Circulating Neutralizing Antibody Research Articles

Longitudinal and Cross-Sectional Evaluation of Two Commercial Swine Breeding Herds to Characterize Neutralizing Antibody Levels following Porcine Epidemic Diarrhea Virus Outbreaks.

Neutralizing antibodies to Porcine Epidemic Diarrhea Virus (PEDV) can be detected by 3 weeks post-infection and remain detectable through at least 24 weeks post-infection. The objective of this study was to evaluate the levels of neutralizing antibodies in sow and piglet serum and sow milk to determine the duration of neutralizing antibodies following PEDV outbreaks. Two farms were selected for the study following outbreaks of PEDV. Monthly, cohorts of sows were sampled and followed through two farrowings. Following each farrowing, samples from piglets and milk were collected. Samples were evaluated for PEDV-neutralizing antibodies by a high-throughput fluorescent neutralization assay. Although neutralizing antibodies to PEDV can be detected throughout 15 months post-outbreak, a decrease in circulating neutralizing antibody levels is noted in farms beginning at six months post-outbreak. With decreasing levels, farms may become more vulnerable to PEDV outbreaks, and practitioners can focus on this time window to implement intervention strategies.

Pulmonary alveolar proteinosis due to mycophenolate and cyclosporine combination therapy in a renal transplant recipient.

Pulmonary alveolar proteinosis (PAP) is an orphan disease characterized by the accumulation of excess of surfactant within alveoli and bronchioles. The primary form of PAP (P-PAP; also referred to as idiopathic or autoimmune) is the most common form. It is mediated through a circulating neutralizing antibody against granulocyte-macrophage colony-stimulating factor. Secondary PAP (S-PAP) can be induced by a host of inciting agents and is far more liable to progress to terminal respiratory failure. We describe a rare case of S-PAP occurring in a renal transplant recipient due to mycophenolate and cyclosporine combination-therapy, which resolved spontaneously following withdrawal of these drugs.

Botulinum neurotoxin neutralizing activity of immune globulin (IG) purified from clinical volunteers vaccinated with recombinant botulinum vaccine (rBV A/B)

The basis for efficacy of the recombinant botulinum vaccine, serotypes A and B (rBV A/B) is that neutralizing antibodies induced by vaccination bind to botulinum neurotoxin complex serotype A, subtype A1 (BoNT/A1) and serotype B, subtype B1 (BoNT/B1) and prevent their actions at cholinergic neurons. The protective capacity of BoNT/A1 and BoNT/B1 neutralizing antibodies derived from the serum of clinical volunteers vaccinated with rBV A/B was evaluated in a guinea pig passive transfer model and a mouse bioassay. Guinea pigs passively immunized to achieve circulating neutralizing antibody concentration (NAC) levels representing the lowest measurable concentrations for BoNT/A1 and BoNT/B1 were protected against an intramuscular (IM) challenge more than 10 times the guinea pig IM median lethal dosage for BoNT/A1 and BoNT/B1. The passively immunized guinea pigs were asymptomatic during the 14-day post-challenge observation period. Control guinea pigs died within 48 h after challenge. Calculation of neutralizing efficiency of antibodies using results from a mouse bioassay indicated that a simple linearly proportional relationship does not exist between NAC level and the amount of BoNT neutralized. Based on this finding, estimates of level of protection must consider variability in BoNT neutralizing efficiency at different NAC levels. The protective capacity of human BoNT/A1 and BoNT/B1 neutralizing antibodies induced by rBV A/B vaccination was verified in a guinea pig passive immunization model. Additionally, estimates of the neutralizing efficiency have been established for BoNT/A1 and BoNT/B1 neutralizing antibodies obtained from clinical volunteers vaccinated with the rBV A/B.

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.

Diphtheria-neutralizing antibody levels in healthy adults from Rio de Janeiro, Brazil

In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no official routine booster dose administered after 15 years of age. This study assessed functional antibody levels against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and, 27.1% (95%, CI: 20.2-34.9) had (3) 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (< 0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently healthy adults remain susceptible to diphtheria.

