International Bordetella pertussis assay standardization and harmonization meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, United States, 19-20 July 2007.

An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19-20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO). The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.

Relationship between serum bactericidal activity and serogroup-specific immunoglobulin G concentration for adults, toddlers, and infants immunized with Neisseria meningitidis serogroup C vaccines.

A new meningococcal group C-CRM(197) conjugate vaccine (MnCC; Meningitec) has been evaluated in multiple clinical trials in the United States and most recently has been approved for routine administration in the United Kingdom. Meningococcal serogroup C (MnC)-specific immunoglobulin G (IgG) antibodies in pre- and postimmunization sera obtained from healthy U.S. adults, toddlers, and infants were quantitated by enzyme-linked immunosorbent assay (ELISA) and by an antibody-dependent, complement-mediated serum bactericidal assay (SBA). Serogroup-specific IgG antibody (micrograms per milliliter) in adults immunized either with the quadrivalent polysaccharide (A, C, Y, and W-135) vaccine or with MnCC showed a strong correlation (r = 0.848 and 0.934, respectively) by linear regression analysis with SBA. Sera from infants immunized with the MnCC (n = 30) and an age-matched unimmunized control group (n = 15) were also analyzed. Linear regression analysis of serum bactericidal and IgG ELISA data from sera obtained at 2 months of age (preimmunization) showed no correlation; however, a high degree of correlation was observed at time points after two (r = 0.877) and three (r = 0.951) immunizations, where significant rises in anti-MnC polysaccharide antibodies occurred relative to the age-matched control group. Infants previously primed with 3 doses of MnCC were given a booster dose of conjugate vaccine at 12 to 15 months of age. The correlation coefficient of ELISA to SBA for combined pre- and postbooster data was r = 0.836 (n = 48 pairs). In conclusion, increases in serum bactericidal activity in immunized adult, toddler, and infant populations were found to correlate very well with increases in serogroup-specific IgG concentrations, whereas the correlation between these two assays in nonimmunized 2-month-old infants was poor. Characterizing the relationship between these methods is important for understanding the significance of antigen-specific antibody concentrations relative to vaccine performance and protection from disease.

Safety and immunogenicity of the PFP vaccine against respiratory syncytial virus (RSV): the western blot assay aids in distinguishing immune responses of the PFP vaccine from RSV infection.

PFP-1 vaccine was evaluated in a randomized, controlled study in 47 RSV seropositive children. Trivalent inactivated influenza virus (TIV) vaccine was the control. Vaccine reactions were monitored, and bloods were obtained before vaccination, 4 weeks after vaccination, and at the end of the RSV season. Respiratory illnesses were evaluated during the outbreak. Neutralizing antibody (Nt Ab) assay to RSV, IgG ELISA to RSV proteins and a Western blot assay were performed. Acute reactions with the PFP vaccine were mild. An early RSV outbreak resulted in infection of 44.4% of the TIV recipients shortly after vaccination. In the PFP vaccine groups, the Nt Ab and ELISA assays did not distinguish between Ab rises due to natural infection versus vaccine; however, the Western blot assay characterized the post-vaccine rises. Two major Western blot profiles were produced: an infection profile (antibodies that recognized the F and G surface glycoproteins and internal proteins) and a vaccine profile (antibodies that recognized only the surface glycoproteins). The PFP vaccinees who were not infected with RSV developed ELISA and Nt Ab responses to the surface glycoproteins that were similar to the TIV vaccines with natural RSV infection. None of the children developed vaccine-enhanced disease. Thus, the PFP-1 vaccine was safe and immunogenic in RSV seropositive children even when vaccine was administered during a RSV outbreak, and the Western blot assay was useful in distinguishing Ab rises caused by RSV infection versus PFP vaccine.

Safety and immunogenicity of a subunit respiratory syncytial virus vaccine in children 24 to 48 months old.

