Immunogenicity, safety and tolerability of 3 lots of 13-valent pneumococcal conjugate vaccine given with routine pediatric vaccinations in the United States.

BACKGROUND: The 7-valent pneumococcal conjugate vaccine (PCV7; serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) has decreased invasive pneumococcal disease incidence. This study was performed to support licensure of a 13-valent pneumococcal conjugate vaccine (PCV13), which expands serotype coverage to include serotypes 1, 3, 5, 6A, 7F and 19A. This study assessed the immunogenicity, safety and manufacturing consistency of PCV13. METHODS: Randomized, double-blind, multicenter trial. Healthy United States infants were randomized 2:2:2:1 to receive 1 of 3 lots of PCV13 or PCV7, along with routine US pediatric vaccines at ages 2, 4 and 6 months (infant series), and 12 months (toddler dose). RESULTS: Among 1709 vaccinated infants, 1 month postinfant series and 1 month posttoddler dose, immunoglobulin G geometric mean concentrations (GMCs) were within 2-fold among the PCV13 lots, meeting equivalence criteria for all 13 serotypes. In a post hoc analysis, based on percent responders at ≥0.35 µg/mL postinfant series and immunoglobulin G GMC ratios postinfant series and posttoddler dose, noninferiority criteria were met for combined PCV13 lots compared with PCV7 for all common serotypes. Posttoddler dose immunoglobulin G GMCs were higher than postinfant series GMCs for all serotypes. Local reactions and fevers were generally mild; incidences of local reactions, systemic events and adverse events were generally similar between groups. CONCLUSIONS: PCV13 can be manufactured in a manner that elicits consistent immune responses. PCV13 provides increased serotype coverage and immunogenicity that is noninferior to PCV7 and has a safety profile similar to PCV7 when given with routine pediatric vaccines.

Immunogenicity and safety of 13-valent pneumococcal conjugate vaccine in children previously immunized with 7-valent pneumococcal conjugate vaccine.

BACKGROUND: The 7-valent pneumococcal conjugate vaccine (PCV7) has proven highly effective in preventing diseases caused by Streptococcus pneumoniae; however, in some regions, serotype coverage is limited. A recently licensed 13-valent PCV (PCV13) was developed to provide additional coverage globally. Children previously vaccinated with PCV7 could benefit from supplemental vaccination with PCV13 to provide protection against the 6 additional serotypes in PCV13. This open-label study evaluated the immunogenicity and safety of administering PCV13 to healthy children previously vaccinated with PCV7. METHODS: Children between 15 months and 2 years of age (group 1) received 2 doses of PCV13; children between 2 and 5 years (group 2) received 1 dose. Antibodies (immunoglobulin G) against the polysaccharide antigens in PCV13 were measured before vaccination and 1 month after the final dose. Solicited local and systemic adverse events (AEs) were collected for 7 days postvaccination. Unsolicited and serious AEs were collected throughout. RESULTS: A total of 284 subjects (group 1: n = 109; group 2: n = 175) had blood available for testing. Antipneumococcal immunoglobulin G geometric mean fold rises ranged from 2- to 19-fold for the PCV7 serotypes and from approximately 2- to 124-fold for the 6 additional serotypes. Additionally, postvaccination titers in excess of 0.35 µg/mL, the serologic correlate of immunity against pneumococcus for children, occurred in ≥98% of subjects in both groups for 12 of the 13 serotypes in PCV13. Slightly lower percentage of subjects, 94.5% and 92% of subjects in group 1 and group 2, respectively, had postvaccine titers for serotype 3 exceeding the serologic correlate of immunity. Reactogenicity was typically mild and self-limited, and unsolicited AEs reported were generally consistent with common childhood illnesses. CONCLUSION: PCV13 was safe and immunogenic when administered to children who had previously received PCV7, and can be used for supplemental vaccination to provide additional protection against the 6 additional serotypes.

An efficient helper-virus-free method for rescue of recombinant paramyxoviruses and rhadoviruses from a cell line suitable for vaccine development.

