Structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 23F (American type 23)

The specific capsular polysaccharide of Streptococcus pneumoniae serotype 23F (American type 23) is composed of a repeating tetrasaccharide unit containing D-glucose (one part), D-galactose (one part), L-rhamnose (two parts), glycerol (one part), and phosphate (one part). By composition analysis, optical rotation, partial hydrolysis, periodate oxidation, methylation, and high-resolution 1H and 13C nuclear magnetic resonance studies, the elucidated unambiguous structure was in agreement with our earlier proposal but is at variance with structures proposed later by other authors. The structure of the type 23F pneumococcal polysaccharide is (formula; see text).     

不同来源HL60细胞用于调理吞噬杀菌试验的比较

目的通过比较不同来源的HL60细胞分化后细胞的表面标志、活性及其对肺炎链球菌调理吞噬杀菌指数的动态变化,评价HL60细胞的来源对肺炎链球菌调理吞噬杀菌能力的影响。方法使用流式细胞仪连续监测来源于美国ATCC和中国科学院上海细胞研究所的HL60细胞在分化后1～7d的表面标志CDllb、CD35和CD71的表达情况,并观察活细胞、凋亡细胞和死亡细胞的比例;同时使用两种不同来源的细胞检测09CS和B两份质控血清对肺炎链球菌血清型6B、7F、14和23F菌株的调理吞噬杀菌指数,同时观察09CS对13价结合疫苗血清型1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F和23F菌株的调理吞噬杀菌指数。结果两种不同来源HL60细胞分化3～6d细胞的表面标志、活细胞比例均可达到国际实验室的要求指标;两份质控血清对检测菌株的调理吞噬杀菌指数均能稳定于质控范围之内;两种细胞检测的09CS对13价肺炎链球菌结合疫苗血清型菌株的调理吞噬杀菌指数具可比性。结论两种来源不同的HL60细胞分化3-6d均能用于评价肺炎链球菌疫苗免疫血清的调理吞噬杀菌试验,这为调理吞噬杀菌试验及其标准化在国内的建立提供了便利。     

Effect of vaccination against pneumococcal infection in children with type 1 diabetes mellitus

Vaccination with polysaccharide pneumococcal vaccine "Pneumo 23" (Sanofi Pasteur, France) was performed in 31 children with type 1 diabetes mellitus (DM1) as well as in 19 children with respiratory tract diseases (asthma, chronic pneumonia), which formed comparison group. Fourty-three unvaccinated children with DM1 were included in the control group. Dynamics of IgG levels to mixture of pneumococcal polysaccharides (PS) included in the vaccine as well as to PS of serotypes 3, 6B, 9N, 23F, and to cell wall polysaccharides of Streptococcus pneumoniae were assessed. Using ELISA method, significant increase of IgG levels to mixture of PS and to PS of pneumococcal serotype 3 was detected. Although intensity of immune response to vaccination in children with respiratory diseases was significantly higher compared to children with DM1 (mean geometric titer of antibodies, proportion of patients with high antibody titers, and with 4-fold seroconversion). Development of methods to strengthen immune response in children with DM1 vaccinated against pneumococcal infection is required.     

Serum requirements necessary for the opsonophagocytosis ofescherichia coli O73:K92:H1

TheEscherichia coli O73:K92:H1 serotype, which possesses a capsular antigen immunochemically similar to the capsule of the group C meningococcus, is demonstrated in this study to be resistant to phagocytosis by normal human PMNs and serum and to be dependent upon immune antibody and presumably the classical complement pathway for opsonization. Using both a luminol-dependent chemiluminescence assay and an in vitro bactericidal system, we examined, in both the absence and presence of complement, the opsonic activity of IgM ang IgG antisera. Of the various antisera tested, only those sera cross-reactive with the K92 capsular antigen were found to be opsonic both in vivo and in vitro, while somatic O or lipid A antisera demonstrated no activity. In in vitro studies with capsular IgM and IgG antisera, only IgG demonstrated opsonic activity without complement, whereas IgM required complement for opsonization of O73:K92:H1. These data demonstrate that antisera directed toward capsular antigens are opsonic for this phagocytosis-resistantE. coli, and that complement is a necessity for opsonization in the absence of sufficient capsular IgG antibodies.     

