The association between influenza vaccine effectiveness and egg-based manufacturing technology: literature review and US expert consensus.

BACKGROUND: Influenza is associated with significant disease burden in the US and is currently best controlled by vaccination programs. Influenza vaccine effectiveness (VE) is low and may be reduced by several factors, including egg adaptations. Although non-egg-based influenza vaccines reportedly have greater VE in egg-adapted seasons, evidence for egg adaptations' reduction of VE is indirect and dissociated, apart from two previous European consensuses. METHODS: This study replicated the methodology used in a 2020 literature review and European consensus, providing an updated review and consensus opinion of 10 US experts on the evidence for a mechanistic basis for reduction of VE due to egg-based manufacturing methods. A mechanistic basis was assumed if sufficient evidence was found for underlying principles proposed to give rise to such an effect. Evidence for each principle was brought forward from the 2020 review and identified here by structured literature review and expert panel. Experts rated the strength of support for each principle and a mechanistic basis for reduction of VE due to egg-based influenza vaccine manufacture in a consensus method (consensus for strong/very strong evidence = ≥ 3.5 on 5-point Likert scale). RESULTS: Experts assessed 251 references (from previous study: 185; this study: 66). The majority of references for all underlying principles were rated as strong or very strong supporting evidence (52-86%). Global surveillance, WHO candidate vaccine virus selection, and manufacturing stages involving eggs were identified as most likely to impact influenza VE. CONCLUSION: After review of extensive evidence for reduction of VE due to egg-based influenza vaccine manufacture, influenza experts in the US joined those in Europe in unanimous agreement for a mechanistic basis for the effect. Vaccine providers and administrators should consider use of non-egg-based influenza vaccine manufacture to reduce the risk of egg adaptations and likely impact on VE.

Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis.

BACKGROUND: Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS: Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS: Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS: The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.

CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation.

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.

Macrolide efflux in Streptococcus pneumoniae is mediated by a dual efflux pump (mel and mef) and is erythromycin inducible.

Macrolide resistance in Streptococcus pneumoniae due to efflux has emerged as an important worldwide clinical problem over the past decade. Efflux is mediated by the genes of the genetic element mega (macrolide efflux genetic assembly) and related elements, such as Tn1207.1. These elements contain two adjacent genes, mef (mefE or mefA) and the closely related mel gene (msrA homolog), encoding a proton motive force pump and a putative ATP-binding cassette transporter homolog, and are transcribed as an operon (M. Del Grosso et al., J. Clin. Microbiol. 40:774-778, 2004; K. Gay and D. S. Stephens, J. Infect. Dis. 184:56-65, 2001; and M. Santagati et al., Antimicrob. Agents Chemother. 44:2585-2587, 2000). Previous studies have shown that Mef is required for macrolide resistance in S. pneumoniae; however, the contribution of Mel has not been fully determined. Independent deletions were constructed in mefE and mel in the serotype 14 macrolide-resistant strains GA16638 (erythromycin [Em] MIC, 8 to 16 microg/ml) and GA17719 (Em MIC, 2 to 4 microg/ml), which contain allelic variations in the mega element. The MICs to erythromycin were significantly reduced for the independent deletion mutants of both mefE and mel compared to those of the parent strains and further reduced threefold to fourfold to Em MICs of <0.15 microg/ml with mefE mel double mutants. Using quantitative reverse transcription-PCR, the expression of mefE in the mel deletion mutants was increased more than 10-fold. However, in the mefE deletion mutants, the expression of mel did not differ significantly from the parent strains. The expression of both mefE and mel was inducible by erythromycin. These data indicate a requirement for both Mef and Mel in the novel efflux-mediated macrolide resistance system in S. pneumoniae and other gram-positive bacteria and that the system is inducible by macrolides.

Cationic antimicrobial peptide resistance in Neisseria meningitidis.

