Mapping the specificity of an anti-feline immunodeficiency virus monoclonal antibody using a filamentous phage displayed peptide library.

Recent developments in peptide technology enable the use of random peptide libraries for identifying linear amino acid sequences (mimotopes) which can mimic conformational epitopes without necessarily exhibiting amino acid sequence homology with the native linear sequence. In this study a 15-mer random peptide library displayed on the surface of a filamentous phage has been used to characterise the conformational epitopes recognised by a monoclonal antibody raised against the envelope protein gp120 of feline immunodeficiency virus (FIV). Three mimotopes were identified that reacted with the selecting antibody in an immunoblot assay. Sequence analysis revealed that, whereas the three mimotopes had several amino acids in common, there was no significant homology with the primary amino acid sequence of gp120 although some amino acids were shared between the variable region (V3) and the three mimotopes. Petide mimtopes of complex retroviral glycoproteins may have potential uses as novel vaccines and for the serological diagnosis of FIV.     

随机肽库筛选赭曲霉毒素A模拟表位及其应用

目的从噬菌体随机肽库中筛选模拟赭曲霉毒素A表位的噬菌体粒子,并以其替代毒素建立免疫学检测方法。方法以抗赭曲霉毒素A的单克隆抗体为配基,免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机七肽库,以ELISA方法鉴定阳性克隆,同时进行DNA测序确定插入七肽的氨基酸序列。结果经过4轮淘选,共筛选到11株能与该抗体特异性结合的阳性克隆,且该结合能被赭曲霉毒素A阴断,模拟表位的共有序列为:异亮氨酸—精氨酸—脯氨酸—蛋氨酸—缬氨酸—X—X,X为任意氨基酸。以其中阻亲和力最强的克隆(P2)建立了竞争ELISA检测方法,线性范围为200~8000pg/ml,检测下限为150pg/ml。结论噬菌体展示技术可成功筛选到赭曲霉毒素A模拟表位,筛选到的噬菌体粒子可作为毒素的替代品建立免疫学分析方法。     

Hemoglobin as a predictor of response to iron therapy and its use in screening and prevalence estimates

The use of hemoglobin as a predictor of response to iron therapy, for screening, and for prevalence estimates was studied. An Fe supplementation trial was performed in Quito, Ecuador, in which 412 pregnant women were randomly assigned to treatment and control groups. Women in the treatment group received 390 mg ferrous sulfate/d for 2 mo. The prevalence of Fe deficiency as defined by response to therapy was found to be 60.8%. Sensitivity and specificity were calculated at various cutoff points of hemoglobin. The estimates of sensitivity and specificity allow for the use of hemoglobin in screening for Fe deficiency anemia and in the estimation of the prevalence in populations with characteristics similar to those found in the sample of pregnant women in Quito. Hemoglobin was shown to be a good predictor of response to Fe treatment and a good estimate of prevalence of Fe deficiency when prevalence is high.     

Identification of a peptide capable of inducing an HIV-1 Tat-specific CTL response

Although Tat-specific CTL responses are elicited in HIV-infected patients and in non-human primate models, specific CTL epitopes within Tat have not been identified. In this study, we mucosally immunized mice with recombinant, full-length Tat protein or individual Tat-specific, overlapping peptides to map putative H-2d-restricted, Tat-specific CTL epitopes. Standard chromium release assays from splenocytes of immunized animals identified a peptide (QPKTACTNC) capable of inducing Tat-specific CTL responses. This newly-identified epitope lies within a region of low sequence variability among HIV-1 subtypes, suggesting its potential use in a multicomponent AIDS vaccine.     

A two-codon mutant of cholera toxin lacking ADP-ribosylating activity functions as an effective adjuvant for eliciting mucosal and systemic cellular immune responses to peptide antigens

Vaccination with peptide antigens is an effective strategy against mucosal viral infections. We tested a two-codon mutant of cholera toxin (CT-2*) lacking ADP-ribosylating activity and toxicity as a mucosal adjuvant for T cell epitope peptides for intranasal immunization of mice. Efficient induction of helper and cytotoxic T lymphocyte responses associated with TH1 cytokine production were observed in the systemic and mucosal compartments including nasal, gut, and vaginal associated lymphoid tissues. Single or multiple dosing with the peptide antigen and CT-2* induced strong memory immunity without tolerance. These results demonstrate CT-2* as a suitable mucosal adjuvant for priming antigen-specific cellular immune responses.     