Oral immunization with recombinant Lactococcus lactis confers protection against respiratory pneumococcal infection

In the present work, we evaluated if oral immunization with the pneumococcal protective protein A (PppA), expressed in the cell wall of Lactococcus lactis (L. lactis PppA+), was able to confer protective immunity against Streptococcus pneumoniae. Mice were immunized orally with L. lactis PppA+ for 5 consecutive days. Vaccination was performed one (nonboosted group) or 2 times with a 2 week interval between each immunization (boosted group). Oral priming with L. lactis PppA+ induced the production of anti-PppA IgM, IgG, and IgA antibodies in serum and in bronchoalveolar (BAL) and intestinal (IF) lavage fluids. Boosting with L. lactis PppA+ increased the levels of mucosal and systemic immunoglobulins. Moreover, the avidity and the opsonophagocytic activity of anti-PppA antibodies were significantly improved in the boosted group. The presence of both IgG1 and IgG2a anti-PppA antibodies in serum and BAL and the production of both interferon gamma and interleukin-4 by spleen cells from immunized mice indicated that L. lactis PppA+ stimulated a mixture of Th1 and Th2 responses. The ability of L. lactis PppA+ to confer cross-protective immunity was evaluated using challenge assays with serotypes 3, 6B, 14, and 23F. Lung bacterial cell counts and hemocultures showed that immunization with L. lactis PppA+ improved resistance against all the serotypes assessed, including serotype 3, which was highly virulent in our experimental animal model. To our knowledge, this is the first demonstration of protection against respiratory pneumococcal infection induced by oral administration of a recombinant lactococcal vaccine.     

Structural characterization of Streptococcus pneumoniae serotype 9A capsule polysaccharide reveals role of glycosyl 6-O-acetyltransferase wcjE in serotype 9V capsule biosynthesis and immunogenicity

The putative capsule O-acetyltransferase gene wcjE is highly conserved across various Streptococcus pneumoniae serotypes, but the role of the gene in capsule biosynthesis and bacterial fitness remains largely unclear. Isolates expressing pneumococcal serotype 9A arise from precursors expressing wcjE-associated serotype 9V through loss-of-function mutation to wcjE. To define the biosynthetic role of 9V wcjE, we characterized the structure and serological properties of serotype 9V and 9A capsule polysaccharide (PS). NMR data revealed that both 9V and 9A PS are composed of an identical pentasaccharide repeat unit, as reported previously. However, in sharp contrast to previous studies on 9A PS being devoid of any O-acetylation, we identified O-acetylation of α-glucuronic acid and α-glucose in 9A PS. In addition, 9V PS also contained -CH(2) O-acetylation of β-N-acetylmannosamine, a modification that disappeared following in vitro recombinatorial deletion of wcjE. We also show that serotyping sera and monoclonal antibodies specific for 9V and 9A bound capsule PS in an O-acetate-dependent manner. Furthermore, IgG and to a lesser extent IgM from human donors immunized with serotype 9V PS displayed stronger binding to 9V compared with 9A PS. We conclude that serotype 9V wcjE mediates 6-O-acetylation of β-N-acetylmannosamine. This PS modification can be selectively targeted by antibodies in immunized individuals, identifying a potential selective advantage for wcjE inactivation and serotype 9A emergence.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides. II. Increased susceptibility to fatal pneumococcal sepsis due to absence of anti-polysaccharide IgG3 is corrected by induction of anti-polysaccharide IgG1

