The effect of immunization with a 14-kDa streptococcal antigen on primate T cell and B cell responses

A streptococcal antigen (SA) of 185 kDa was isolated from Streptococcus mutans and this antigen induced in vitro helper, suppressor and contrasuppressor activities with primate peripheral blood lymphocytes. The 185-kDa SA was then treated by sodium dodecyl sulfate and yielded a 4-kDa SA which was capable of eliciting only helper activity. We have now cleaved the 185-kDa SA with cyanogen bromide, in an attempt to identify suppressor and contrasuppressor determinants. A 14-kDa SA was separated from the cyanogen bromide digest and its ability to elicit T cell and B cell functional activities was tested in rhesus monkeys. Whereas the 185-kDa SA (and 4-kDa SA) elicited high serum anti-SA antibodies and the CD4 cells showed an increase in DNA synthesis, this was not demonstrable with the 14-kDa SA. However, the 14-kDa SA, unlike the 185-kDa SA, activated a significant proportion of CD4 and CD8 cells to bind the Vicia villosa lectin (VV) and this is a characteristic feature of contrasuppressor cells. We then studied the effect of sequential immunization of monkeys with the 14-kDa SA, followed by the 185-kDa SA. The results of this showed suppression of the CD4 proliferative response, in the presence of a normal antibody production. We suggest that the split tolerance between the T cell proliferative and B cell differentiating functions might be interpreted on the basis of suppressor CD8 cells inhibiting the CD4 proliferative phase and the VV-adherent CD8 cells contrasuppressing B cell antibody formation.     

Helper and suppressor functions of human mononuclear cells depleted of antigen-binding T8+ cells.

We have utilized the antigen-binding function of a subset of T8+ cells to remove these cells in vitro from human peripheral blood mononuclear cells. This was carried out by treating the cells with streptococcal antigen (SA), monoclonal anti-SA antibody and complement. The concentration of SA binding to T8+ cells differs with the HLA-DR type of the cells: 1 ng SA binds to DRw6+ cells and elicits T helper activity, whereas 1000 ng SA elicits T suppressor activity, in an assay for antibody-forming cells. After depletion of the antigen-binding cells by the SA-specific complement-dependent killing technique, the helper function of the DRw6+ cells was lost but suppression was elicited not only by 1000 ng but also by 1 ng SA. Similarly, DRw6- cells which bind 1000 ng SA to elicit helper activity and 1 ng to elicit suppression, when depleted of the SA-binding cells, resulted in loss of helper activity but again, suppression could be elicited by both 1000 and 1 ng SA. We suggest that treatment of mononuclear cells with antigen, the specific antibody and complement removes the T8+ antigen-binding cells which present antigen to T helper cells and results in the loss of helper function. Suppressor function is however, not only retained with the original concentration of SA but also expressed with that required to elicit helper function in the untreated cells. These findings are consistent with our hypothesis that the antigen-binding and presenting T8+ cells also function as contrasuppressor cells. Thus, the T8+ subset of cells have a dual function, to present antigen and to activate T helper cell function, and to prevent suppressor cells from inhibiting the helper cells.     

Protection against experimental cholera by oral or parenteral immunization.

Comparisons were made between the antigenic potency and protective capacity of several cholera toxin derivatives. Rabbits were immunized parenterally with 50 microgram of cholera toxin, A subunit, B subunit, procholeragenoid, or Wyeth glutaraldehyde toxoid 20101. Examination of the antibody response curves revealed that cholera toxin elicited serum antitoxin responses that rose more quickly than in the subunit-immunized animals; however, antitoxin levels were of the same magnitude after 10 weeks. Parenteral immunization with procholeragenoid evoked antibody titers that were similar to the toxin, whereas Wyeth toxoid yielded only one-tenth the level of antitoxin. Oral immunization with procholeragenoid as well as Wyeth toxoid resulted in lower serum antitoxin titers than that achieved with parenteral immunization, despite the oral administration of 10 times the parenteral dose. Analysis of protection against live-cell challenge revealed that parenteral administration of procholeragenoid provided the best protection against fluid accumulation. Oral immunization with procholeragenoid also was very effective, whereas oral immunization with B subunit or Wyeth toxoid resulted in minimal protection. Also, the A subunit provided surprisingly more protection than did cholera toxin.     

