Class, amounts, and affinities of anti-dinitrophenyl antibodies in chickens. II. Production of a restricted population of high affinity 7S antibodies by injection of antigen emulsified in adjuvant.

The response of chickens given a single intramuscular injection of maximally coupled dinitrophenylated-gamma-bovine beta-globulin in either Freund's complete (FCA) or incomplete (FIA) adjuvants was characterized by an initial synthesis of 7S and 17S antibodies followed by the exclusive and persistent production of 7S antibodies. The 17S antibodies were not detected either 3 to 4 weeks after a single injection or after an intravenous boost 16 months later. Injections of low doses of antigen in FCA induced the synthesis of 7S antibodies of high affinity at least by 4 months. Analyses of the Sips plots generated from equilibrium dialysis data indicated that a shift in the distribution of 7S antibody affinities occurred because of the production of a restricted population of high affinity antibodies. The changes in the binding properties of antibody during the immune response from chickens given antigen in FIA were less apparent, although qualitatively similar, to those found in birds given antigen in FCA. Three possibilities were presented to explain the effect of adjuvant on the class and affinity of the antibody: a) the requirement of a second signal for B cell differentiation, b) the presence of subpopulation of B cells, and c) somatic mutation events.     

Direct and indirect plaque forming cells in extrapulmonary lymphoid tissue following local vs systemic injection of soluble antigen

AKR strain mice were immunized with solubilized SRBC stroma either by direct injection into the lower respiratory tract or intravenously via the tail vein. The number of plaque forming cells (PFC) in the draining plumonary lymph node (tracheobronchial node) and spleen were determined by direct (IgM) and indirect IgG₁, IgG_2b, IgA) plaque assays.Intravenously administered antigen induced an initially strong IgM response in the spleen which was subsequently followed by antibody of the IgG₁, IgG_2b, and IgA classes of immunoglobulins. The tracheobronchial lymph node contained a minimal number PFC representing all four types of immunoglobulins studied. Conversely, following a single local injection of antigen directly into the lower respiratory tract, the tracheobronchial node responded with relatively high concentrations of PFC of all classes. The response in the spleen, although higher than background, was barely detectable. The splenic response to locally administered antigen was, however, considerably augmented as a result of a second local injection given 45 days after the initial stimulation. Under these conditions, IgG₁ IgG_2b, and IgA were represented in both tissue sites by sharp increases in the number and a decrease in the time of appearance of their respective antibody forming cells. Comparable changes were not noted for the case of IgM.Serum hemagglutination titres following a single injection by either route did not vary significantly during the time course of the experiment (28 days). The sera from locally immunized mice were treated with the reducing agent dithiothreitol and hemagglutination titres, before and after treatment, were compared. The major serum activity observed during the first 10 days following injection was affected by reduction and could therefore be assigned to high molecular weight antibody (19S, 13S). Subsequent titres (Days 13–26) were less susceptible to DTT and are considered to represent low molecular weight immunoglobulins (7S).     

Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10⁷ thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls.Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.     

The immune system of juvenile thick-lipped grey mullet, Chelon labrosus Risso: antibody responses to soluble protein antigens

Juvenile thick-lipped grey mullet, Chelon labrosus , believed to be about 6–7 months old, possessed well developed lymphoid organs including a clearly differentiated thymus with distinct cortical and medullary zones. However, unlike older fish, the juvenile mullet usually failed to produce antibody in response to a single injection of classical thymus-dependent antigen (using the soluble proteins human gamma globulin or keyhole limpet haemocyanin). Prior priming of the juvenile fish with antigen was found to potentiate antibody production following challenge with a second dose of the antigen in adjuvant, priming by oral administration being equally as effective as priming by injection. Neither juvenile nor adult mullet produced any significant level of antibody against ovalbumin.
The results suggest that, despite their apparently well differentiated lymphoid organs, juvenile mullet still show a certain level of immaturity in their antibody responses to soluble proteins; also that immunization can improve their ability to respond.     