A subunit vaccine for respiratory syncytial virus (RSV) consisting of purified fusion glycoprotein (designated PFP-1) was tested in children 24 to 48 months old. Two doses of 20 micrograms (n = 13) and 50 micrograms (n = 10) were compared with a saline (n = 24) placebo control group. Local and systemic reactions, reported within 96 hours postvaccination, were mild, transient, and did not differ significantly from the control cohort. Long term follow-up through at least one, and in some cases two, RSV seasons showed no serious RSV illness in vaccinees at any time. There was, therefore, no evidence of disease enhancement postvaccination. In the 20-micrograms cohort, 92% responded to vaccination by a 4-fold increase in enzyme-linked immunosorbent titer to the F glycoprotein and 42% had a 4-fold or greater rise in neutralizing titer to the A2 virus. In the 50-micrograms cohort 100% responded by enzyme-linked immunosorbent to the F glycoprotein and 70% responded by A2-neutralizing titers. The neutralizing titers in the vaccinated cohorts were similar to those seen previously in adults. These data show the ability of the subunit vaccine to boost existing immunity and to prime for a response to natural virus exposure in children who were seronegative at the time of vaccination.

Second-year surveillance of recipients of a respiratory syncytial virus (RSV) F protein subunit vaccine, PFP-1: evaluation of antibody persistence and possible disease enhancement.

In a previous study, children 18 to 36 months of age and seropositive for respiratory syncytial virus (RSV) were vaccinated with an RSV subunit vaccine (PFP-1) consisting of the viral fusion protein. Vaccines developed substantial increases in anti-fusion and neutralizing antibody and exhibited protection against RSV infection through one RSV epidemic, in comparison to controls. This present study of the same cohort was undertaken to determine the persistence of antibody responses and immunity to reinfection, as well as to monitor for enhanced disease upon subsequent RSV infection during the second RSV season after vaccination. Vaccinees continued to have greater ELISA specific anti-fusion (F) antibody responses than controls up to 18 months after vaccination. Neutralizing antibody titres were not as durable, and the attack rates for RSV in the second winter season after vaccination (25% in vaccines versus 42% in controls) were not significantly different (p = 0.23). Nevertheless, 'high-responder' subgroups may have had residual protection into the second postvaccination year. Enhanced illness did not occur. PFP-1 is immunogenic and appears safe, but yearly reimmunization may be necessary to maintain immunity to RSV infection.

The pulmonary immune response of Balb/c mice vaccinated with the fusion protein of respiratory syncytial virus.

We have investigated the efficacy of vaccination with the purified fusion (F) protein of respiratory syncytial virus (RSV) on aluminium hydroxide adjuvant in Balb/c mice. The purpose of the study was to define the role of the local pulmonary mononuclear cell (PMC) infiltrate in the clearance of virus from the lower respiratory tract. Balb/c mice immunized with F protein were able to inhibit the replication of virus in the lungs as early as 4 days after intranasal challenge. In contrast, unimmunized mice required 8 days. Examination of humoral immune mechanisms demonstrated that vaccination with the purified protein induced moderate titres of serum neutralizing antibody. In addition, immunization induced low to moderate levels of antigen-dependent killer cell activity. To examine the immunological events responsible for virus clearance in vivo, PMC infiltrates were isolated after virus challenge and tested directly for protective capacity. After virus challenge, the F protein-immune mice were able to recall the cytolytic cells to the pulmonary tissues. The results further suggested that the local antigen-dependent killer activity was mediated by cytolytic T cells of the CD8 phenotype. Adoptive transfer studies were also conducted to identify further the role the PMC infiltrate had in protective immunity. Adoptive transfer of F protein-educated PMC into naive syngeneic recipients suggested that the pulmonary infiltrates contained the cellular constituents necessary for protective immunity. Both humoral and cellular immune elements were present.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced pulmonary pathology associated with the use of formalin-inactivated respiratory syncytial virus vaccine in cotton rats is not a unique viral phenomenon.

The specificity of viral antigens in the formalin-inactivated, alum-precipitated respiratory syncytial virus (FI-RSV) vaccine in augmenting the pulmonary inflammatory response was evaluated. Cotton rats were immunized with a FI-RSV vaccine derived from Vero cells, a monkey cell line, or HEp-2 cells, a human cell line. The FI-RSV/Vero and the FI-RSV/HEp-2 vaccines were prepared similarly to the original Lot-100 FI-RSV vaccine that was associated with enhanced disease in the mid-1960s field trials. Each vaccine was administered intramuscularly at various doses and intervals. At 1, 4 or 7 weeks after the last vaccine dose, cotton rats were challenged with 10(6) plaque-forming units of live RSV grown in HEp-2 cells. For controls, FI-parainfluenza, FI-HEp-2 and alum vaccines, and live RSV primary infection were used. For measuring virus replication and histopathology, lungs were harvested at 4 and 8 days postchallenge. A dose-response relationship to vaccine dose was observed for ELISA, neutralizing and antifusion antibodies. All animals given three doses or two of the higher doses of FI-RSV/Vero vaccine developed significant neutralizing antibody, were protected against pulmonary virus replication and had similar low levels of histopathology compared with live RSV and controls. Two immunizations of the lowest dose of FI-RSV/Vero vaccine did not induce neutralizing antibody, did not provide protection of the lung against RSV and did not enhance the pulmonary cellular response. However, FI-RSV/HEp-2 vaccine was associated with significant enhanced pulmonary histopathology despite inducing high titres of neutralizing antibody and protecting the lungs against RSV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the G glycoprotein gene of human respiratory syncytial virus in Salmonella typhimurium.