早发性牙周炎患者血清抗牙龈卟啉菌表面抗原抗体滴度和亲和力的研究(英文)

目的：检测早发性牙周炎患者血清抗牙龈卟啉菌脂多糖IgG抗体的滴度和亲和力，并探讨抗体滴度水平和亲和力之间的相关性。 方法： 受试者为15名早发性牙周炎患者、16名成人牙周炎患者和14名牙周健康者。检测了血清中抗牙龈卟啉菌脂多糖IgG抗体滴度和亲和力。血清IgG抗体滴度采用酶联免疫吸附试验（ELISA）测定；其亲和力通过二乙醇胺分离，ELISA测定。 结果： 早发性牙周炎患者和成人牙周炎患者的血清中抗牙龈卟啉菌脂多糖IgG抗体水平显著高于牙周健康者（P<0.01），而在早发性牙周炎患者和成人牙周炎患者之间无显著差异（P>0.05）。早发性牙周炎患者抗体亲和力显著高于成人牙周炎患者和牙周健康组（P<0.01），而成人牙周炎患者和牙周健康组之间无显著差异（P>0.05）。 结论： 早发性牙周炎患者和成人牙周炎患者的抗体滴度水平和亲和力之间不存在相关性。IgG抗体亲和力可以作为诊断早发性牙周炎的有效参数。     

Antibody response to immunization with purified GD3 ganglioside and GD3 derivatives (lactones, amide and gangliosidol) in the mouse

GD3 is the ganglioside most abundantly expressed on the cell surface of human melanoma, and treatment with a monoclonal antibody recognizing GD3 has induced major responses in a small proportion of patients. However, we have been unable to induce production of GD3 antibodies in melanoma patients by active immunization with GD3-expressing melanoma cells or purified GD3. In this report we describe attempts to increase the immunogenicity of GD3 in the mouse by chemical modification. GD3 lactone I and II, GD3 amide and GD3 gangliosidol were synthesized, and the humoral immune response to these derivatives was compared with the response to unmodified GD3. The GD3 derivatives were more immunogenic than GD3. At a low dose all congeners induced an IgM response, with antibody titers higher than those elicited by low-dose GD3. The gangliosidol and amide derivatives also induced an IgG response. IgM antibodies induced by immunization with GD3 lactone I cross-reacted with purified GD3 and GD3-expressing melanoma cells. Titers of GD3 cross-reactive antibodies were slightly higher than after immunization with GD3 itself at the same low dose. IgM and IgG antibodies induced by the other congeners did not cross-react with GD3.     

The role of a human antigen specific T8+ cell subset in antigen presentation, helper function and contrasuppression