The effect of Bordetella pertussis on the antibody response in mice to type III pneumococcal polysaccharide.

The effect of an i.p. injection of Bordetella pertussis on the primary humoral immune response in mice to the thymus-independent antigen SIII has been studied. Suppression of the antibody response occurred when pertussis cells were injected at the same time as an optimal immunizing dose of SIII. In contrast, the antibody response to high doses of SIII was enhanced by B. pertussis. When SIII alone was injected, only 19S antibody was detected. However, when B. pertussis was administered with either optimal or high doses of SIII, 7S as well as 19S antibody against SIII was produced.     

Preparation of conjugates of 19s and 7s globulins of antitularemic sera for the determination of specific fluorescence ofPasteurella tularensis

Pasteurella tularensis was detected by means of conjugates of 19S and 7S globulins from antitularemic serum with fluorescein isothiocyanate (FITC). The serum was obtained by short-term immunization of rabbits. The capacity of both fractions to produce the immunofluorescence reaction with pure culturesof Pasteurella tularensis and the agglutinin titre of these fractions are compared here. It was found that the 19S globulin fraction which contained most agglutinating antibodies showed only weak immunofluorescence. The 7S fraction in which the agglutinin content was very low as compared with the 19S globulin fraction gave more intense fluorescence than the 19S fraction. On the basis of this finding the opinion is advanced that the agglutinating antibodies need not be the actual carrier of fluorescence due to fluorescent antibodies (FA) in the immunofluorescence reaction ofPasteurella tularensis. The different degrees of labelling with FITC of the individual globulin fractions as observed during simultaneous conjugation are also discussed. The 19S globulin fraction is regularly conjugated to a higher degree than the 7S component. The view is presented that the avirulent strains lack a certain part of the antigen structure as compared with virulent or vaccine strains (with residual virulence), the part being responsible for the positive reaction of immunofluorescence with preparations of fluorescent antibodies.     

Effect of specific antibodies on active immunity development in those inoculated with an antirabies vaccine

The study of the effect of gamma globulin introduced in different doses (0.5 and 0.25 ml/mg) in combination with Fermi rabies vaccine (observations on humans were made) and with cerebral rabies vaccine inactivated by UV irradiation (in animal experiments) demonstrated that the injection of the higher doses of gamma globulin resulted in lower geometrical mean of antibody titers. Therefore, in combined administration of rabies vaccine and gamma globulin for postexposure rabies prevention it is advisable to reduce the dose of gamma globulin by one-half.     

Attempted Passive Immunization of Young Calves Against Eimeria bovis

SYNOPSIS. Two experiments attempted to produce passive immunity against Eimeria bovis coccidiosis in Holstein-Friesian calves. Immune serum concentrated by a freezing technique, or serum globulin obtained by a precipitating technique from immune calves, was injected intravenously or intraperitoneally into young calves. Four calves received concentrated immune serum injected intravenously on the day of oral inoculation with sporulated oocysts and again 7 and 14 days later. Four calves were given intravenous injections with some of the same serum on the 7th and 14th days after inoculation and 4 others were given a single similar injection with the same serum 14 days after inoculation.
Three calves in a second experiment received intraperitoneal injections of serum globulins in increasing amounts every 3 days for 2 weeks. The calves were then orally inoculated with sporulated oocysts one week after the last globulin injection. Some calves receiving immune serum had an anaphylactoid reaction characterized by increased respiration rate, dyspnea, coughing, and salivation; however, all affected calves recovered spontaneously within 2 hours. Calves receiving serum globulin had no reactions.
Coccidiosis developed in all of the calves in spite of the injection of immune serum or globulin presumed to carry the immune factor. There was no detectable difference in the rate of oocyst discharge or in clinical symptoms between treated and control calves; therefore, no evidence of passive immunity was observed.     