Differentiation of a passive vaccine and the humoral immune response toward infection: analysis of phage displayed peptides

Antibody-genes undergo molecular events that produce unique binding-sites that recognize specific epitopes, thus, leading to B-cell clonal variation. As a result, different binding-site structures (paratope internal images) are produced even when two distinct B-cells bind one and the same epitope. Paratope structural variation can be exploited to enable one to evaluate antibody-diversity in a single polyclonal serum sample. This is accomplished through the selection of antibody-specific peptides isolated from combinatorial phage displayed peptide libraries. As an example, we demonstrate the analysis of macaque sera containing passively administered antibodies, given as a therapeutic vaccine and antibodies actively produced by the virus-infected monkeys.     

少年接种乙型肝炎疫苗的免疫应答及其抗—HBs的动态观察

The immune response and its dynamic changes of anti-HBs in 98 Juveniles vaccinated with three doses of native plasma derived HB vaccine (have been were studied and) followed up for three years. The results showed that the cumulative positive rate and its mean S/N values of anti-HBs at T1 were 70.41% and 19.89 respectively, at T3, T6, T12, T24 all 98% and 65.54, 41.75, 53.95, 23.79 respectively. However out of 98 mentioned recipients 18 subjects become negative for anti-HBs at T36.     

Effects of interferons on immune response to a synthetic peptide malaria sporozoite vaccine in non-immune adults

A Plasmodium falciparum sporozoite vaccine, composed of a synthetic dodecapeptide (NANP)3 coupled to tetanus toxoid (TT), was injected, at weeks 0 and 8, into non-immune volunteers in two randomized double-blind placebo-controlled trials. In the first trial, 37 volunteers received the vaccine simultaneously with placebo (group 1), 0.5 x 10(6-) (group 2), or 1.5 x 10(6) U (group 3) of recombinant human interferon-alpha (= IFN-alpha). In the second trial, 35 other volunteers received the vaccine with placebo (group 4), 0.25 x 10(6) (group 5), or 1.0 x 10(6) IU (group 6) of interferon-gamma (= IFN-gamma). Immunizations were well tolerated and resulted in seroconversion rates (greater than or equal to 4-fold increase of antibody titre in immunofluorescence or enzyme-linked immunosorbent assays) of 67-100% of volunteers. IFN-alpha significantly enhanced the IgG antibody titres in ELISA to malaria peptide.     

The immune response to a meningococcal polysaccharide vaccine in an African village

The antibody response to group C meningococcal polysaccharide vaccine was studied in a Nigerian village. Household clustering of poor responders to immunization was detected. Age had a marked effect on antibody response, maximal titres being obtained only in those over the age of 10 years. Children with malaria parasitaemia had a lower antibody response than those without parasitaemia and subjects with the genotype AA had a lower antibody response than those with the genotype AS. The antibody response to the vaccine was not influenced by mild degrees of malnutrition but children with clinical marasmus or kwashiorkor were excluded from the study.     

Comparison of immune responses to a native viral antigen and a synthetic peptide derived from it: implications for vaccine development.

Murine immune responses to the hepatitis B surface antigen (HBsAg) and a synthetic peptide derived from it were compared at the humoral level. Six of nine strains used responded to either peptide or HBsAg, though restriction profiles were not superimposable. Two of three strains non-responsive to HBsAg produced an antibody response on immunization with peptide which was cross-reactive with both peptide and HBsAg. In in vitro lymphocyte stimulation assays, lymphocyte from all six peptide-immunized mouse strains could be induced to proliferate on challenge with HBsAg. However, of the HBsAg-immunized groups, lymphocytes from only three of six responder strains proliferated on in vitro HBsAg challenge. Cumulatively, these results suggest that a vaccine formulation that includes both protein antigens and synthetic peptides derived from these proteins may be more effective at eliciting an immune response in a broader cross-section of target population.     