Regulation of the human immune response was studied by sequential separation of subsets of T cells, followed by assessment of their helper and suppressor functions in a series of reconstitution experiments. T8+ lymphocytes were separated by panning on streptococcal antigen (SA) coated plates into T8+ SA-adherent cells (T8+SA+) and T8+ SA-non-adherent cells (T8+SA-). The helper and suppressor functions of the T8+SA+ and T8+SA- cells, reconstituted with T4+ helper cells were then studied by a direct antibody forming cell assay. T4+ cells will not induce helper activity by 1000 ng SA alone but require the accessory function of monocytes (Mo). However, replacing Mo by T8+SA+ cells will elicit a similar helper activity by T4+ cells and SA as that induced by Mo. In addition to the antigen-specific presentation and induction of helper activity, the T8+SA+ subset displays the properties of antigen-specific contrasuppressor cells. Thus, reconstitution of T4+ cells and T8+SA- (suppressor cells) with T8+SA+ and 1000 ng SA induces helper and no suppressor activity. Substitution of Mo for the T8+SA+ cells converts the helper to a predominantly suppressor-cell function. T8+SA- cells elicit suppression with 1 ng SA in the absence of accessory cells and reconstitution with Mo, T8+SA+ or T4+ cells failed to affect the suppressor activity. Total reconstitution of the four principle subsets of T4+, T8+SA+, T8+SA- cells and Mo elicited similar antigen dose-dependent responses as those of the unseparated mononuclear cells. It seems that all four cell subsets are required for optimal immunoregulation. We suggest that the T8+SA+ can present antigen to T4+ helper cells and induce helper activity, but in addition these cells can prevent the suppressor subset of T8+ cells from inhibiting T4+ helper cells and function as contrasuppressor cells. The mechanism of these functions is not known but HLA class II antigens might play an essential role in antigen binding, presentation and contrasuppression of the T8+SA+ cells, as the latter are significantly enriched in Ia+ cells.     

Primary humoral antibody response to Coxiella burnetii, the causative agent of Q fever.

Of 147 patients with acute Q fever diagnosed during a major outbreak in Birmingham, England, in early summer 1989, 41 provided sets of sera which allowed us to make a detailed analysis of the primary humoral immune response. Antibody titers specific for Coxiella burnetii were measured by the complement fixation test and by an immunoglobulin M (IgM)- and IgG-specific indirect immunofluorescence test. The relative avidity of specific IgGs was determined by the indirect immunofluorescence test with and without treatment of antigen-antibody complexes with 8 M urea. The IgG subclass responses after primary infection and their avidities were also determined for a limited number of paired serum specimens. Specific IgM titers persisted for more than 6 months in the majority of cases and were therefore not a sufficient criterion for the diagnosis of recent infection. However, for serial samples the antibody titer ratios (IgG/IgM) and the ratios (IgG titer with treatment/IgG titer without treatment) that indicated relative avidity changed significantly, depending on the time postinfection. Within the IgG class, the C. burnetii-specific antibody response over time was almost exclusively represented by subclass 1 molecules, which thus showed affinity maturation.     

Evaluation of antibody parameters as potential correlates of protection or enhancement by experimental vaccines to equine infectious anemia virus.

We previously demonstrated in trials of a variety of experimental vaccines to equine infectious anemia virus (EIAV) a remarkable spectrum of efficacy ranging from sterilizing protection to severe enhancement of virus replication and disease, depending on the immunization strategy used. This range of vaccine efficacy observed in vivo offers a unique opportunity for evaluating potential in vitro immune correlates of protection and enhancement. We describe here a comprehensive analysis and comparison of EIAV envelope-specific antibody responses elicited by attenuated, inactivated whole virus and envelope subunit vaccines to EIAV, and we evaluate the potential of in vitro antibody assays as correlates of protection or enhancement. Thus vaccine-induced serum antibody responses in experimentally immunized ponies at the day of challenge were assayed using a panel of quantitative, qualitative, and functional in vitro assays, including end-point titer of total and isotypic IgG, serum antibody avidity, conformational dependence, and serum neutralization. The results of these studies revealed substantial differences in the EIAV envelope-specific antibody responses elicited by the different vaccines, indicating the importance of envelope glycoprotein antigen presentation in determining the specificity of vaccine immunity. Although no single in vitro parameter provided a statistically significant correlate of protection or enhancement, the use of multiple parameters (titer, avidity index, and conformation ratio) could be used as a reliable correlate of vaccine protection and that the level of vaccine protection was closely associated with the development of mature antibody responses. These studies demonstrate the importance of using multiple antibody assays to evaluate lentiviral vaccine responses and emphasize the need for the development of new in vitro antibody assays that may provide more insight into vaccine protection and enhancement.     

The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration.