Recombinant C fragment of botulinum neurotoxin B serotype (rBoNTB (H C)) immune response and protection in the rhesus monkey

Botulinum neurotoxin B (BoNTB) is a distinct protein subtype of a family of neurotoxins with the potential for use in biological warfare or terrorist attacks. This study is one in a series evaluating the immunogenicity and protective effects of recombinant vaccines against the different subtypes of botulinum toxin. The recombinant subunit vaccines encoding the C fragment portion (∼50 kDa) of the toxins are produced in the yeast, Pichia pastoris. In this study, groups of rhesus monkeys were vaccinated with three doses (1 and 5 μg per dose) of rBoNTB(H c) vaccine. Total and neutralizing antibody titers were determined at various times during and postvaccination. Two groups of vaccinated monkeys plus non-vaccinated controls were actively challenged with B toxin by aerosol exposure. All monkeys receiving vaccine were protected from the toxin and no clinical signs of disease were observed, while controls displaying classic signs of botulism succumbed to the toxin challenge. Two additional groups of monkeys receiving the same vaccine regiment as the first two groups had significant levels of circulating neutralizing antibody titers up to 24 months postvaccination. This non-human primate study demonstrated the short- and long-term immunity afforded by the rBoNTB(H c) vaccine.

Increased revascularization efficacy after administration of an adenovirus encoding VEGF(121).

Adenovirus-mediated VEGF gene delivery is being evaluated in clinical trials as a treatment for patients with vascular diseases that stem from ischemia, such as diffuse coronary artery disease and peripheral vascular disease. Although adenoviral vectors are one of the most widely utilized vectors to deliver therapeutic genes to cells, they also have a major limitation in that their inherent immunogenicity leads to the production of neutralizing antibodies that block effective repeat administration. Although this may be true of intravenous, intranasal, and other routes of administration, recent studies have indicated that it may be possible to effectively readminister adenovirus to skeletal muscle. The present study found improved efficacy after administration of AdVEGF(121.10), an E1/E3-deleted adenovirus encoding human VEGF(121) under the control of a CMV promoter in a rat hindlimb ischemia model. As expected, repeat administration of adenovirus resulted in a marked increase of circulating neutralizing antibody, yet nanogram quantities of VEGF protein were still detectable within the hindlimb skeletal muscle after a second administration of vector. The amount of VEGF protein produced after repeat administration translated into improved efficacy as evidenced by increased blood flow as measured by laser Doppler, increased vessel number upon post-mortem angiography, and an increased number of CD31-positive vessels. These findings have important implications for increasing the efficacy of adenovirus-mediated gene therapy in the treatment of peripheral vascular disease and coronary artery disease.

Evaluation of Tick-Borne Encephalitis DNA Vaccines in Monkeys

Tick-borne encephalitis is usually caused by infection with one of two flaviviruses: Russian spring summer encephalitis virus (RSSEV) or Central European encephalitis virus (CEEV). We previously demonstrated that gene gun inoculation of mice with naked DNA vaccines expressing the prM and E genes of these viruses resulted in long-lived homologous and heterologous protective immunity (Schmaljohn et al., 1997). To further evaluate these vaccines, we inoculated rhesus macaques by gene gun with the RSSEV or CEEV vaccines or with both DNA vaccines and compared resulting antibody titers with those obtained by vaccination with a commercial, formalin-inactivated vaccine administered at the human dose. Vaccinations were given at days 0, 30, and 70. All of the vaccines elicited antibodies detected by ELISA and by plaque-reduction neutralization tests. The neutralizing antibody responses persisted for at least 15 weeks after the final vaccination. Because monkeys are not uniformly susceptible to tick-borne encephalitis, the protective properties of the vaccines were assessed by passive transfer of monkey sera to mice and subsequent challenge of the mice with RSSEV or CEEV. One hour after transfer, mice that received 50 μl of sera from monkeys vaccinated with both DNA vaccines had circulating neutralizing antibody levels <20–80. All of these mice were protected from challenge with RSSEV or CEEV. Mice that received 10 μl of sera from monkeys vaccinated with the individual DNA vaccines, both DNA vaccines, or a commercial vaccine were partially to completely protected from RSSEV or CEEV challenge. These data suggest that DNA vaccines may offer protective immunity to primates similar to that obtained with a commercial inactivated-virus vaccine.