Bacterial polysaccharides (PS) are type 2 T-independent Ags that elicit Abs restricted in isotype to IgM and predominantly IgG2 in humans and IgM, and IgG3 in mice. Humans with IgG2 subclass deficiency are susceptible to sinus and pulmonary infections with PS-encapsulated bacteria. We previously developed an IgG3-deficient mouse by disrupting the gamma3 H chain constant region gene via targeted mutagenesis. Mutant mice lacking IgG3 were backcrossed for 10 generations to wild-type (WT) BALB/c mice to generate BALB/c mice that have complete absence of IgG3. WT mice immunized with type 3 Streptococcus pneumoniae capsular PS made anti-PS IgM, IgG3, and small quantities of IgG1, which opsonized S. pneumoniae for killing by polymorphonuclear leukocytes. These mice were protected against death from lethal doses of type 3 S. pneumoniae. In contrast, IgG3(-/-) mice made similar titers of anti-PS IgM and IgG1 as WT mice but no IgG3, and had poorly opsonic sera with significantly increased mortality after S. pneumoniae challenge. Immunization of IgG3(-/-) mice with type 3 S. pneumoniae PS conjugated to carrier protein CRM(197)-elicited IgM and high-titer IgG1 Abs, restored serum opsonization, and gave protection from mortality after S. pneumoniae, challenge comparable to WT mice. We conclude that mice lacking the dominant IgG3 subclass made to bacterial PS are more susceptible to fatal S. pneumoniae sepsis than WT mice, but that IgG1 induced by a S. pneumoniae glycoconjugate can adequately protect against S. pneumoniae sepsis. This model suggests that IgG subclass of anti-PS Ab is an important component of immunity to encapsulated bacteria.     

应用单克隆抗体测定人弓形虫IgM抗体的研究

为了检测人血清弓形虫ＩｇＭ抗体,采用抗人ＩｇＭ单克隆抗体和特异性抗弓形虫单克隆抗体建立捕获ＥＬＩＳＡ法,并与ＰＣＲ方法进行了比较。结果检测１０６５份献血员血清,检出阳性３例,用ＰＣＲ方法检测呈阳性结果;检测２３例类风湿病人血清及２份弓形虫ＩｇＧ抗体阳性血清均为阴性反应。说明该方法不受类风湿因子（ＲＦ）和特异性ＩｇＧ抗体的干扰,同时也表明捕获ＥＬＩＳＡ检测人血清中弓形虫ＩｇＭ抗体特异性,敏感性良好。     

Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I Antibody concentrations,avidity and functional activity

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide.     

Antibodies reacting to mimotopes of Simian virus 40 large T antigen,the viral oncoprotein,in sera from children

Recent data indicate that the Simian virus 40 (SV40) infection appears to be transmitted in humans independently from early SV40-contaminated antipolio vaccines. Serum antibodies against SV40 large T antigen (Tag) were analyzed in children/adolescents and young adults. To investigate antibodies reacting to SV40 Tag antigens, serum samples ( n = 812) from children and young adults were analyzed by indirect ELISAs using specific SV40 Tag mimotopes. Mimotopes were synthetic peptides corresponding to SV40 Tag epitopes. In sera ( n = 412) from healthy children up to 17 years old, IgG antibodies against SV40 Tag mimotopes reached an overall prevalence of 15%. IgM antibodies against SV40 Tag were detected in sera of children 6–8 months old confirming and extending the knowledge that SV40 seroconversion occurs early in life. In children/adolescents affected by different diseases ( n = 180) SV40 Tag had a prevalence of 18%, being the difference no significant compared to healthy subjects ( n = 220; 16%) of the same age. Our immunological data indicate that SV40 circulates in children and young adults, both in healthy conditions and affected by distinct diseases. The IgM detection in sera from healthy children suggests that the SV40 infection/seroconversion occurs early in life (>6 months). Our immunological data support the hypothesis that SV40, or a closely related still unknown polyomavirus, infects humans. The SV40 seroprevalence is lower than common polyomaviruses, such as BKPyV and JCPyV, and other new human polyomaviruses. In addition, our immunological surveillance indicates a lack of association between different diseases, considered herein, and SV40.     