We have investigated the immunological consequences of feeding a protein antigen to previously immunized animals. BALB/c mice were systemically primed with ovalbumin (OVA) in complete Freund's adjuvant (CFA) and fed with high (10 mg/g body weight), medium (1 mg/g body weight) or low (1 microgram/g body weight) doses of OVA once (Day 1, 7 or 14) or sequentially for 5 days (Days 1-5, 7-11, 14-18). The specific IgG antibody response was suppressed only by early feeds of high-dose OVA (Days 1-5). Medium-dose OVA fed on Day 14 or low-dose OVA fed at any stage after immunization enhanced the IgG antibody response. In contradistinction, systemic delayed-type hypersensitivity responses (DTH) were usually suppressed by early feeds of high or medium doses of OVA but never after feeding low-dose OVA. The results suggest that systemic DTH and IgG antibody responses to oral antigen are subject to different control mechanisms in previously primed animals. Such responses depend on the immune status of the animal and are controlled by antigen dose, time and frequency of feeding. The immunological effects observed are also demonstrable following adoptive transfer of spleen cells collected 14 days after multiple feeds of high-dose OVA to immunized mice. Our findings suggest that oral hyposensitization after systemic immunization is regulated by (suppressor) spleen cells which are activated by gut-processed antigen.     

Estradiol induced alterations of the immune system--I. Enhancement of IgM production

Estrogens have been postulated as natural modulators of the immune system. In this study we have evaluated the effect of estradiol on in vivo immunoglobulin production in male rats. Administration of either a 75 microgram/kg or 750 microgram/kg dose of estradiol resulted in a dose-related increase of anti-sheep red blood cell antibody titers during a primary antibody response. These same animals when dosed with estradiol during a secondary antibody response showed an earlier appearance of the peak antibody titer in estradiol-treated rats as compared to controls. Rats dosed with estradiol only during the secondary antibody response period also showed a dose-related increase in Ab titer over control values but no shift in the time of appearance of the peak titer. Treatment of sera with 2-mercaptoethanol to evaluate IgG titers showed that 2-mercaptoethanol treatment both reduced the titer to control values and eliminated the estradiol-induced shift in the appearance of the peak antibody titer in rats given estradiol during the primary and secondary antibody response periods. Sera from animals dosed with estradiol during the secondary antibody response period only also showed titers reduced to control levels after 2-mercaptoethanol treatment of the sera. Estradiol-treated rats also had an increase in antibody titers to polysaccharide from Type III pneumococci, a T-cell independent antigen. While total antibody responses were increased following estradiol, there were no differences in the number of antibody-producing cells between control and estradiol-treated animals. The results of these studies suggest that estradiol exerts a direct effect on B cells resulting in increased synthesis of IgM antibodies. We cannot rule out, however, some modulation through regulatory T-cells.     

Mucosal immune responses to meningococcal group C conjugate and group A and C polysaccharide vaccines in adolescents

Previous studies in children have shown that Haemophilus influenzae type b (Hib) polysaccharide conjugate vaccines can reduce nasopharyngeal carriage of H. influenzae and provide herd immunity and suggest that this effect is mediated through mucosal antibodies. As this phenomenon may operate in other invasive bacterial infections which are propagated by nasopharyngeal carriage, mucosal antibody responses to meningococcal C conjugate and A/C polysaccharide vaccines were investigated. A total of 106 school children aged 11 to 17 years were randomized to receive a single dose of either conjugate or polysaccharide vaccine in an observer-blind study. Before and at 1, 6, and 12 months after immunization, samples of unstimulated saliva were collected and assayed by enzyme-linked immunosorbent assay for group C polysaccharide-specific immunoglobulin A (IgA), IgA1, IgA2 and secretory component, IgG antibodies, and total IgG and IgA. A subset of serum samples were also assayed for specific IgA and IgG antibodies. The concentrations of specific IgA and IgG in saliva were expressed both as nanograms per milliliter and as nanograms per microgram of total IgA or IgG. One month after immunization, significant increases in antibody titers (both IgA and IgG) were observed in saliva in both groups. There were significant subsequent falls in antibody titers by 6 months. Anti-meningococcal C-specific secretory component and IgA antibody titers were closely correlated (r = 0.85, P < 0.001), but there was no significant correlation between salivary and serum IgA titers, suggesting that IgA antibodies are locally produced. Significant correlation was found between salivary and serum IgG titers (r = 0.52, P < 0.01), suggesting that salivary IgG may be serum derived. Compared with polysaccharide vaccine, the conjugate vaccine induced significantly higher salivary IgG responses (P < 0.05), although there were no significant differences between salivary IgA responses to the two vaccines. The conjugate vaccine induced greater salivary IgG responses than a polysaccharide vaccine. Both vaccines induced significant salivary IgA antibodies. Further studies are needed to establish the functional significance of these mucosal responses.     