Recovery of recombinant, negative-strand, nonsegmented RNA viruses from a genomic cDNA clone requires a rescue system that promotes de novo assembly of a functional ribonucleoprotein (RNP) complex in the cell cytoplasm. This is accomplished typically by cotransfecting permissive cells with multiple plasmids that encode the positive-sense genomic RNA, the nucleocapsid protein (N or NP), and the two subunits of the viral RNA-dependent RNA polymerase (L and P). The transfected plasmids are transcribed in the cell cytoplasm by phage T7 RNA polymerase (T7 RNAP), which usually is supplied by infection with a recombinant vaccinia virus or through use of a stable cell line that expresses the polymerase. Although both methods of providing T7 RNAP are effective neither is ideal for viral vaccine development for a number of reasons. Therefore, it was necessary to modify existing technology to make it possible to routinely rescue a variety of recombinant viruses when T7 RNAP was provided by a cotransfected expression plasmid. Development of a broadly applicable procedure required optimization of the helper-virus-free methodology, which resulted in several modifications that improved rescue efficiency such as inclusion of plasmids encoding viral glycoproteins and matrix protein, heat shock treatment, and use of electroporation. The combined effect of these enhancements produced several important benefits including: (1) a helper-virus-free methodology capable of rescuing a diverse variety of paramyxoviruses and recombinant vesicular stomatitis virus (rVSV); (2) methodology that functioned effectively when using Vero cells, a suitable substrate for vaccine production; and (3) a method that enabled rescue of highly attenuated recombinant viruses, which had proven refractory to rescue using published procedures.

Genetic stability of live, cold-adapted influenza virus components of the FluMist/CAIV-T vaccine throughout the manufacturing process.

FluMist is a live-attenuated, trivalent influenza vaccine (LAIV) recently approved for intranasal administration. To demonstrate genetic stability during manufacture of the vaccine viruses in LAIV and a similar vaccine in development (CAIV-T), full genome consensus sequences were determined at multiple manufacturing stages for four influenza type A and five type B strains. The critical cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) mutations were preserved in the virus manufacturing intermediates. Moreover, sequence identity was observed for all vaccine intermediates of the same strain. Minor sequence differences were noted in the shared gene segments of the vaccine viruses and their common progenitor master donor virus (MDV) and several of the hemagglutinin (HA) and neuraminidase (NA) genes contained nucleotide differences when compared to the wild-type parent. Nonetheless, all vaccine viruses retained the ca, ts, and att phenotypes. Thus, genetic and phenotypic stability of the vaccine viruses is maintained during the manufacture of LAIV/CAIV-T vaccines.

Role of V protein RNA binding in inhibition of measles virus minigenome replication.

Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein-RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3' terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein-RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238-266 were necessary for efficient RNA binding and overlapped with residues (238-278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238-247) that blocked RNA binding and repression indicating that these two activities were related.

Genetic and phenotypic stability of cold-adapted influenza viruses in a trivalent vaccine administered to children in a day care setting.

The genetic and phenotypic stability of viruses isolated from young children following intranasal administration of the trivalent live-attenuated influenza virus vaccine (LAIV, marketed in the United States as FluMist) was evaluated by determination of genomic sequence and assessment of the cold-adapted (ca), temperature-sensitive (ts) and attenuated (att) phenotypes. The complete genomic sequence was determined for 56 independent isolates obtained from children following vaccination (21 type A/H1N1, 12 A/H3N2, 1 A/H3N1 and 22 type B viruses), 20% of which had no nucleotide misincorporations compared with administered vaccine. The remaining isolates had from one to seven changes per genome. None of the observed misincorporations resulted in predicted amino acid codon substitutions at sites previously shown to contribute to the ca, ts or att phenotypes, and all vaccine-derived isolates retained ca and ts phenotypes consistent with the observation that none of the vaccine recipients displayed distinctive symptoms. The results indicate that LAIV strains undergo very limited genetic change following replication in vaccine recipients and that those changes did not affect vaccine attenuation.