Peter R. Paradiso

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)

Respiratory syncytial virus vaccines

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract illness (LRI) in infants and children worldwide and causes significant LRI in the elderly and in immunocompromised patients. The goal of RSV vaccination is to prevent serious RSV-associated LRI. There are several obstacles to the development of successful RSV vaccines, including the need to immunize very young infants, who may respond inadequately to vaccination; the existence of two antigenically distinct RSV groups, A and B; and the history of disease enhancement following administration of a formalin-inactivated vaccine. It is likely that more than one type of vaccine will be needed to prevent RSV LRI in the various populations at risk. Although vector delivery systems, synthetic peptide, and immune-stimulating complex vaccines have been evaluated in animal models, only the purified F protein (PFP) subunit vaccines and live attenuated vaccines have been evaluated in recent clinical trials. PFP-2 appears to be a promising vaccine for the elderly and for RSV-seropositive children with underlying pulmonary disease, whereas live cold-passaged (cp), temperature-sensitive (ts) RSV vaccines (denoted cpts vaccines) would most probably be useful in young infants. The availability of cDNA technology should allow further refinement of existing live attenuated cpts candidate vaccines to produce engineered vaccines that are satisfactorily attenuated, immunogenic, and phenotypically stable.

Safety and immunogenicity of four doses of Neisseria meningitidis group C vaccine conjugated to CRM197 in United States infants

BACKGROUNDnFollowing widespread use of conjugate pneumococcal vaccine, Neisseria meningitidis likely will become the leading cause of bacterial sepsis and meningitis in US children. This report describes the safety and immunogenicity in US children of four consecutive doses of a meningococcal group C vaccine conjugated to CRM197 via reductive amination (MnCC).nnnMETHODSnOne hundred six healthy 2-month-old infants received MnCC at 2, 4 and 6 months of age in a randomized, controlled double blind study; children in the other treatment arm were given a 7-valent conjugate pneumococcal vaccine. Parents reenrolled 64 of these children at 12 to 15 months to receive a fourth dose of MnCC. Routine childhood vaccines, including DTP, were coadministered. Temperatures and symptoms were recorded for 3 days after each immunization. Serum enzyme-linked immunosorbent assay IgG and bactericidal antibodies were measured prevaccination and before and 1 month after Doses 3 and 4.nnnRESULTSnModerate to severe local reactions, defined as erythema or induration > or =2.4 cm or pain that interfered with limb movement was reported after 0 to 3.2% of MnCC injections, depending on the reaction and dose. Fever occurred in 23 to 37% of children, but the contribution of MnCC to the febrile reactions is unknown. Geometric mean concentrations of IgG antibody to group C meningococcal polysaccharide were 3.72 microg/ml after Dose 3 and 8.03 microg/ml after the booster. Geometric mean functional serum bactericidal antibody titers after Doses 3 and 4 were 1:463 and 1:2341, respectively. One hundred percent of children had a serum bactericidal antibody titer of > or =1:64 after three doses and > or = 1:128 after the booster.nnnCONCLUSIONSnThe MnCC vaccine had an acceptable safety profile and generated high titers of bactericidal antibody in immunized US infants and toddlers. It appears to be an attractive candidate vaccine for the prevention of serogroup C meningococcal disease in young children.

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.

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.

Expression of the F glycoprotein gene from human respiratory syncytial virus inEscherichia 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.

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.

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

SummaryThe 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.

Combination Vaccines for Diphtheria, Tetanus, Pertussis, and Haemophilus influenzae Type b

The ability to combine the standard DTP and Haemophilus b conjugate vaccine considerably simplifies the childhood immunization schedule and process. In addition to reducing the number of immunizations by half, the combination product reduces administrative aspects associated with vaccination including tracking. Simplification of the immunization process should have a positive impact on the vaccine delivery and utilization. Perhaps more importantly, an ability to create combination vaccines will be critical for inclusion of new antigens appropriate for infant vaccines. The combination of DTP and HbOC reduces the number of immunizations routinely given at 2, 4, and 6 months of age by half. Since it is unlikely that parents or pediatricians will accept more than two shots per visit, this reduction is critical. As new vaccines are licensed for such important childhood pathogens as Streptococcus pneumoniae and respiratory syncytial virus, designing stable combination products will become even more critical. Having stated that, we must also not lose site of the fact that combination products must meet the criteria for stability, safety, and efficacy comparable to the separately delivered products. These considerations are not trivial. In the development of Tetramune (DTP-HbOC), stability of the product and consistency of the immune response were critical design parameters for both the preclinical and clinical research. Likewise, the experience with other DTP-Haemophilus b combinations has shown that simple mixing of products prior to injection can reduce the immune response in ways that are not necessarily predictable. In contrast, the response to each of the components of Tetramune was in fact higher than when the vaccines were given separately. This increased response to all of the antigens was not anticipated based on the vaccine composition and points to the need for not only physical characterization of new combinations, but also clinical testing of final combined products before they are introduced for routine use.

Mapping of a fusion related epitope of the respiratory syncytial virus fusion protein.

The region of the fusion glycoprotein of respiratory syncytial virus which reacts with a neutralizing and fusion inhibiting monoclonal antibody, was mapped using a deductive method derived from analysis of Western blot reactivity of proteolytic fragments. Reaction of the whole fusion protein was found to be so conformationally dependent, that complete digestion of the protein with a variety of proteases resulted in fragments which were not sufficiently reactive to permit mapping. For this reason, polyclonal antibodies to synthetic peptides which spanned the fusion protein sequence, were used to map the position of large peptides derived from partial digests, and these peptides were then analysed for their ability to react with the monoclonal antibody. Comparison of the peptides which were reactive with the monoclonal antibody to those which were not, identified a region of non-overlap between residues 283 and 327 in the F1 subunit of the fusion protein. Synthesis of a peptide within this region confirmed the placement of the epitope.