High Level Antibody Avidity is Achieved in HIV-Seropositive Recipients of an Inactivated Split Adjuvanted (AS03A) Influenza Vaccine

Purpose

More severe influenza disease and poor vaccine immunogenicity is reported in HIV-infected patients. We measured antibody avidity after influenza vaccination in HIV patients to assess vaccine efficacy.

Methods

Two dosing strategies (Group1: single dose, n?=?28. Group2: single dose plus booster, n?=?36) with an AS03_A-adjuvanted H1N1₂₀₀₉ pandemic influenza vaccine (Arepanrix, GSK) were assessed in HIV patients. Serum hemagglutination inhibition (HAI) titers and antibody avidity reported as an avidity index (AI) were measured at days 21 and 42 and at 6 months.

Results

Baseline HIV parameters were similar among all participants. Eighteen participants had measurable baseline HAI titers. In these subjects, AI was at ~9 at baseline and was not significantly increased by one or two vaccine doses. In those without detectable baseline antibodies, immunization induced modest antibody titers [Group1 HAI, 61 (26–144); Group2 HAI, 46 (28–76)] with high AI after one dose at day 21 [Group1 AI, 8.8 (7.3–10.7); Group2 AI, 8.9 (7.8–10.1)]. A second dose of vaccine generated significantly higher HAI titers at day 42 [Group1 HAI, 41 (18–90); Group2 HAI, 92 (64–132)] and persisted to 6 months [Group1 HAI, 9 (6–13); Group2 HAI, 19 (13–30)]. All subjects who produced detectable HAI titers after vaccination generated high antibody avidity (AI, 9–10), which persisted up to 6 months.

Conclusion

In participants initially seronegative, two doses of vaccine enabled a greater percentage of subjects to respond to the vaccine and elicited higher HAI titers. All subjects who produced detectable HAI titers also rapidly generated high AI in the short and long term. We demonstrate that high avidity antibodies can be achieved after vaccination and support a two-dose immunization strategy for HIV-positive subjects.     

Solid phase enzyme-linked immunosorbent assay (ELISA) for anti-sheep erythrocyte antibody in mouse serum

The solid phase enzyme-linked immunosorbent assay (ELISA) was developed to measure IgM and IgG anti-SRBC antibody titers in mouse serum. Sheep erythrocytes, which have a surface negative charge, were attached directly to the bottom of an aminoplate well (96-well flat bottom, Sumitomo Bakelite Co.) which is charged positively to provide a solid phase for the ELISA antigen. Serum samples titrated were obtained from mice 5 and 10 days after SRBC immunization. Alkaline phosphatase-labeled goat anti-mouse IgM and/or IgG preparations were used as second antibody. Alkaline phosphatase activity in a well was measured by the Kind and King method [Kind, P. R. N. & King, E. J. (1954). J. clin. Path., 7, 322-326]; the optical density (OD) value of quinone as a final product was measured at 492 nm. (1) A linear relationship was observed between the antiserum concentration and OD value over a wide enough dilution range to assay antibody titers of serum samples; (2) IgM and IgG fractions from antiserum showed almost the same antibody titer as did the reconstituted samples; (3) a good correlation was observed between the serum IgM titer and the number of IgM hemolytic plaque-forming cells (PFCs) in spleen 5 days after the immunization, and was also observed between serum IgG antibody titer and the number of IgG PFCs 5 and 10 days after. Therefore, the ELISA described here requires no fixative for preparation of cell-coated plates, and serum IgM and IgG anti-SRBC antibody titers can be measured without any fractionation technique.     