Live rubella vectors can express native HIV envelope glycoproteins targeted by broadly neutralizing antibodies and prime the immune response to an envelope protein boost

Following HIV infection, most people make antibodies to gp120 and gp41, yet only a few make broadly neutralizing antibodies that target key antigenic sites on the envelope glycoproteins. The induction of broadly neutralizing antibodies by immunization remains a major challenge of HIV vaccine research. Difficulties include: variable protein sequence, epitopes that depend on the native conformation, glycosylation that conceals key antigenic determinants, and the assembly of Env trimers that mimic viral spikes. In addition, more potent immunogens may be needed to initiate the response of germline antibody precursors and drive B cell maturation toward antibodies with broad neutralizing activity.We have expressed HIV Env glycoproteins by incorporation into live attenuated rubella viral vectors. The rubella vaccine strain RA27/3 has demonstrated its safety and potency in millions of children. As a vector, it has elicited potent and durable immune responses in macaques to SIV Gag vaccine inserts. We now find that rubella/env vectors can stably express Env core derived glycoproteins ranging in size up to 363 amino acids from HIV clade C strain 426c. The expressed Env glycoproteins bind broadly neutralizing antibodies that target the native CD4 binding site. The vectors grew well in rhesus macaques, and they elicited a vaccine “take” in all animals, as measured by anti-rubella antibodies. By themselves, the vectors elicited modest antibody titers to the Env insert. But the combination of rubella/env prime followed by a homologous protein boost gave a strong response. Neutralizing antibodies appeared gradually after multiple vaccine doses. The vectors will be useful for testing new vaccine inserts and immunization strategies under optimized conditions of vector growth and protein expression.     

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naïve subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and FcγRI (CD64) receptor (P ≤ 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P = 0.0356 and P = 0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-α, IL-12p40 and IFN-γ (P ≤ 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection.     

HSP65 serves as an immunogenic carrier for a diabetogenic peptide P277 inducing anti-inflammatory immune response in NOD mice by nasal administration

Mucosal administration of autoantigen HSP65 can induce anti-inflammatory immune response and decrease organ-specific inflammation and disease in several models of autoimmunity, such as arthritis and atherosclerosis. We have been interested in whether the HSP65 serves as an immunogenic carrier for a diabetogenic peptide P277 can also induce anti-inflammatory immune response in NOD mice by mucosal administration. Thus, the dual functions of anti-type 1 diabetes of HSP65 and P277 will be obtained. To test this hypothesis, we examined the effect of intranasal vaccination with P277 tandem repeat sequences carried by HSP65 in the absence of adjuvants on autoimmune diabetes in NOD mice. We found a significant decrease in the incidence of diabetes, inhibition of insulitis, reduction in IgG2a isotype antibodies to P277 and proinflammatory cytokines IFN-γ and IL-2 secretion, increased IgG1, IgG2b subclass antibodies to P277 and anti-inflammatory cytokines IL-10 and IL-4 secretion, and reduced proliferation in nasal administration of the fusion protein HSP65-6 × P277. Our results demonstrate that HSP65 may serve as a particularly advantageous carrier for P277-based vaccines and mucosal administration may be a therapeutic approach for treatment of type 1 diabetes.     

Development of a screening instrument to detect physical abuse and its use in a cohort of pregnant women in Sri Lanka

The objective of this study was to develop and validate a screening instrument (Abuse Assessment Questionnaire) to estimate the prevalence of physical abuse in a cohort of pregnant women in a district of Sri Lanka. The samples of 1200 pregnant women were identified by using a cluster sampling technique. Public health midwives (primary healthcare workers) were selected as interviewers and the antenatal clinic was identified as the setting to identify physical abuse. The reliability and validity (sensitivity 85.7%; specificity 89.7%) of the screening instrument proved to be high. The prevalence of physical abuse in categories 'ever-abuse', 'current abuse' and 'current pregnancy'were 18.3%, 10.6% and 4.7% respectively. In addition, 'current sexual abuse' was reported by 2.7% of women. The prevalence rates indicate that the physical abuse of women is a significant public health problem. The Abuse Assessment Questionnaire, administered by public health midwives, proved valuable in detecting physical abuse in pregnant women. If this instrument is used universally to screen Lankan women for physical abuse in antenatal clinics, it has good potential for early detection and intervention.     