Plasmodium berghei: relationship between protective immunity and anti-sporozoite (CSP) antibody in mice

Protective immunity and production of anti-sporozoite (CSP) antibody was studied in A/J mice injected with X-irradiated sporozoites using different immunization schedules and antigen doses. Data were also obtained on the immunogenicity of X-irradiated as compared to nonirradiated sporozoites. After a single immunization (1.5 × 10⁵ or 7.5 × 10⁴ X-irradiated sporozoites) a number of animals was completely protected when challenged, but the percentage of protected mice varied considerably from experiment to experiment. Maximal protection was obtained 7 days after the immunization. When the first injection of parasites was followed by a single booster administered 3, 4 or 5 days later, protection was considerably enhanced and the results more consistent. After a single injection of 1.5 × 10⁵ or 7.5 × 10⁴ sporozoites, CSP antibody was detectable from the 19th and 23rd day, respectively, i.e., at a time point when protection was diminishing. This antibody persisted only for a short period. When a single booster was given soon after the first injection, CSP antibody was present in the sera of all the mice from the ninth day on and persisted for greater than 80 days. A single dose of X-irradiated sporozoites injected into rats, induced antibody (CSP) formation which reached a peak after 2 weeks and persisted at this level for more than 3 months. However in rats injected with viable sporozoites, the antibody titers fell rapidly and became undetectable after 4 weeks.From these data we can conclude that (a) the immune response induced by attenuated X-irradiated sporozoites is considerably longer-lasting than that induced by viable sporozoites; (b) CSP antibodies are not detectable during the early stages of the immune response; and (c) protective immunity precedes the presence of detectable serum and antibodies.     

Specific biochemical marker-based techniques for the identification of damage to Douglas-fir seed resulting from feeding by the western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera: Coreidae)

Specific biochemical marker-based techniques were tested for their ability to distinguish between seeds of Douglas-fir, Pseudotsuga menziesii (Mirbel) Franco, that were filled or unfilled (aborted) at maturity and those that were damaged or emptied by the western conifer seed bug, Leptoglossus occidentalis Heidemann. A polyclonal antibody raised against salivary gland extracts from L. occidentalis successfully identified residual salivary proteins on Western blots containing proteins from Douglas-fir seeds that had sustained various degrees of seed bug feeding damage. In a single blind experiment, the polyclonal antibody correctly identified 100% of undamaged control, 97% of unfilled control (aborted), and 98% of seed bug damaged seeds. Polyclonal antibodies raised against insoluble alfalfa crystalloid storage protein (11S globulin) detected the depletion of 11S globulin and the subsequent appearance of its hydrolyzed fragments in the soluble protein fraction of Douglas-fir seeds that were fed-upon by the seed bug. Feeding by L. occidentalis nymphs caused ca. 98% depletion of insoluble protein, but only ca. 53% reduction in the amount of soluble protein in seeds that appeared empty on radiographs. By comparison, unfilled (aborted) seeds contained significantly less insoluble and soluble protein than empty seeds that were fed-upon by L. occidentalis; moreover, no crystalloid (11S globulin) breakdown products were generated. The biochemical markers described in this study are reliable tools that can be used to identify conifer seeds that have sustained light to severe damage from L. occidentalis feeding.     

The morphological characteristics of globulin production during the vaccinal process induced by tularemia and brucellosis vaccines

Significant data on the dynamics of globulin production in guinea pigs in the process of immunogenesis after the injection of Francisella tularensis vaccine strain or conjugated brucellosis vaccine have been obtained by means of immunofluorescence and the enzyme immunoassay. The number of globulin-producing cells in lymphoid organs (the spleen, regional and remote lymph nodes) differs, depending on the injected antigen. The relationship between the character of immunomorphological changes in lymphoid organs and the dynamics of the increase of antibody titers in the peripheral blood of the animals after their immunization with conjugated brucellosis vaccine and the injection of avirulent F. tularensis has been established.     