The detection of intracellular bluetongue virus particles within ovine erythrocytes

1. 1. We report here a simplified method for detecting viruses and other antigenic agents in red blood cells (RBCs). Using a nonionic detergent to prepare cytoskeletons, the interior of RBCs can be scanned rapidly using immunoelectron microscopy. 2. 2. In this study, RBCs from bluetongue (BLU) virus-infected sheep were adsorbed directly onto Formvar-coated, gold electron microscope grids. 3. 3. Cytoskeletons were prepared and then probed using a monoclonal antibody to VP 7, a structural BLU-virus protein and Protein-A gold. 4. 4. Of the ca 32,000 RBCs that were examined from BLU virus-infected sheep, 34 (0.106%) contained labelled BLU virus particles. 5. 5. No labelled particles were observed in any of ca 8000 RBCs taken prior to BLU virus inoculation of sheep. 6. 6. If the antigenic BLU virus particles (which may be viral cores) are in fact infectious, this method of sequestration of virus within RBCs could contribute to the prolonged viremia typical of this arboviral disease, which is known to occur concurrently with circulating neutralizing antibody.

The immune response to vaccinia virus infection in mice: analysis of the role of antibody

Immune response to primary intraperitoneal infection with vaccinia virus (strain IHD-J) was studied in C3H/Hej mice. Antibodies reactive with virus structural proteins were detected 6 days and neutralizing antibodies 8 days after infection. Although serum antibodies from infected mice bound to vaccinia virus infected cells, these antibodies were ineffective in complement mediated lysis of infected cells and were only moderately active in experiments with antibody dependent cellular cytotoxicity (ADCC). Immunoblotting analysis showed that serum antibody reacted with a number of structural proteins of both intracellular and extracellular forms of vaccinia virus. Immunoprecipitation results showed antibody binding of nonstructural proteins and glycoproteins. Correlation of the kinetics of NK and CTL activities in infected mice with neutralizing antibodies indicated that the cellular functions clearly precede the appearance of serum neutralizing antibody. The resolution of primary infection in mice thus appears to be mediated by functions of cellular immunity while resistance to reinfection may be dependent on circulating neutralizing antibody.

Skin reactions to vaccinia virus infection in the rabbits immunized with vaccinia-soluble early antigen.

Rabbits were immunized with vaccinia-soluble early antigen (Es antigen) in Freund's complete adjuvant (FCA). Circulating neutralizing antibody was not induced, but CF antibody against the soluble antigens of vaccinia-infected cells was induced. By intradermal inoculation of vaccinia virus, erythema developed early at the site of inoculation and enlarged up to 3 days post infection (p.i.). No multiplication of the viruses in the erythematous areas was indicated on day 3 p.i. The erythematous areas showed histologically severe changes involving infiltration of numerous cells accompanied with necrotic and hemorrhagic lesions in a part of the dermis. Eosinophilic leukocytes were dominant and there were also some lymphocytes in the cell infiltration. The role of these kinds of cells was discussed in connection with cell-mediated immunity in the mechanism of excluding vaccinia virus from the immune rabbits.

Excretion of foot-and-mouth disease virus in oesophageal-pharyngeal fluid and milk of cattle after intranasal infection