Identification of mechanisms conferring an enhanced immune response in mice induced by CVC1302-adjuvanted killed serotype O foot-and-mouth virus vaccine

The adjuvant CVC1302 was previously shown to efficiently enhance the immunogenicity of killed foot-and-mouth disease virus (FMDV) in mice and piglets. However, the underlining mechanism of action of CVC1302 remains unclear, especially at local injection sites and draining lymph nodes. Since the FMDV vaccine is administrated intramuscularly in field settings, we studied local immune responses to FMDV following intramuscular injection in mice, and found that CVC1302-adjuvanted killed FMDV (KV-CVC1302) induced secretion of several chemokines in murine muscle tissues, including MCP-1, MIP-1α, and MIP-1β. The number of monocytes recruited to the site of injection was significantly higher in mice immunized with KV-CVC1302 compared with mice immunized with killed FMDV alone (KV). iTAQ-based quantitative proteomic assays were additionally employed to explore the molecular mechanisms of CVC1302 action in the draining lymph nodes. A total of 35 proteins were identified as being differentially expressed among the control group, KV-immunized group and KV-CVC1302-immunized group at 10 days post immunization (dpi). Proteins exhibiting differential expression were mainly involved in signal transduction, apoptosis, endocytosis and innate immune responses. Pathway analysis demonstrated that AMPK, phospholipase D, cAMP, Rap1, and MAPK signaling pathways were potentially induced by the immunopotentiator CVC1302. Understanding the local mechanism of CVC1302 action at injection sites and draining lymph nodes will provide new insights into the development of FMDV vaccines.     

Effect of priming/booster immunisation protocols on immune response to canine parvovirus peptide induced by vaccination with a chimaeric plant virus construct

Expression of a 17-mer peptide sequence from canine parvovirus expressed on cowpea mosaic virus (CPMV) to form chimaeric virus particles (CVPs) creates vaccine antigens that elicit strong anti-peptide immune responses in mice. Systemic (subcutaneous, s.c.) immunisation and boosting with such CVP constructs produces IgG(2a) serum antibody responses, while mucosal (intranasal, i.n.) immunisation and boosting elicits intestinal IgA responses. Combinations of systemic and mucosal routes for priming and boosting immunisations were used to examine their influence on the level, type and location of immune response generated to one of these constructs (CVP-1). In all cases, s.c. administration, whether for immunisation or boosting, generated a Th1-biased response, reflected in a predominantly IgG(2a) serum antibody isotype and secretion of IFN-gamma from in vitro-stimulated lymphocytes. Serum antibody responses were greatest in animals primed and boosted subcutaneously, and least in mucosally vaccinated mice. The i.n. exposure also led to IFN-gamma release from in vitro-stimulated cells, but serum IgG(2a) was significantly elevated only in mice primed intranasally and boosted subcutaneously. Peptide- and wild-type CPMV-specific IgA responses in gut lavage fluid were greatest in animals exposed mucosally and least in those primed and boosted subcutaneously or primed subcutaneously and boosted orally. Lymphocytes from immunised mice proliferated in response to in vitro stimulation with CPMV but not with peptide. The predominant secretion of IFN-gamma from all immunising/boosting combinations indicates that the route of vaccination and challenge does not alter the Th1 bias of the response to CVP constructs. However, optimal serum and intestinal antibody responses were achieved by combining s.c. and i.n. administration.     

Osteopontin is an autoantigen of the somatostatin cells in human islets: identification by screening random peptide libraries with sera of patients with insulin-dependent diabetes mellitus.

Random peptide libraries (RPLs) screening with IDDM sera has identified 5 disease-specific 'mimotopes' displayed on phage (phagotopes). We characterised one phagotope (CH1p), by raising a rabbit antibody against the peptide insert on phage, which was employed in immunohistochemistry, Western blotting and cDNA libraries screening. The CH1p mimotope was detected in somatostatin cells of human islets and experimentally raised anti-osteopontin antibodies or human sera positive for the phagotope, detected a similar subpopulation of islet cells. The screening of cDNA library identified a clone corresponding to human osteopontin. In summary, RPLs proved to be successful in the identification of a novel islet-related autoantigen (osteopontin), whose significance in disease remains to be established.