fundamental and technological implications for health and disease.     

Production of a monoclonal antibody against SARS-CoV spike protein with single intrasplenic immunization of plasmid DNA

Severe acute respiratory syndrome (SARS) is a highly infectious disease caused by a novel coronavirus (SARS-CoV). Specific monoclonal antibodies (mAbs) against the SARS-CoV are vital for early diagnosis and pathological studies of SARS. Direct intrasplenic inoculation of plasmid DNA encoding antigen is an effective and fast approach to generate specific mAb when the protein antigen is difficult to prepare or dangerous in use. In this study, we selected one fragment of SARS-CoV spike protein (S1-₃) as antigenic determinant by immunoinformatics. Single intrasplenic immunization of plasmid DNA encoding S1-₃ induced anti-spike protein antibodies. We established one hybridoma cell line secreting specific mAb and evaluated this mAb with murine leukemia virus pseudotyped with SARS-CoV spike protein (MLV/SARS-CoV). The mAb could recognize the spike protein on the MLV/SARS-CoV-infected Vero E6 cells albeit with no neutralizing effect on the infectivity of the pseudotype virus. Our results show that a single-shot intrasplenic DNA immunization is efficient for the production of specific mAb against SARS spike protein, and a linear epitope of the spike protein is recognized in this study.     

Protection of newborn animals through maternal immunization

Providing protective immunity to neonatal animals in early life is associated with numerous challenges regarding vaccine safety and efficacy. A much simpler approach is maternal vaccination, either before or during pregnancy, to provide the neonate with passively transferred immunity. In humans, the medical, societal and legal risks of immunizing pregnant women are important considerations in undertaking this approach. By contrast, maternal vaccination has been successfully employed in the animal health industry for decades. These veterinary vaccines have proven to be safe and efficient. Although only passively transferred antibodies have been extensively studied, other immunological mechanisms may be equally important in providing maternally derived immunity.     

Effect on the production of soluble endoglin from human choriocarcinoma cells by preeclampsia sera

Citation Aoki Y, Yamamoto T, Fumihisa C, Nakamura A, Asanuma A, Suzuki M. Effect on the production of soluble endoglin from human choriocarcinoma cells by preeclampsia sera. Am J Reprod Immunol 2012; 67: 413–420 Problem The soluble endoglin (sEng) is an antiangiogenic protein that may inhibit TGF‐β1 signaling and endothelial nitric oxide synthase activation in endothelial cells. The levels of sEng increased in sera obtained from preeclampsia. The factors that increase the sEng in preeclampsia have not been known well. To investigate the factors that may increase sEng in preeclampsia, we examined the effect of preeclampsia sera on the production of sEng from human choriocarcinoma (JEG‐3) cells. Methods Serum samples were taken from women with normal pregnancy and from those with preeclampsia. JEG‐3 cells were cultured with serum for 24 hrs, and the sEng levels in supernatants and expression of sEng and Hemo oxygenase‐1 (HO‐1) mRNA in cells were measured. Results The addition of preeclampsia sera into JEG‐3 cells led to increased release of sEng and expression of Eng mRNA. Preeclampsia sera inhibited the expression of HO‐1 mRNA in JEG‐3 cells. Conclusion The results suggest that preeclampsia sera may increase the protein production of sEng and mRNA expression of Eng from JEG‐3 cells like trophoblast without hypoxia and that in addition to hypoxia, preeclampsia sera may play a role of high level of serum sEng in preeclampsia patients. Decreased HO‐1 activity may relate to increased sEng release.     

A simple method for demonstrating specificity of antibody production by single cells

A simple method is described for identifying possible double-antibody-producing spleen cells. This utilises the slide chamber adaptation of the haemolytic plaque technique and chicken and sheep red blood cells which are morphologically easy to distinguish and against which mice do not from cross-reacting antibodies. Of 1600 plaques examined only 2 were found to involve haemolysis of both chicken and sheep red blood cells and it is possible that these both resulted from two antibody-forming cells rather than one.