The incidence and clinical significance of antibodies to interferon-a in patients with solid tumors

It is well known that natural and recombinant proteins can cause antibody formation in the host. We have studied the incidence of binding and neutralizing antibodies in carcinoid patients (n=327). All together 204 patients received interferon-α 2b (Intron-A), median does 15 MU range 9–35 MU/week subcutaneously and 51% of the patients developed binding antibodies by immunoassay and 17% showed positive neutralization assay but high titer antibodies (>800 NU/ml) were only found in 4% of the patients. The median time until the development of binding antibodies was 26 months and neutralizing antibodies 25 months. Twenty-nine patients received interferon-α 2a (Roferon), median does 18 MU/week subcutaneously and 45% developed binding antibodies, 38% had positive neutralization assay and 28% presented high titer antibodies. Binding and neutralizing antibodies occurred at the same time after median six months of treatment. Patients treated with Wellferon (n=45) and leukocyte interferon (n=48), median dose of 15 MU/week subcutaneously did not develop any neutralizing antibodies. The majority of the interferon-α 2 antibodies were of the IgG isotype. The clinical relevance of the development of high titer neutralizing antibodies was evaluated in the patients. All together 17 patients developed high titer neutralizing antibodies and of these 12 patients showed loss of antitumor response measured as increased level of tumor markers and of tumor progression. In nine of these patients a switch to human leukocyte interferon reinstituted an antitumor response. Neutralizing antibodies against recombinant interferon-α 2a and 2b might occur in patients with carcinoid tumors. The incidence of high titer neutralizing antibodies is significantly higher in patients treated with interferon-α 2a compared to interferon-α 2b. A significant number of patients lost the antitumor effect during development of neutralizing antibodies at high titers, but human leukocyte interferon can be used as rescue treatment.     

A semi-quantitative serological method to assess the potency of inactivated rabies vaccine for veterinary use

Potency is one of the most important indexes of inactivated vaccines. A number of methods have been established to assay the potency, of which the NIH test and single-dose mouse protection test are the “prescribed methods”. Here, we report a method to semi-quantitatively assay the potency of an inactivated rabies vaccine, which uses fewer animals and takes less time to complete. Depending on the quality requirements of a vaccine (e.g. minimum potency), a rabies reference vaccine is, for example, diluted to the minimum potency, and 50 μL of the dilution is taken to inoculate 10 mice. The same amount of the test rabies vaccine is inoculated into another 10 mice. After two weeks, all mice are bled and serum samples are assayed for viral neutralizing antibody by the fluorescent antibody virus neutralization (FAVN) test. By comparing the median and interquartile range of antibody titers of the reference vaccine with those of the test vaccine, the test vaccine potency can be semi-quantitatively judged as to whether it is in accord with the required quality. The reliability of this method was also confirmed in dogs. The procedure can be recommended for batch potency testing during inactivated rabies vaccine production.     

Chicken embryo related (CER) cell line for quantification of rabies neutralizing antibody by fluorescent focus inhibition test.

Levels of rabies virus neutralization antibody in sera from vaccinated dogs and cattle were either measured by mouse neutralization test (MNT) or by rapid fluorescent focus inhibition test (RFFIT), performed on CER monolayers. The two tests were compared for their ability to detect the 0.5 International Units/ml (I.U.) recommended by the World Health Organization (WHO) as the minimum response for proof of rabies immunization. A significant correlation was found between the two tests (n=211; r=0.9949 in dogs and 0.9307 in cows, p<0.001), good sensitivity (87.5%), specificity (94.7%) and agreement (96.6%) as well. RFFIT method standardized on CER cell system for neutralizing antibodies detection turns the diagnosis easier and less expensive, specially when a great number of samples must be tested from endemic areas as commonly found in Brazil.     

Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies

We previously reported that high-titered neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope can block the establishment of a simian immunodeficiency virus (SIV)/HIV chimeric virus (SHIV) infection in two monkeys following passive transfer (R. Shibata et al., Nat. Med. 5:204-210, 1999). In the present study, increasing amounts of neutralizing immunoglobulin G (IgG) were administered to 15 pig-tailed macaques in order to obtain a statistically valid protective neutralization endpoint titer in plasma. Using an in vitro assay which measures complete neutralization of the challenge SHIV, we correlated the titers of neutralizing antibodies in plasma at the time of virus inoculation (which ranged from 1:3 to 1:123) with the establishment of infection in virus-challenged animals. Ten of 15 monkeys in the present experiment were virus free as a result of neutralizing IgG administration as monitored by DNA PCR (peripheral blood mononuclear cells and lymph node cells), RNA PCR (plasma), virus isolation, and the transfer of lymph node cell suspensions (10(8) cells) plus 8 ml of whole blood from protected animals to na?ve macaques. The titer of neutralizing antibodies in the plasma calculated to protect 99% of virus-challenged monkeys was 1:38.     

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (K(D)) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection.     

A novel assay to detect low-titred antibodies to interferon beta in multiple sclerosis patients

Neutralizing antibodies (NAbs) may compromise interferon (IFN) clinical efficacy in patients with multiple sclerosis (MS) receiving IFN-beta treatment. When bioassays are used for anti-IFN-beta antibody detection, they are unable to discriminate between NAbs or other interfering substances with anti-IFN activity. Here we report the development of an anti-IFN-beta Western blot method that facilitates the detection of IFN low-titred antibodies and characterizes such low neutralizing activity as specifically due to the presence of particular IFN antibodies. The assay was characterized using serum samples from patients with MS treated with IFN-beta. It was developed by adding anti-IFN-positive antibody sera to Dynabeads M-280 tosylactivated followed by Western blot analysis. All sera samples from MS patients with IFN-betala NAbs (< or = 50 t1/10) proved to be antibody-positive using this new method and, more importantly, four of 27 binding antibody-negative sera samples were scored as IFN antibody-positive. The method was found to be rapid, specific and sensitive and consistent with respect to well-established antiviral neutralization or commercial enzyme-linked immunosorbent assays.     

ELISA法检测白喉抗体的研究和初步应用

以白喉类毒素为包被抗原,经方阵滴定确定其包被浓度。通过对百白破三联疫苗免疫的小鼠血清中白喉抗体的小鼠-Vero细胞法效价与按几何平均滴度计算的ELISA效价进行比较,验证ELISA法检测白喉抗体的可行性。该试剂用人免疫球蛋白白喉抗体国家标准品检测的敏感度为0.05EU/ml,检测864份健康人血清,阳性率为50%,检测89份2~14岁儿童血清,阳性率为91%,与免疫规划的实际情况相符,可用于疫苗免疫效果的观察。     

Comparison of JEV neutralization assay using pseudotyped JEV with the conventional plaque-reduction neutralization test

We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV-PV). JEV-PV-based neutralization assay offers several advantages compared with the current standard plaque-reduction neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque reduction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.     

A novel process for the production of a veterinary rabies vaccine in BHK-21 cells grown on microcarriers in a 20-l bioreactor

We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.     

Neutralizing antibody responses in Africa green monkeys naturally infected with simian immunodeficiency virus (SIVagm)

Abstract: This study assessed the magnitude and cross-reactivity of the neutralizing antibody response generated by natural SIV infection in wild-caught African green monkeys. Neutralizing antibodies of variable potency, sometimes exceeding a titer of 1:1,000, were detected in 20 of 20 SIV-seropositive African green monkeys in Kenya. Detection of those neutralizing antibodies was dependent on the strain of virus and the cells used for assay, where the most sensitive detection was made with SIVagml532 in Sup T1 cells. Potent neutralization of SIVagml532 was seen with contemporaneous autologous serum. Potent neutralization was also detected with laboratory-passaged SIVmac251 and SIVsmB670, but not with SIVsmE660 and two additional strains of SIVagm. Serum samples from rhesus macaques (Macaca mulatta) experimentally infected with either SIVmac251 or SIVsmE660 were capable of low-level neutralization of SIVagm. These results indicate that natural infection with SIV can generate strain-specific neutralizing antibodies in African green monkeys. They also indicate that some neutralization determinants of SIVagm are partially shared with SIV strains that arose in sooty mangabys and were subsequently transmitted to rhesus macaques.     