Cationic antimicrobial peptides (CAMPs) are important components of the innate host defense system against microbial infections and microbial products. However, the human pathogen Neisseria meningitidis is intrinsically highly resistant to CAMPs, such as polymyxin B (PxB) (MIC > or = 512 microg/ml). To ascertain the mechanisms by which meningococci resist PxB, mutants that displayed increased sensitivity (> or =4-fold) to PxB were identified from a library of mariner transposon mutants generated in a meningococcal strain, NMB. Surprisingly, more than half of the initial PxB-sensitive mutants had insertions within the mtrCDE operon, which encodes proteins forming a multidrug efflux pump. Additional PxB-sensitive mariner mutants were identified from a second round of transposon mutagenesis performed in an mtr efflux pump-deficient background. Further, a mutation in lptA, the phosphoethanolamine (PEA) transferase responsible for modification of the lipid A head groups, was identified to cause the highest sensitivity to PxB. Mutations within the mtrD or lptA genes also increased meningococcal susceptibility to two structurally unrelated CAMPs, human LL-37 and protegrin-1. Consistently, PxB neutralized inflammatory responses elicited by the lptA mutant lipooligosaccharide more efficiently than those induced by wild-type lipooligosaccharide. mariner mutants with increased resistance to PxB were also identified in NMB background and found to contain insertions within the pilMNOPQ operon involved in pilin biogenesis. Taken together, these data indicated that meningococci utilize multiple mechanisms including the action of the MtrC-MtrD-MtrE efflux pump and lipid A modification as well as the type IV pilin secretion system to modulate levels of CAMP resistance. The modification of meningococcal lipid A head groups with PEA also prevents neutralization of the biological effects of endotoxin by CAMP.

Outer membrane protein (OMP) based vaccine for Neisseria meningitidis serogroup B.

A family of outer membrane lipoproteins of Neisseria meningitidis, LP2086, has been shown to induce serum bactericidal activity against a broad variety of meningococcal strains. Two sub-families of serologically distinct LP2086 proteins (A and B) have been identified. In the present study, we have shown that polyclonal anti-serum against rLP2086 is protective in vivo in an infant rat passive-protection model. Additionally, the LP2086 protein is displayed on the surface of 91% meningococcal strains as measured in a whole cell ELISA using polyclonal anti-sera raised against these proteins. We also demonstrate based on the reactivity of anti-rLP2086 antibody with recombinantly expressed C- and N-terminal fragments of rLP2086 in a Western blot assay that the C-terminal fragment of LP2086 dictates sub-family specificity and the N-terminal fragment determines the family specificity. A formulation containing family A and B of LP2086 potentially would provide broad protection against a majority of Neisseria meningitidis strains.

The MisR/MisS two-component regulatory system influences inner core structure and immunotype of lipooligosaccharide in Neisseria meningitidis.

Lipooligosaccharide (LOS) of Neisseria meningitidis is the major inflammatory mediator that contributes to meningococcal pathogenesis. Variable attachments to the HepII residue of the LOS inner core together with the alpha-chain heterogeneity result in immunologically distinct LOS structures, which may be selected for during human infection. Lpt-3, a phosphoethanolamine (PEA) transferase, and LgtG, a glucosyltransferase, mediate the substitution of PEA or glucose at the O-3 position of HepII in L3 or L2 LOS immunotypes, respectively. Inactivation of a two-component response regulator, encoded by NMB0595, in N. meningitidis strain NMB resulted in the loss of all PEA decorations on the LOS inner core expressed by the NMB0595 mutant. When compared with the parental strain NMB that predominantly expresses L2 immunotype LOS and other minor LOS structures, the NMB0595 mutant expresses a pure population of a novel LOS structure completely substituted at the HepII O-3 position with glucose, but lacking other PEA decorations on the inner core. Quantitative real time PCR experiments showed increased transcription of lgtG in the NMB0595 mutant, and no significant change in lpt-3 transcription. Inactivation of lgtG resulted in LOS inner cores without glucose, but these structures, even though the lpt-3 transcription was unaffected, also lacked the O-3-linked PEA. Consistently, a double mutation of lgtG and misR in strain NMB yielded a LOS structure without PEA or Glc substitution of HepII. These data indicated a new pathway for the regulation of LOS inner core structure in N. meningitidis through an environmental sensing two-component regulatory system, named misR(NMB0595)/misS(NMB0594) for regulator and sensor of the meningococcal inner core structure.