The attachment protein, G, of human respiratory syncytial virus (RSV) is an M(r) 84K to 90K species which has a high content of N-linked and O-linked carbohydrates. The unglycosylated form of this protein was expressed by inserting a full-length cDNA copy of the mRNA from the A2 strain of RSV into a prokaryotic expression vector under the control of the lambda PL promoter. Salmonella typhimurium cells transformed with the G-containing plasmid synthesized a protein of M(r) 40,000 that specifically reacted with polyclonal and two neutralizing monoclonal antibodies raised against the native RSV G glycoprotein. Recombinant G protein was purified by immunoaffinity chromatography using a neutralizing monoclonal antibody. Cotton rats immunized with the recombinant G protein produced serum antibodies to the G glycoprotein that neutralized RSV in vitro. The study demonstrates that the G protein of RSV can be expressed in bacteria and that at least one neutralizing epitope is not structurally dependent on carbohydrates.

Lack of detectable enhanced pulmonary histopathology in cotton rats immunized with purified F glycoprotein of respiratory syncytial virus (RSV) when challenged at 3-6 months after immunization.

The cotton rat model has been used to evaluate the potential for immunogens to induce respiratory syncytial virus (RSV)-enhanced pulmonary histopathology. A recent study evaluated purified F protein in this model when animals were challenged intranasally with RSV 3 or 6 months after immunization. The authors concluded that the purified F protein was associated with the same level of histopathological changes as observed with the positive control, a formalin-inactivated RSV immunogen. Three pathologists have independently evaluated the lung sections from the animals of this study and the results are reported in this article. In contrast to the previously published data, we have found that F protein was associated with a substantially milder and qualitatively different response to that observed with the formalin-inactivated RSV vaccine. We concluded that the minimal histological changes observed and lack of clinical disease make it very difficult to assess the issue of enhanced pulmonary RSV disease with the cotton rat model.

Immunogenicity and safety of respiratory syncytial virus subunit vaccine in seropositive children 18-36 months old.

Twenty-six children (aged 18-36 months) previously hospitalized for respiratory syncytial virus (RSV) infection were randomized to receive 50 micrograms of an RSV subunit vaccine composed primarily of F glycoprotein or saline placebo by intramuscular injection. Serum was obtained at entry and at 1 and 6 months after vaccination for detection of antibody to F glycoprotein and G glycoprotein of subtypes A (Ga) or B (Gb) and of neutralizing antibody (nAb). At 1 month, by comparing the baseline values, vaccinees had statistically significant increases in geometric mean antibody titer (GMT) of more than fourfold to F (P = .0001), Ga (P = .0001), Gb (P = .003), and nAb (P = .009). No differences in GMT were observed between F protein vaccine and placebo recipients at entry, nor between placebo recipients at entry and 1 month. RSV infections were identified in 7 placebo recipients (4 by both viral identification and seroconversion, 3 by seroconversion alone). No vaccine recipient had RSV infection documented in the 6 months after vaccination (P = .003). There were no significant vaccine-related side effects, and no evidence of enhanced respiratory illnesses was observed. The subunit F protein vaccine appears safe and immunogenic and may prevent infection in healthy children primed by prior RSV infection.

Expression of the F glycoprotein gene from human respiratory syncytial virus in Escherichia coli: mapping of a fusion inhibiting epitope.

A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus (strain A2) was inserted into a bacterial expression vector containing the lambda PR promoter. Upon heat induction, Escherichia coli cells harboring the vector produced a 45-kDa peptide which reacted with rabbit polyclonal antiserum to the native F protein. Expression of the F gene resulted in severe inhibition of bacterial growth, which was overcome by deletion of the DNA sequences encoding the F signal peptide. The region of the F protein which reacted with a virus-neutralizing and fusion-inhibiting monoclonal antibody was probed by expressing cDNA fragments encoding different protein domains in E. coli and testing antibody reactivity by Western blot analysis. Analysis of six fragments yielded an overlapping antibody-reactive region between amino acids 253 and 298. Analysis of reactivity with a cassette of synthetic peptides confirmed that the virus-neutralizing epitope mapped between residues 289 and 298 defined by the amino acid sequence M-S-I-I-K-E-E-V-L-A.