不同佐剂对preS2／S乙型肝炎疫苗免疫原性的影响

目的探讨不同佐剂对preS2／S乙型肝炎疫苗免疫原性的影响。方法将BALB／C小鼠随机分为6组：铝屯剂组（S＋AI和S2／S＋A1）、细胞因子佐剂组（S2／S＋CKl、S2／S＋CK2和S2／S＋CK3）及阴性对照组（NG）,用相应抗原且腔免疫各组小鼠,1mL／只,分别于免疫后2、4和6周采血,分离血清,ELISA法检测血清中抗-HBs抗体和preS2圭体水平;wsT一1法检测淋巴细胞增殖情况。结果免疫后2周,铝佐剂组（S＋A1和S2／S＋A1）抗体水平较高,阳转i达100％,而细胞因子佐剂组抗-HBs抗体水平均较低,S2／S＋CK2及S2／S＋CK3组抗体阳转率为0,与NG组比较2异无统计学意义（P〉0．05）;免疫后4周,细胞因子佐剂组抗-HBs抗体水平升高明显,铝佐剂组S2／S＋A1抗体水习显著高于S＋A1（P〈0．05）;免疫后6周,细胞因子佐剂组抗体水平高于铝佐剂组,其中S2／S＋CKl组抗体水平较高免疫后2周,各组小鼠血清中抗-preS2抗体均达到较高水平,各组间差异均无统计学意义（P〉0．05）;免疫后4月和6周,抗体水平均有所下降,但下降不明显;铝佐剂组S＋AI和S2／S＋A1抗-preS2抗体水平差异无统计学意义（P0．05）。免疫后4周,各组小鼠淋巴细胞刺激指数铝佐剂组（S＋A1和S2／S＋A1）低于细胞因子佐剂组,且差异有统t学意义（P〈0．05）,其中S2／S＋CKl组淋巴细胞增殖水平较高。结论不同佐剂的preS2／S乙型肝炎疫苗均能诱导较高水平的抗-HBs和抗-preS2抗体,细胞因子佐剂组淋巴细胞增殖水平显著高于铝佐剂组,为新型乙型肝炎疫l和治疗性乙型肝炎疫苗的研究提供了实验依据。     

The anti-idiotypic response to the experimental administration of the inactivated or live influenza virus]

The injection of inactivated and live influenza virus into rabbits induces the formation of anti-idiotypic antibodies, appearing after anti-influenza hemagglutinins, in the blood. The presence of immune complexes antibody--anti-idiotypic antibody in the blood of the animals has been established. The booster immunization of the animals with influenza virus antigens produces a rise in the levels of both idiotypic and anti-idiotypic antibodies. The injection of autologous anti-idiotypic globulin into the primed animals ensures the induction of idiotypic and anti-idiotypic revaccinal reactions.     

Adenovirus antibody measured by the passive hemagglutination test

Lefkowitz, Stanley S. (Variety Children's Research Foundation, Miami, Fla.), Julia A. Williams, Bernard E. Howard, and M. Michael Sigel. Adenovirus antibody measured by the passive hemagglutination test. J. Bacteriol. 91:205-212. 1966.-Rabbits immunized intravenously with adenovirus type 5 antigen were tested for antibody titers by use of the passive hemagglutination test (PHA). Primary and secondary responses were studied, and the class of antibody was determined by means of density gradient centrifugation and reduction with 2-mercaptoethanol (ME). It was found that the PHA was 10 to 100 times more sensitive than complement-fixation and neutralization tests for the detection of antibodies to adenovirus. The immunological response to primary immunization was dependent on the dose of antigen, with antibody appearing in as early as 3 days. After secondary stimulation with the same antigen, there was a rapid response which appeared to be less dose-dependent. It was found that a heavy 19S antibody sensitive to ME was produced early and was followed by a lighter, presumably 7S, ME-resistant antibody. Upon secondary stimulation, both 7S and 19S antibody increased to levels greater than those of the primary injection.     