Minimal concentration of human IgM and IgG antibodies necessary to protect mice from challenges with live O6 Escherichia coli

This work evaluated the ability of human anti-lipopolysaccharide O6 IgM and IgG antibodies to protect mice challenged with Escherichia coli serotype O6 : K2ac. Purified IgM-effluent, purified IgG, pools of normal human serum (NHS), or control group were injected into mice 18 h before challenges with O6 E. coli. Interleukin 6 and tumor necrosis factor alpha were quantified in the sera of test and control groups. All mice receiving purified IgM-effluent (66.6 mg L(-1) of anti-lipopolysaccharide O6 IgM antibodies) and NHS survived. Purified IgG (1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies) protected 87.5% of the animals. The control group showed no protective ability. The minimal concentration of anti-lipopolysaccharide O6 IgM antibodies, able to protect 50% of the animals was 33.3 mg L(-1) of purified IgM-effluent, whereas purified IgG was able to protect 50% of the animals with only 1.1 mg L(-1) of anti-lipopolysaccharide O6 IgG antibodies. Serum from animals pretreated with purified IgM-effluent and purified IgG before challenges with lipopolysaccharide O6 did not have detectable pro-inflammatory cytokines. Hepatocytes of the control group were completely invaded by bacteria, whereas none was found in animals pretreated with purified IgM-effluent and purified IgG. Higher concentrations of anti-lipopolysaccharide O6 IgM antibodies as compared to anti-lipopolysaccharide O6 IgG antibodies were needed to protect mice from challenges with E. coli O6 serotype.     

Salivary Immune Responses to the 7-Valent Pneumococcal Conjugate Vaccine in the First 2 Years of Life

Background

The CRM197-conjugated 7-valent pneumococcal vaccine (PCV7) is protective against vaccine serotype disease and nasopharyngeal carriage. Data on PCV7-induced mucosal antibodies in relation to systemic or natural anticapsular antibodies are scarce.

Methods

In a randomized controlled setting, children received PCV7 at age 2 and 4 months (2-dose group), at age 2, 4 and 11 months (2+1-dose group) or no PCV7 (control group). From 188 children paired saliva samples were collected at 12 and 24 months of age. From a subgroup of 15 immunized children also serum samples were collected. IgG and IgA antibody-levels were measured by multiplex immunoassay.

Results

At 12 months, both vaccine groups showed higher serum and saliva IgG-levels against vaccine serotypes compared with controls which sustained until 24 months for most serotypes. Salivary IgG-levels were 10–20-fold lower compared to serum IgG, however, serum and saliva IgG-levels were highly correlated. Serum and salivary IgA-levels were higher in both vaccine groups at 12 months compared with controls, except for serotype 19F. Higher salivary IgA levels remained present for most serotypes in the 2+1-dose group until 24 months, but not in the 2-dose group. Salivary IgA more than IgG, increased after documented carriage of serotypes 6B, 19F and 23F In contrast to IgG, salivary IgA-levels were comparable with serum, suggesting local IgA-production.

Conclusions

PCV7 vaccination results in significant increases in salivary IgG and IgA-levels, which are more pronounced for IgG when compared to controls. In contrast, salivary anticapsular IgA-levels seemed to respond more to natural boosting. Salivary IgG and IgA-levels correlate well with systemic antibodies, suggesting saliva might be useful as potential future surveillance tool.     

Prevalence of antibodies to specific infectious agents in bovine fetuses from a slaughterhouse in Minnesota

Sera from 486 bovine fetuses, approximately 60 to 270 days of gestation, were collected at slaughter and tested for the presence of immunoglobulins (Ig). One hundred ten (27%) of the sera were positive for IgG and/or IgM. The earliest age at which fetuses tested positive for IgM and IgG was estimated to be 100 and 120 days, respectively. Ig concentration increased with increased age of the fetus. Sera that were positive for Ig were tested for the presence of specific antibodies to five different infectious agents. Bovine parvovirus antibodies were found in 99 of 110 sera (90%) by hemagglutination inhibition (HI) test. However, only 35 (31.8%) of these sera were positive by serum neutralization (SN) test. Antibodies to parainfluenza-3 virus were detected in 30 sera (27%) by HI test and in 20 sera (18%) by SN test. Five (4%) sera contained SN antibodies to bovine viral diarrhea virus. Only one (0.9%) serum sample contained SN antibodies to infectious bovine rhinotracheitis virus. None of the sera had antibodies against five Leptospira spp. Results of this study suggest that bovine parvovirus may be a potential cause of reproductive problems in cattle.     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