Effect of passive antibody on the immune response of cotton rats to purified F and G glycoproteins of respiratory syncytial virus (RSV).

The effect of passively transferred RSV immune serum on the antibody response to a single dose of purified RSV fusion (F) and large (G) glycoproteins was studied in cotton rats. Passively transferred antibody that achieved serum antibody levels similar to those seen in newborn human infants resulted in a seven- to eightfold suppression of the neutralizing antibody response of cotton rats to low doses of purified F and G glycoproteins (0.2-1.7 micrograms) and a twofold suppression to higher doses of these antigens (5-15 micrograms). This suppression of the antibody response was accompanied by a reduction in the protective efficacy of the F and G purified glycoprotein vaccine. These results suggest that parenteral immunization with RSV antigens could be less immunogenic in seropositive human infants, but that this suppressive effect might be partially overcome with increased antigen dose.

Occurrence of groups A and B of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children.

Over 15 years respiratory syncytial virus (RSV) isolates from 1209 hospitalized and ambulatory children were examined for strain group and in a subset for subgroup to determine the associated epidemiologic and clinical characteristics. Three patterns of yearly outbreaks existed: (1) strong predominance of group A strains (9 years with 83%-100% A strains), (2) relatively equal proportions of group A and B strains (4 years), and (3) strong predominance of group B strains (78%-85%) in 2 years, separated by a decade. The first pattern of highly dominant A strains occurred in cycles of 1 or 2 consecutive years with a single intervening year in which B strains were greater than or equal to 40% of the isolates. Subgroups A1 and A2 predominated, while B2, 3, and 4 occurred almost equally. A greater clinical severity for Group A strains was suggested by children with group A infections requiring intensive care significantly more often (15.4 vs. 8.3%, P = .008). Further, strongly dominant A strain years were associated with higher proportions of RSV admissions requiring intensive care (16.6% vs. 5.5%, P less than .01). Strains of subgroups A2 and B4 were more frequently found in hospitalized patients and A1 in outpatients, and the 2 years with the highest rates of intensive care admissions were those in which subgroup A2 dominated.

Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease.

A formalin-inactivated respiratory syncytial virus (RSV) vaccine tested 22 years ago failed to protect infant vaccinees against RSV infection or disease. Instead, lower respiratory tract disease was enhanced during subsequent infection by RSV. Enhancement of pulmonary pathology is also observed when cotton rats are immunized with formalin-inactivated RSV and subsequently infected with this virus. A major question that must be addressed for each new paramyxovirus vaccine is whether the immunogen possesses the capacity to potentiate disease. In the present study, we evaluated a newly developed purified F and G glycoprotein vaccine over a wide dosage range for immunogenicity, efficacy and capacity to potentiate pulmonary pathology in cotton rats. In addition, a formalin-inactivated RSV vaccine, which served as a positive control for enhancement of pulmonary pathology, was evaluated simultaneously. The results of these comparisons indicate that the purified F and G glycoprotein vaccine was highly immunogenic and was efficacious even in animals that developed low levels of serum-neutralizing antibodies. Furthermore, the F and G vaccine did not induce potentiation of pulmonary pathology. In contrast, formalin-inactivated RSV potentiated RSV pulmonary histopathology, but there was a sparing of potentiation at high and low doses. Both the formalin-inactivated RSV and purified F and G preparations induced a high level of serum antibodies capable of binding to purified F and G glycoproteins but both sets of antibodies had significantly reduced neutralizing activity. These results are encouraging because they suggest that purified paramyxovirus glycoproteins might be used safely as a vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus.