Antibody Response to a Human Diploid Cell Rabies Vaccine

An experimentally killed rabies virus vaccine prepared in a human diploid cell strain (WI-38)—Wyeth rabies vaccine (WRV)—was used by various injection schedules in two separate studies to define more closely in human volunteer subjects an effective vaccination schedule for pre- or postexposure immunization, particularly for donors of rabies-hyperimmune plasma. To permit valid comparisons between our results and those of other workers, antibody levels achieved were expressed in terms of international units (IU) per milliliter of serum. Antibody response of previously nonvaccinated persons were only modest after a single dose of WRV, never reaching a level higher than 0.80 IU/ml over a 56-day testing period. Moreover, antibody was not detected at 0.16 IU/ml before the 14th day, either after a single dose or after two doses given 3 days apart. The best response followed four doses of WRV given within 4 weeks. This schedule resulted in the highest rate of seroconversion to the ≥6 IU/ml antibody level required of potential rabies-immune plasma donors. Giving the first vaccine dose in aluminum hydroxide diluent did not enhance the antibody response. There was a definite suggestion in the various injection schedules that higher and more sustained antibody levels were reached when the interval between the first and second vaccine doses was longest. The greater immunogenicity of WRV as compared with duck embryo vaccine was best demonstrated by the fact that a single booster dose of duck embryo vaccine to previously vaccinated individuals resulted in only a sevenfold antibody rise during the following 56 days, whereas a booster dose of WRV elicited a 69-fold rise. Al(OH)₃ in the first dose of WRV had no effect, but the enhancing effect of a longer interval between vaccine doses was noted once again; 20 of 20 subjects who received three doses of WRV with 4 weeks between doses developed good levels of rabies antibody, and 19 exceeded 6 IU/ml.     

Antenatal prophylaxis of Rh isoimmunization: 28-weeks'-gestation service program

Two (0.18%) of 1086 Rh-negative primigravidas or multigravidas treated similarly in all previous pregnancies, who were given a single injection of Rh immune globulin (300 μg) at 28 weeks'' gestation and subsequently were delivered of Rh-positive babies, had demonstrable Rh isoimmunization at the time of that injection and must be considered “logistic” failures of antenatal prophylaxis. The remaining 1084 (who were treated again after delivery) had no evidence of Rh isoimmunization at delivery and none of the 512 screened at 6 months after delivery appeared to be immunized. If the 28th-week injection had not been protective, one would have expected 14 of the 1084 to have been demonstrably Rh isoimmunized and evidence of Rh isoimmunization to have persisted in 6 of the 512 observed 6 months after delivery.Six of 719 Rh-negative multigravidas who had not received Rh immune globulin after previous pregnancies or had been treated only after delivery showed evidence of Rh isoimmunization despite a single injection of Rh immune globulin at 28 weeks in a subsequent pregnancy. In three of the six the cause was most likely “sensibilization” due to previous exposure to Rh-positive blood or an untreated Rh-positive pregnancy. in 3 of the remaining 716 (0.42%) there may have been true failure of antenatal Rh prophylaxis administered at the 28th week. One would have expected this figure to be 12 of 716 if antenatal Rh prophylaxis at 28 weeks'' gestation were totally unsuccessful.It is concluded that a single intramuscular injection of Rh immune globulin, 300 μg, is 88% effective in preventing Rh isoimmunization during pregnancy in Rh-negative primigravidas and in multigravidas treated antenatally in all previous pregnancies, and is 75% effective in preventing Rh isoimmunization in Rh-negative multigravidas untreated during previous pregnancies. The majority of failures are due to Rh isoimmunization during pregnancy prior to antenatal prophylaxis at 28 weeks.     

Suppression of growth of a mouse plasmacytoma by the IgG2 fraction of syngeneic antitumor globulin