Antibody responses of monkeys to oral and local immunization with Streptococcus mutans.

Monkeys were immunized with Streptococcus mutans by a number of routes in an attempt to elicit exclusively a secretory immunoglobulin A (IgA) response. Antibody responses were detected by a sensitive radioimmunoassay. Monkeys primed subcutaneously and boosted submucosally with formolized cells of S. mutans had high serum IgG, IgA, and IgM radioimmunoassay titers and only slight salivary IgG titers. Instillation of killed cells of S. mutans into the right parotid salivary duct elicited good IgG, IgA, and IgM responses in both the right parotid saliva and serum, but only a weak IgM response was detected in the left parotid saliva. Administration of killed cells of S. mutans in enterically coated capsules did not elicit a detectable antibody response or have a discernible effect on the antibody response to subsequent immunization by instillation. No increase in antibody titer was detected in the serum or whole saliva from monkeys orally immunized with enterically coated capsules containing viable S. mutans or in the serum, whole saliva, or intestinal contents from monkeys immunized with uncoated capsules containing killed cells of the same organism. These results do not support the concept that oral immunization with S. mutans is effective in stimulating a generalized secretory IgA response in primates.     

IgE-isotype-specific suppressor cells in the mouse: characterization using tetraparental chimera mice of high (DBA/2) and low (SJL) IgE responder embryos

Tetraparental chimera mice were developed by aggregation of IgE high responder (DBA/2) and IgE low responder (SJL) embryos. Anti-dinitrophenyl (DNP) IgE antibody response in such mice (SJL----DBA/2) upon challenge with DNP-keyhole-limpet hemocyanin (KLH) in alum was clearly suppressed, while anti-DNP IgG antibody response was not. High-titer anti-DNP IgE and IgG antibody response developed in F1 hybrid mice of SJL and DBA/2 (SDF1) mice. The experimental results suggest that high IgE antibody production is the dominant trait, and the IgE-specific suppressor gene in SJL mice is autosomal recessive. IgE-specific suppressor T cells in SJL mice actively suppressed IgE antibody formation by DBA/2 immuno-competent cells across the histocompatibility barrier. Hapten-specific B cells and carrier-specific T cells were prepared in SJL----DBA/2 and SDF1 mice by immunization with DNP-KLH or ovalbumin (OA) in alum and transferred to irradiated SDF1 mice followed by challenge with DNP-OA. Hapten-specific B cells and carrier-specific helper T cells clearly developed in SDF1 mice. Recipient mice transferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells showed high-titer anti-DNP IgE and IgG antibody responses. OA-primed SJL----DBA/2 spleen cells cotransferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells completely abolished secondary anti-DNP IgE antibody response. The data suggest that carrier-specific helper T cells for IgE and IgG antibody responses are distinct. The regulatory role of IgE-isotype-specific suppressor cells were considered to be the interference of cooperative cellular interaction between IgE B cells and carrier-specific, IgE-specific helper T cells.     

The primary and secondary antibody response to Escherichia coli O6 lipopolysaccharide analysed at the humoral and cellular level: Amount and avidity of the antibodies in relation to protective capacity

The primary and secondary antibody response in inbred CBA mice against Escherichia coli O6 lipopolysaccharide was investigated. The avidity was found to be inversely related to the immunization dose. An optimally immunogenic dose resulted in a maximal IgM production on day 4 and a significant IgG production around day 30. Concomitant to the IgG formation there was an increase in avidity both at the serum level and at the level of the IgM-secreting cells.