We describe the covalent attachment of palmitate to the fusion glycoprotein of respiratory syncytial virus and the identification of the attachment site. Labeling of respiratory syncytial virus-infected Vero cells with [3H]palmitate, followed by the purification and subsequent analysis of the fusion glycoprotein in conjunction with polyacrylamide gel electrophoresis, demonstrated that the fatty acid is covalently attached to the F1 subunit of the fusion glycoprotein. The bound palmitate was sensitive to 1 M hydroxylamine at neutral pH. In addition, the release of palmitate label by reduction with sodium borohydride showed that the palmitate is linked to the protein through a thioester bond. Isolation of a radiolabeled peptide from a tryptic digest of the protein and subsequent amino-terminal sequence analysis revealed that the cysteine residue (amino acid residue 550) within the anchor sequence, located at the carboxyl terminus of the F1 subunit, is the covalent attachment site for palmitate.

Respiratory syncytial virus-specific antibody responses in immunoglobulin A and E isotypes to the F and G proteins and to intact virus after natural infection.

We studied the antibody response to the fusion (F) and attachment (G) proteins of respiratory syncytial virus and to purified intact virus in the respiratory secretions of 29 infants and children. The goal of the study was to determine whether the immune response to either of the glycoproteins occurred predominantly in the immunoglobulin A (IgA) as opposed to the IgE isotype, which would indicate that one protein subunit would be a better candidate as a potential vaccine. Antibody responses were determined by using an enzyme-linked immunosorbent assay with purified F and G proteins and sucrose gradient-purified intact virus as targets. Infants and children were capable of developing an antibody response in both the IgA and IgE isotypes to each target antigen. The magnitude of the antibody response to the F protein was essentially similar to that to the intact virus, while responses to the G protein were diminished in infants. A slightly more favorable ratio of IgA to IgE responses was observed against the F protein in comparison to the G protein. While neither protein subunit had the ideal characteristics of inducing an IgA response in the absence of an IgE response, the F protein seems to be a better candidate for use as a vaccine, on the basis of better IgA/IgE ratios.

Interprotein disulfide bonding between F and G glycoproteins of human respiratory syncytial virus.

The envelope glycoprotein G, of human respiratory virus was purified by immunoaffinity chromatography using a monoclonal antibody reacting with G glycoprotein. The purified material was analyzed for its protein patterns and by western blot for its reactivity with specific monoclonal antibodies. In addition to the G specific proteins at 90 and 55 kilodalton (kDa) range, high molecular weight species were coeluted with G protein. Three high molecular weight species were noticed: one (140 kDa) reacting with fusion protein (F) monoclonal antibody and two other species (230 and 195 kDa) reacting with both fusion protein and G protein monoclonal antibodies. The protein reacting only with F monoclonal antibody consists of fusion protein dimer. Western blot and two dimensional gel electrophoretic analysis revealed that each of the other two complexes is composed of two moles of F protein and one mole of G protein. These two complexes differ in their molecular sizes depending on whether G is in the form of 90 or 55 kDa. Upon heat denaturation, fusion protein monomer (70 kDa) is released from the complex, leaving the two complexes, consisting of one mole of F protein and one mole of G protein (160 and 125 kDa species respectively). Disulfide-reducing agents are required to break the monomers of F and G complexes. These results provide a direct evidence for the presence of envelope glycoprotein complexes linked by interprotein disulfide bonding. This may have implications on the structural and functional properties of envelope glycoproteins.

Evidence that the fusion protein of respiratory syncytial virus exists as a dimer in its native form. Brief report.

The quaternary structure of respiratory syncytial virus (RSV) fusion protein has been studied. Crosslinking studies were done to stabilize the noncovalently associated proteins. These stable, heat-resistant, covalently linked complexes were analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis. In situ crosslinking studies demonstrated that the fusion protein of RSV exists as a dimer in its native form on the surface of infected cells. The purified protein was also found to be present predominantly as a dimer. In addition, the results suggest that F1 subunits may play a role in the dimerization of the fusion protein.

Economic comparison of enzyme immunoassay and virus isolation procedures for surveillance of arboviruses in mosquito populations.

Cost-effectiveness analysis of an enzyme immunoassay (EIA) for the surveillance of arboviruses was conducted. The EIA was compared with conventional virus isolation and serologic identification procedures (virus isolation procedures; VIP). Under most circumstances, EIA was more cost-effective than VIP. Costs for processing mosquito pools by VIP increased with the number of viruses included in the surveillance program and with the prevalence rate of each virus. In contrast to VIP, the prevalence rate did not affect costs for processing pools by EIA. In general, EIA was the most cost-effective procedure, followed by cell culture and mouse bioassays. In a 5-year cost-effectiveness analysis of a model surveillance program in which EIA and cell culture bioassays were used, the EIA again proved to be the most cost-effective assay procedure under most circumstances.