Summary The effects of syngeneic antitumor antibody on transplanted plasmacytoma cells have been examined. Globulin was prepared from ascites fluids produced in Balb/c mice injected with MOPC 315 tumor cells and bearing the solid tumor. Normal Balb/c mice were given inoculations of tumor cells that had been incubated with the antitumor globulin obtained at various intervals after immunization, or with portions of such globulins. These materials were prepared to express the IgG2 class antibody by three procedures: precipitation with heterologous anti-mouse IgG1, passage through columns of Sepharose anti-IgG1, or adsorption to and elution from heat- and formalin-killed protein A-bearing staphylococci. The original antitumor globulins showed differences with time relative to the second injection of the immunizing tumor, in that a number of the earlier pools led to some suppression of tumor growth, and a number of the later pools led to some enhancement. Of the globulins obtained later, preparations expressing the IgG2 class of antibody by precipitation with anti-IgG1 serum caused some suppression of tumor growth. Pronounced suppression of growth was consistently obtained with anti-MOPC 315 globulin freed of IgG1 by passing it through an anti-IgG1 immunoadsorbent, and with IgG2a preparations obtained by elution from the protein A-bearing staphylococci. Of the anti-IgG1 column-treated preparations, the suppressive effect was maximal in globulin obtained 15–26 days after the second immunization. The suppressive effect of these preparations could be removed by absorption with MOPC 315 cells, but not by cells of another Balb/c tumor nor by two other plasmacytomas.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Tuberculosis is an ancient scourge of mankind. According to statistics, there are more than 10 million new cases of tuberculosis each year and the annual death toll for tuberculosis exceeds three millions. The current available BCG is of questionable efficacy, and its protection ranges from 0 to 85%. Therefore, developing a safe and effective vaccine against this scourge is very important. Previous studies have shown that the secreted proteins of Mycobacterium tuberculosis (M. TB) can induc…     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Immune response and protective efficacy for the combination of four tuberculosis DNA vaccines were evaluated in this study. We obtained 1:200 antibody titers against Ag85B 21d after mice were vaccinated for the first time by four recombinant eukaryotic expression vectors containing coding sequences for Ag85B, MPT-64, MPT-63 and ESAT-6. The titers of Ag85B were elevated to 1:102400 after the second injection and decreased to 1:12800 after the third injection. Antibody titers for MPT-64 and MPT-63 reached 1:25600 21 d after the first vaccination, and were then decreased following the second and third injections. No antigen-specific antibody titer against ESAT-6 was detected in sera harvested from immunized mice at any time. These DNA vaccines evoked specific IFN-λ responses in the spleens of vaccinated mice as well. When challenged with M. tuberculosis H37Rv, we found that the lungs of the vaccinated mice produced 99.8% less bacterial counts than that of the empty-vector control group and the bacterial counts were also significantly less than that of the BCG group. Histopathological analyses showed that the lungs of vaccinated mice produced no obvious caseation while over 50%-70% of the pulmonary parenchyma tissue produced central caseation in the vector control group. Our results indicated that the combination of four tuberculosis DNA vaccines may generate high levels of immune responses and result in better animal protection.     

Kinetics of B cell memory development during a thymus "independent" immune response

Some parameters of the development of immunological memory to B. abortus (BA) and sheep erythrocytes (SE) in the mouse have been compared. The thymus-independence of the BA response allowed evaluation of B-cell memory in vivo and in adoptive immune responses. A reduced responsiveness to BA was seen during the first few days after the primary injection, whereas enhanced ability to give responses to SE (thymus dependent) occurred at that time.The ability of primed spleen cells to transfer 19S and 7S memory responses to SE developed in parallel. In contrast, the earliest appearance of 19S memory to BA on Days 5–7 after priming was not yet accompanied by memory for the 7S response, but by Day 10 both 19S and 7S memory were present. At 1–2 months after priming, 100-fold fewer cells than needed for transfer of the primary response still transferred excellent 19S and 7S memory responses to BA. Anti-θ treatment of long-term memory 19S and 7S spleen cells did not affect their ability to respond to challenge even with limiting BA doses. It is suggested, however, that the T-independency of the response to BA applies only to the specific induction by antigen of preexisting B cells into antibody secreting cells, whereas optimal B cell memory formation to any antigen may be a separate T-dependent function.Serial spleen cell transfers into lethally irradiated recipients at 1–2 week intervals with antigen challenge at each transfer, appeared to interfere with the development of memory to BA, particularly for the 7S response. No such effect was seen on the responses to SE.