A micro-neutralization system for detection of s-CRN antibody to herpes simplex virus

It was learned that the ordinary micro-neutralization system with herpes simplex virus (HSV) gave a composite result of the initial neutralization and the effect of antibody on subsequent growth of unneutralized virus. In the case of slow-reacting complement-requiring neutralizing (s-CRN) antibody, which was detected by incubating virus-serum mixtures at 4 C for 3 days before addition of complement, the titer obtained was lower than expected from the result of the plaque reduction test. This was thought ascribable to its low ability to prevent viral breakthrough caused by growth of unneutralized virus. This was overcome by adding an appropriate amount of hyperimmune antibody at 3 hr after addition of cells. The endpoint of s-CRN antibody so determined was but slightly lower than that obtained by the plaque reduction test. Early (1-week) rabbit sera, which were negative in the ordinary micro-neutralization test, titered 1:2,560 to 1:5,120 when tested by this method. When the 3-day sensitization in the cold was substituted by 5-hr incubation at 37 C, the titer obtained was 2 to 4-fold lower; in this case, however, the whole process could be finished within 3 days. Also, s-CRN antibody reactive with type 2 HSV in homologous and heterologous sera could be detected by the same method using type 1 hyperimmune serum as the additional antibody.     

Analysis of antibody response in goats to caprine herpesvirus 1

Serum samples of goats experiencing natural and experimental infections and/or reactivation of caprine herpesvirus 1 (CpHV.1) were analysed with neutralization and Western blotting (WB) tests. WB immunological patterns resulted differently and related to neutralizing titers. In serum samples having neutralizing titer 1:2-1:4, antibodies to two proteins of Mw of 150 and 34 kDa were present. Antibodies against several proteins, two of those being characterized by monoclonal antibodies as gB and gC, were visualized by WB in sera having titer > or = 1:8. The neutralizing antibody titers and the pattern of antibody reactivity were hypothesized to modulate the reactivation and re-excretion process of CpHV.1.     

Hepatitis A Immunoglobulin: An International Collaborative Study to Establish the Second International Standard

A collaborative study was carried out to assess the suitability of a candidate replacement material for the International Standard for hepatitis A immunoglobulin, which was found to be reactive for HCV RNA, and to calibrate it in International Units. The candidate standard, coded 97/646, was derived from a bulk of 16% immunoglobulin supplied by the Central Laboratory of the Netherlands Red Cross, Amsterdam, and diluted 1 in 2 in H2O resulting in a final immunoglobulin concentration of 8%. Sixteen laboratories from 11 countries participated in the study and contributed data from 64 assays performed using six commercial assay kits and four in-house methods. All assays were analysed as parallel line bioassays comparing assay response with log concentration. The overall mean potency of the candidate replacement immunoglobulin standard, 97/646, relative to the International Standard for hepatitis A immunoglobulin, was 98.6 IU/ml. A freeze-dried serum preparation, 97/648, was also calibrated in this study and had a potency of 22.64 IU/ml. The Second International Standard for hepatitis A immunoglobulin, human, was established by the World Health Organisation Expert Committee on Biological Standardisation in 1998 with a potency of 49 IU per ampoule when reconstituted in 0.5 ml.     

A new microplate neutralization test for typing of herpes simplex virus.

A microplate serum neutralization test for estimation of complement-requiring neutralizing (CRN) antibody was established as the first step for simplification of typing of herpes simplex virus (HSV). When guinea pigs were immunized with type 2 HSV, the late sera could mostly differentiate the types of HSV better than hyperimmune rabbit sera, the CRN titer against the heterologous type 1 HSV being much lower than the homologous titer. Sera of guinea pigs immunized with type 1 HSV showed about the same level of cross reaction against type 2 HSV as did rabbit antisera. Guinea pig sera having minimal levels of cross reaction were selected, and their high dilution (1:160) and complement were added to serial 10-fold dilutions of virus in the microplate titration of virus infectivity. Selective reduction of virus titer by either antiserum could determine the type of HSV. No equivocal intermediate case was found among a number of stock strains including many fresh isolates. The typing result coincided with that determined by a modification of Yang et al's method based on virus titers obtained with Vero and primary chick embryo cells. The typing based on plaquing in chick embryo cells sometimes failed to identify type 1 HSV.     