The virus growth in the pharyngeal area and the virus excretion in milk of susceptible and vaccinated dairy cows after intranasal instillation of foot-and-mouth disease (FMD) virus type O1 were examined. Ten vaccinated cows were purchased through a market. Of these, nine had delivered their first calf. The cows were inoculated 2-9 months after having received the last dose of vaccine. All vaccinated cows resisted the intranasal challenge. The virus multiplied in the pharyngeal area but, compared with two susceptible controls, to a limited extent. No clear relation was found between virus growth and the titre of circulating neutralizing antibody at the time of challenge. Virus was first detected in milk samples of the susceptible cows when generalized FMD lesions had developed on day four; the excretion lasted for 3-4 days. Up to 19 days after inoculation untreated milk of the vaccinated cows was examined for the presence of infectious FMD virus. Samples were inoculated onto cell cultures, fed to susceptible pigs and calves and injected intramuscularly and/or intradermolingually into susceptible steers. No infectious FMD virus could be detected, either in cell cultures or in susceptible animals. The animals did not develop neutralizing antibody against FMD virus and were subsequently shown to be fully susceptible to challenge. The results are discussed with particular reference to current problems regarding the export of milk products from countries where vaccination against FMD is practised to countries free of the disease.

Cytomegalovirus Infection in Guinea Pigs. II. Transplacental and Horizontal Transmission

Transplacental transmission of cytomegalovirus (CMV) was demonstrated in guinea pigs, but transmission only occurred during acute primary maternal infection. Infectious CMV was isolated from 27 of 44 placental tissues and nine of 37 fetal tissues (including brain, lung, and kidney) tested five to 24 days after maternal infection. Fetal infection occurred at different stages of gestation ranging from 28 to 60 days. No virus was detected in the tissues of 43 fetuses examined that were taken from pregnant guinea pigs that had been infected for greater than 30 days. The maternal animals had significant levels of circulating neutralizing antibody concurrent with infectious CMV in the salivary glands at the time of sacrifice. Contact infection was accomplished when infected and uninfected animals of either the same sex or oppsite sexes were paired and housed together for one to three months. Among the seven male contact guinea pigs housed with infected females, five showed virus in their salivary glands, and all seven showed rises in titer of antibody; five of the females became pregnant. In contrast, when infected and uninfected animals of the same sex were paired and housed together, only one of 13 contact animals had virus in the salivary gland, and four of 13 had rises in titer of antibody. Thus sexual contact appeared to be more efficient than environmental contact in the spread of CMV infection.

Live respiratory syncytial virus vaccine administered parenterally.

SummaryA unique live attenuated respiratory syncytial virus vaccine that is administered parenterally is described. Nineteen of 22 initially seronegative children, 7-32 months of age, developed homologous circulating neutralizing antibody against the virus and a portion were shown to have present demonstrable antibody in their nasal secretions. None of the children developed illness that could be ascribed to the vaccine, and there was no evident contagious spread of the vaccine virus to susceptible contacts. The vaccine is of considerable promise and trials to determine protective efficacy are being pursued. Seroepidemiologic investigations revealed that only 33 of 66 children 6-48 months of age and residing in an open population had detectable circulating neutralizing antibody against the virus.

In vivo interference in vesicular stomatitis virus infection

Inactivated defective interfering and complete particles of vesicular stomatitis virus given intracerebrally to adult mice protect them against challenge with homologous virus whether this is given at the same time or several days later. Two separate protective processes appear to be involved. The first, which comes into operation immediately after inoculation, is also effective against heterologous strains of vesicular stomatitis virus, rabies (another rhabdovirus), and a neurotropic strain of foot-and-mouth disease virus. The second, later effect, which is strain specific, appears to be correlated with the appearance of circulating neutralizing antibody. Our results suggest that the protective effect that Holland and his colleagues described using defective interfering particles of vesicular stomatitis virus may also be accounted for by an immunological mechanism rather than one involving interference.

Entry of neutralizing antibody to measles into brain and cerebrospinal fluid of immunized monkeys after osmotic opening of the blood-brain barrier

The blood-brain barrier was osmotically opened to circulating neutralizing antibody to measles in immunized rhesus monkeys. Hypertonic solutions of lactamide or arabinose were perfused into the left hemisphere via the left lingual artery, after the external and common carotid arteries were clamped temporarily. Opening was graded by the amount of brain staining produced by intravascular Evans blue-albumin. Elevated brain antibody concentrations were produced in five of seven animals with grade 2+ barrier opening in the absence of neurological sequelae, suggesting that tight junctions between cerebrovascular endothelia were widened to molecules with a molecular weight of at least 180,000. Tenfold higher antibody concentrations were produced by grade 3+ barrier opening but were accompanied by evidence of brain damage. Brain antibody remained elevated for at least 4 days following osmotic opening, and cerebrospinal fluid concentrations also were tenfold higher during this period, both for grade 2+ and 3+ openings.