A time-dependent immunological memory was noted; a booster dose 28 days after priming gave rise to increased IgM and IgG antibody production as compared to the primary response while a booster dose 10 days after priming did not. The antibody avidity in the secondary response was similar to that in the primary response.

The protective capacity of the immune response seemed to be primarily related to the antibody level. However, high avidity of the IgG antibody population seemed to compensate partially for low amount.

     

Immunoglobulin G avidity testing in serum and cerebrospinal fluid for analysis of measles virus infection.

We studied a variety of patients with measles virus infection by using avidity testing for measles virus-specific immunoglobulin G (IgG) in serum and cerebrospinal fluid samples. For the avidity testing, an Enzygnost measles IgG enzyme-linked immunosorbent assay kit was used with an 8 M urea denaturing method. With this method, low-avidity IgG (acute primary infection, avidity of < 30% within 15 days of the onset of rash) and high-avidity IgG (subacute sclerosing panencephalitis, avidity of > 75%) could be clearly distinguished by using serum samples. One patient, who developed a typical course of measles despite a previous vaccination, showed a positive IgM response with an initial low titer of measles virus-specific IgG of low avidity, but a later sample revealed a high titer of IgG of intermediate (40%) avidity, suggesting previous immunological priming. Two patients with breakthrough infection (secondary vaccine failure), both having central nervous system involvement, showed a positive IgM response with initial high titers of serum IgG of high avidity. In addition, one of the patients had a detectable level of measles-specific IgG in cerebrospinal fluid. In this patient, the avidity of both serum and cerebrospinal fluid IgG decreased during the short follow-up period. This phenomenon has never before been reported. In subacute sclerosing panencephalitis patients, the avidity of cerebrospinal fluid IgG was consistently lower than that of serum IgG. The difference in avidity between cerebrospinal fluid and serum IgG may be used as a direct indicator of intrathecal production of IgG. In conclusion, the avidity testing is simple to perform, reliable, and highly informative in the analysis of measles virus infection.     

中国莱姆病螺旋体PD91外膜蛋白A肽段的克隆表达及其免疫保护性的初步研究

目的:克隆并表达中国莱姆病螺旋体基因型代表菌株伽氏疏螺旋体( Borrelia garinii, B. garinii)PD91外膜蛋白A(OspA)的126~274 aa肽段,并对其免疫保护性进行初步研究。 方法:采用聚合酶链反应(PCR)扩增 B. garinii PD91的126~274 aa OspA肽段基因,克隆至原核表达载体pET-30a上,构建pET-30a-OspA-pep重组质粒,转入大肠埃希菌感受态细胞BL21(DE3)中,利用IPTG诱导表达,表达产物用Ni-IDA树脂层析纯化,采用Western blot分析其免疫原性。将不同剂量的重组OspA-pep(rOspA-pep)蛋白(20、30、40、50、60、80、100 μg)免疫新西兰家兔,采用间接免疫荧光法(IFA)检测免疫前后的抗体滴度,选取产生抗体滴度最高的剂量组为最佳剂量组。用最佳剂量组的免疫兔血清进行体外中和试验以检测rOspA-pep蛋白免疫后血清抗体的体外杀菌能力,同时用最佳剂量的rOspA-pep蛋白免疫新西兰家兔,观察其抗体滴度变化。 结果:重组质粒pET-30a-OspA-pep构建成功并在宿主菌体内高效表达。Western blot表明rOspA-pep蛋白与 B. garinii PD91的多抗有明显的免疫应答。IFA检测结果表明rOspA-pep蛋白免疫后的兔血清IgG抗体滴度明显升高(最高可达1∶2 480),40 μg为rOspA-pep的最佳免疫剂量。体外中和试验结果表明该剂量rOspA-pep蛋白免疫家兔后产生的抗体对10 6个/ml的 B. garinii和阿弗西尼疏螺旋体( Borrelia afzelii, B. afzelii)型代表菌株PD91和FP1的中和率达100%,对10 7个/ml的FP1的中和率为100%,对10 7个/ml的PD91的中和率为60%。用40 μg rOspA-pep在1 d和30 d免疫新西兰家兔2次后,其抗体达到高峰,持续时间为3~4个月,之后抗体滴度逐渐下降。 结论:中国莱姆病螺旋体基因型代表菌株 B. garinii PD91的126~274 aa OspA肽段具有较好的免疫原性,其诱导的抗体有较好的体外中和能力,可作为中国二代亚单位疫苗的候选成分。     