肺炎球菌1型荚膜多糖多克隆抗体制备与夹心ELISA法建立及其在多糖浓度测定中的应用

目的：利用肺炎球菌1型全菌体制备多克隆抗体,并且利用该抗体建立肺炎1型荚膜多糖夹心酶联免疫吸附分析法（ Enzyme-linked immunosorbent assay ,ELISA）,用于检测发酵和纯化过程中的多糖浓度。方法用灭活的1型肺炎链球菌免疫家兔6周,获得高滴度的抗多糖血清,经过亲和层析纯化,获得高纯度的兔抗肺炎1型多糖抗体IgG。以纯化IgG作为包被抗体,加入多糖样品,再以生物素化的抗体作为检测抗体,建立夹心ELISA法检测肺炎1型多糖浓度。确定标准曲线的最佳线性范围,并对该方法进行特异性、准确性和精密度验证。结果兔免疫血清经过双向免疫扩散检测抗体滴度可达1∶32;该方法的线性检测范围为1．56～50 ng/mL;最低检测限为3．13 ng/mL。在标准品中混入其他型别多糖或培养基,回收率分别为102％和108％;该方法批内精密度和批间精密度分别为6．08％和7．01％。结论建立的夹心ELISA方法,其特异性、准确性和精密度均良好,可以特异地检测肺炎球菌1型多糖浓度。     

Induction of epitope-specific neutralizing antibodies against West Nile virus

Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.     

HL-60分化细胞表面标志、活性及其对肺炎链球菌调理吞噬杀菌能力的动态变化

目的分析HL-60分化细胞的表面标志、活性及其对肺炎球菌调理吞噬杀菌能力的动态变化。方法以流式细胞仪连续监测分化1～7 d的HL-60细胞表面标志CD11b、CD35和CD71的表达以及活细胞、凋亡细胞和死亡细胞的比例,同时用09CS、QC2、B、C和F 5份质控血清以调理吞噬杀菌试验检测肺炎链球菌血清型6B、7F、14和23F的杀菌滴度。结果分化3～6 d的HL-60细胞表面标志、活细胞比例可达到实验室要求,5份质控血清的调理吞噬杀菌滴度稳定而且在质控范围之内。结论分化3～6 d的HL-60细胞可以用于评价肺炎链球菌疫苗免疫血清的调理吞噬杀菌试验,为调理吞噬杀菌试验的建立和标准化提供了依据。     

Comparison of the opsonic and complement triggering activity of human monoclonal IgG1 and IgM antibody against group B streptococci.

We have compared the opsonic and complement-triggering activity of transfectoma-derived, class-switched human IgG1 and IgM mAb (HumAb) against types Ia, II and III group B streptococci (GBS). These antibodies appear to be directed against the common group B cell wall Ag of these organisms. The HumAb IgM promotes uptake of type Ia and II GBS at concentrations as low as 37 ng/ml and type III GBS at concentrations of 150 ng/ml in the presence of human neonatal complement. In contrast, the IgG1 GBS HumAB showed no detectable opsonic activity in concentrations up to 600 ng/ml. When the concentration of HumAb IgG1 is raised to 2.5 micrograms/ml, significant opsonic activity against GBS is detected and when the concentration is approximately 40 micrograms/ml, the opsonic activity peaked at a slightly higher level than that with the HumAb IgM. Thus, approximately 100- fold higher concentrations of the IgG1 than the IgM HumAb are required for optimal opsonization. The opsonic activity of the IgM and IgG1 HumAb are closely related to their ability to consume complement and deposit C3 on the surface of type Ia, II, and III GBS (r = 0.959). We believe that the marked opsonic and protective activity of the IgM GBS HumAb is due to its enhanced avidity and ability to trigger the complement system. Further studies are indicated to determine the feasibility of employing human IgM antibody preparations in the immunotherapy of neonatal GBS disease.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.