Role of maternal antibody in pneumonia and bronchiolitis due to respiratory syncytial virus.

Fifteen infants with pneumonia caused by respiratory syncytial virus (RSV) and 19 infants with bronchiolitis caused by RSV were studied for the influence of homologous, circulating neutralizing antibody on the severity of their illness. All infants were under nine months of age. Although maternal neutralizing antibody did not prevent infection with RSV and illness, the severity of pneumonia caused by RSV was inversely related to the level of neutralizing antibody. The severity of bronchiolitis caused by RSV was unrelated to maternal antibody levels. Chest roentgenograms showed pneumonia to be slightly more severe than bronchiolitis. Neither the severity of illness nor the presence of maternal neutralizing antibody was related to the development of complement-fixing antibody.

Induction of skin resistance to vaccinia virus in rabbits by vaccinia-soluble early antigens.

The immunological role of vaccinia-soluble early antigen was examined in rabbits. The antigens were prepared from HeLa cells infected with a conditional lethal mutant of vaccinia virus, which induces in these cells early antigens including those responsible for surface immunofluorescence of infected cells, but not viral DNA and late protein syntheses. Immunization of rabbits with the antigens in Freund's complete adjuvant induced complement-fixing antibody but neither detectable circulating neutralizing antibody nor any detectable level of inhibitory substance or interferon in the skin of the animals. When immunized animals were inoculated intradermally with vaccinia virus, multiplication of virus in the skin was greatly inhibited, being accompanied by an earlier appearance as well as an accelerated wane of the local reactions. The resistance could not be transferred passively by the serum of immunized animals to normal rabbits. Immunization of rabbits with the antigens without the adjuvant not only failed to inhibit but, contrariwise, enhanced the multiplication of intradermally inoculated vaccinia virus, inducing heavy skin lesions and exalted virus multiplication.

Depression of immune response to Moloney leukaemia virus by malarial infection.

MALARIAL infection depresses the immune response to a variety of antigens in mice1–6 and man7,8. It has been postulated that malarial immunodepression is implicated in the development of a human neoplasm, Burkitt's lymphoma9, the geographical distribution of which coincides with that of holoendemic malaria10. In Swiss mice, malarial infection has been shown to increase the rate of spontaneous lymphomagenesis11; and in adult Balb/c mice to potentiate the induction of lymphomas by the Moloney leukaemia virus, MLV (ref. 12). Here we present evidence that the latter effect is accompanied by a reduction in the detectable levels of circulating neutralizing antibody to MLV, and in particular by the absence of IgG neutralizing antibody.

Immune Response of Pigs to Inactivated Foot-and-Mouth Disease Vaccines: Response to Emulsion Vaccines

The serological response of pigs to inactivated single and double emulsion foot-and-mouth disease (FMD) vaccines has been examined. The slopes of the dose-response regression lines for types A, O and C vaccines were low and only small amounts of antigen were required to elicit good immune responses. These vaccines resulted in granuloma and abscess formation at the site of injection. The extent of this reaction was dependent on the volume of vaccine administered. A reduction in the dose volume from 2 ml. to 0.25 ml. did not influence the immune response. The intradermal inoculation of 0·1 ml. of a type A double emulsion vaccine stimulated acceptable concentrations of circulating neutralizing antibody with minimal tissue reaction. The duration of immunity produced by emulsion vaccines was good, with neutralizing antibody concentrations remaining above the 50% protection level for 210 days in the case of a type O vaccine and for 280 days for types C and A vaccines. Pigs immunized with an emulsion vaccine showed good anamnestic responses to both emulsion and saponin adjuvauted vaccines. IgM was present for 42 to 63 days after primary inoculation, while IgG appeared at about 21 days after vaccination.