Induction of protective serum meningococcal bactericidal and diphtheria-neutralizing antibodies and mucosal immunoglobulin A in volunteers by nasal insufflations of the Neisseria meningitidis serogroup C polysaccharide-CRM197 conjugate vaccine mixed with chitosan

Thirty-six healthy volunteers received either a single intramuscular injection of Neisseria meningitidis serogroup C polysaccharide (MCP)-CRM197 conjugate vaccine in alum or two nasal insufflations 28 days apart of the same vaccine powder, without alum, mixed with chitosan. Nasal immunization was well tolerated, with fewer symptoms reported than after intramuscular injection. The geometric mean concentrations of MCP-specific immunoglobulin G (IgG) after one nasal immunization were 3.25 microg/ml in na?ve subjects and 14.4 microg/ml in subjects previously immunized parenterally, compared with 4.30 microg/ml in na?ve subjects immunized intramuscularly. The geometric mean titer of serum bactericidal antibody (SBA) rose 24-fold after two nasal immunizations in na?ve subjects and was comparable to parenteral immunization (1,080 versus 1,625). All subjects achieved SBA titers associated with protection after two nasal immunizations: even those with titers of <8 at entry. A single nasal immunization boosted the SBA titer to > or =128 in 96% of previously immunized subjects, and two immunizations achieved this level in 92% of naive subjects. MCP-specific IgG levels were approximately 70% IgG2 and approximately 20% IgG1 after nasal or intramuscular immunization. Increases in CRM197-specific IgG and diphtheria toxin-neutralizing activity were observed after nasal or intramuscular immunization, with balanced IgG1/IgG2 and higher IgG4. Significant MCP-specific secretory IgA was detected in nasal wash only after nasal immunization and predominantly on the immunized side. Simple nasal insufflation of existing MCP-CRM197 conjugate vaccines in chitosan offers an inexpensive but effective needle-free prime and boost against serogroup C N. meningitidis and diphtheria.     

Modulation of desmoglein-3-specific T cell response and pathogenic antibody generation in mice

Pemphigus is a severe blistering disease of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein-3 (Dsg3) and desmoglein-1 (Dsg1). The antibody titer and the distinct isotype patterns correlated with the disease activity. To identify their functional properties and pathogenic potential, we immunized C57BL/6 and Balb/c mice with recombinant Dsg3 fusion protein plus complete Freund's adjuvant (CFA) or Aluminum Hydroxide hydrate (Alum). After immunization, the cytokine profiles on T cells, the antibody titers, and the isotypes were analyzed. The pathogenicity of different autoantibody isotypes was evaluated by antibody passive transfer approach. It was found that Th1 type cytokine interferon gamma (IFN gamma) was elevated in the CFA-treated group, while Th2 type cytokine interleukin-4 (IL-4) was increased in the Alum-treated group. IgG1 expression was persistent in the Alum group while IgG2a was predominant in the CFA group. Neonatal mice transferred with sera from the Alum group, but not the CFA group, developed skin lesions clinically and histologically with IgG deposition on the epidermal keratinocytes. Our findings suggest that distinct T cell responses could be switched after active immunization combined with different adjuvants, resulting in distinct anti-Dsg3 antibody isotypes with different pathogenic activities in disease development. These findings shed new light on the immunopathogenesis of PV and offer a new therapeutic strategy for this potentially fatal disorder.