表达HPV16 L1抗原的1型重组AAV载体免疫效果的研究

为研究1型重组腺病毒伴随病毒(Adeno-associated virus type 1,AAV1)载体作为HPV16预防性疫苗的可行性,构建含密码子优化型HPV16L1基因(mod.HPV16L1)的1型重组AAV载体rAAV1-mod.HPV16L1,将纯化的rAAV1-mod.HPV16L1以肌注和滴鼻途径分别免疫C57BL/6小鼠,使用体外中和实验检测血清中的特异性中和抗体.结果显示,rAAV1-mod.HPV16L1单针肌注及滴鼻免疫均可诱导特异性血清中和抗体,但二组抗体动态变化趋势不同,肌注组血清中和抗体滴度显著高于滴鼻组.rAAV1-mod.HPV16L1单针肌注免疫可诱导强而持久的血清中和抗体,是理想的候选HPV16预防性疫苗.     

Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.     

Stoichiometry of antibody neutralization of human immunodeficiency virus type 1

The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single antibody molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.     

Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays

A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.     

Properties of natural 19S antibodies in normal pig serum against the ΦX 174 and T2 phages

The physicochemical properties of natural phage-neutralizing antibodies were studied. Natural neutralizing antibodies against phages ΦX 174 and T 2 were found in the 19 S fraction isolated from normal pig serum by gel filtration on a Bio-Gel P-300 column. The 7 S fraction of normal pig serum possessed no neutralizing activity. The neutralizing activity of the 19 S fraction against phage ΦX 174 was not modified either by inactivation or by the addition of neutralizing cofactor; its activity against phage T 2 was lost by inactivation, but was restored by the addition of cofactor. The neutralizing activity of the 19 S fraction of normal pig serum was completely destroyed by 2-mercaptoethanol and was not restored by the subsequent addition of antibody cofactor. The results of attempts to release phage ΦX 174 from the neutralization complex with normal porcine serum 19 S macroglobulin antibody by the dilution method were the same as those of attempts to dissociate phage from the complex with 7 S type hyperimmune antibody. The virus particle was firmly and irreversibly adsorbed to both types of antibody and was not released by dilution. It is concluded from the results that neutralization of phage ΦX 174 by 19 S macroglobulin molecule of antibody is a simple, irreversible process, for which the thermolabile nonspecific serum components are not required.     

狂犬病病毒糖蛋白重组人腺病毒的构建及免疫效果的初步研究

构建表达狂犬病病毒弱毒SRV9糖蛋白(GP)的重组人5型腺病毒,检测其对小鼠的免疫效果。将狂犬病病毒SRV9株GP基因的完整开放阅读框克隆到腺病毒表达系统中的穿梭质粒多克隆位点,构建重组穿梭质粒pac-Ad5CMV-Gs9,以罗氏转染液介导线性化骨架质粒和重组穿梭质粒共转染293AD细胞,细胞病变后取培养物进行PCR鉴定并电镜观察,在293AD细胞上测定病毒滴度。以106 TCID50重组腺病毒腹腔接种昆明小鼠,免疫后不同时段采尾静脉血通过荧光抗体病毒中和试验(FAVN)检测小鼠血清狂犬病中和抗体效价。正确构建重组穿梭质粒pacAd5CMV-Gs9;获得表达狂犬病病毒SRV9株GP蛋白的缺陷型重组人5型腺病毒;病毒滴度达到106 CFU/mL以上;腹腔接种小鼠14d后均产生了抗狂犬病中和抗体,有效保护率达90%。成功获得了表达狂犬病病毒GP基因的重组腺病毒,该腺病毒免疫小鼠可产生保护性中和抗体,为进一步开发新型兽用狂犬病疫苗奠定了物质基础。