Steve Rockman

Circulating Precursor CCR7loPD-1hi CXCR5+ CD4+ T Cells Indicate Tfh Cell Activity and Promote Antibody Responses upon Antigen Reexposure

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

Intranasal vaccination with ISCOMATRIX adjuvanted influenza vaccine.

Mucosal delivery of inactivated vaccines that are able to elicit protective immune responses against respiratory diseases has been a long time goal of vaccinologists. Such vaccines would enable a more appropriate means of vaccination against respiratory diseases than those currently delivered by a parenteral route. The intranasal delivery of inactivated influenza vaccine plus the ISCOMATRIX (IMX) adjuvant, simply mixed together, was able to induce serum haemagglutination inhibition (HAI) titres in mice far superior to those obtained with unadjuvanted vaccine delivered subcutaneously. Furthermore, the IMX adjuvanted vaccine delivered intranasally induced mucosal IgA responses in the lung, nasal passages and large intestine, together with high levels of serum IgA. Intranasal delivery of IMX adjuvanted influenza vaccine in sheep gave antibody responses in both serum and nasal secretions that surpassed the levels obtained with unadjuvanted vaccine administered subcutaneously. These observations suggest that it may be possible to induce effective immunity to influenza in humans by intranasal vaccination with an IMX adjuvanted inactivated vaccine.

The antigenic architecture of the hemagglutinin of influenza H5N1 viruses

Human infection with the highly pathogenic avian influenza A virus H5N1 is associated with a high mortality and morbidity. H5N1 continues to transmit from poultry to the human population, raising serious concerns about its pandemic potential. Current influenza H5N1 vaccines are based upon the elicitation of a neutralizing antibody (Ab) response against the major epitope regions of the viral surface glycoprotein, hemagglutinin (HA). However, antigenic drift mutations in immune-dominant regions on the HA structure allow the virus to escape Ab neutralization. Epitope mapping using neutralizing monoclonal antibodies (mAb) helps define mechanisms of antigenic drift, neutralizing escape and can facilitate pre-pandemic vaccine design. This review explores the current knowledge base of the antigenic sites of the H5N1 HA molecule. The relationship between the epitope architecture of the H5N1 HA, antigenic evolution of the different H5N1 lineages and the antigenic complexity of the H5N1 virus lineages that constitute potential pandemic strains are discussed in detail.

Scientific investigations into febrile reactions observed in the paediatric population following vaccination with a 2010 Southern Hemisphere Trivalent Influenza Vaccine.

During the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in the number of febrile reactions reported in the paediatric population in Australia shortly after vaccination with the CSL 2010 trivalent influenza vaccine (TIV) compared to previous seasons. A series of scientific investigations were initiated to identify the root cause of these adverse events, including in vitro cytokine/chemokine assays following stimulation of adult and paediatric whole blood, as well as mammalian cell lines and primary cells, profiling of molecular signatures using microarrays, and in vivo studies in rabbits, ferrets, new born rats and rhesus non-human primates (NHPs). Various TIVs (approved commercial vaccines as well as re-engineered TIVs) and their individual monovalent pool harvest (MPH) components were examined in these assays and in animal models. Although the scientific investigations are ongoing, the current working hypothesis is that the increase in febrile adverse events reported in Australia after vaccination with the CSL 2010 SH TIV may be due to a combination of both the introduction of three entirely new strains in the CSL 2010 SH TIV, and differences in the manufacturing processes used to manufacture CSL TIVs compared to other licensed TIVs on the market. Identification of the causal component(s) may result in the identification of surrogate assays that can assist in the formulation of TIVs to minimise the future incidence of febrile reactions in the paediatric population.

Role of viral RNA and lipid in the adverse events associated with the 2010 Southern Hemisphere trivalent influenza vaccine

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL 2010 SH trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. In an accompanying study, we described the contribution to these adverse events of the 2010 SH influenza strains as expressed in the CSL 2010 SH TIV using in vitro cytokine/chemokine secretion from whole blood cells and induction of NF-κB activation in HEK293 reporter cells. The aim of the present study was to identify the root cause components that elicited the elevated cytokine/chemokine and NF-κB signature. Our studies demonstrated that the pyrogenic signal was associated with a heat-labile, viral-derived component(s) in the CSL 2010 SH TIV. Further, it was found that viral lipid-mediated delivery of short, fragmented viral RNA was the key trigger for the increased cytokine/chemokine secretion and NF-κB activation. It is likely that the FS reported in children <5 years were due to a combination of the new influenza strains included in the 2010 SH TIV and the CSL standard method of manufacture preserving strain-specific viral components of the new influenza strains (particularly B/Brisbane/60/2008 and to a lesser extent H1N1 A/California/07/2009). These combined to heighten immune activation of innate immune cells, which in a small proportion of children <5 years of age is associated with the occurrence of FS. The data also demonstrates that CSL TIVs formulated with increased levels of splitting agent (TDOC) for the B/Brisbane/60/2008 strain can attenuate the pro-inflammatory signals in vitro, identifying a potential path forward for generating a CSL TIV indicated for use in children <5 years.

Evaluation of the bioactivity of influenza vaccine strains in vitro suggests that the introduction of new strains in the 2010 Southern Hemisphere trivalent influenza vaccine is associated with adverse events.

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. The present study describes the outcomes of a series of in vitro experiments directed at elucidating the root cause. The scientific investigations found that a subset of paediatric donors displayed elevated cytokine/chemokine responses to the CSL 2010 SH TIV but not to previous CSL TIVs nor other 2010 SH TIVs. The induction of elevated cytokines/chemokines in paediatric whole blood correlated with elevated NF-κB activation in a HEK293 cell reporter assay. The data indicate that the introduction of the B/Brisbane/60/2008 strain within the CSL manufacturing process (such as occurred in the preceding 2009/10 NH season) appears to have raised the pyrogenic potential of the CSL 2009/10 NH TIV but that this was insufficient to elicit FS in children <5 years. The 2010 SH season coincided with the first introduction of the H1N1 A/California/07/2009 in combination with the B/Brisbane/60/2008 strain. Our data demonstrates that the introduction of the H1N1 A/California/07/2009 (and to a much lesser degree, H3N2 A/Wisconsin/15/2009) in combination with B/Brisbane/60/2008 (as expressed through the CSL method of manufacture) combined and likely compounded the bioactivity of the CSL 2010 SH TIV. This was associated with stronger immune responses, which in a proportion of children <5 years were associated with FS. The assays and systems developed during these investigations should greatly assist in determining the bioactivity of new influenza strains, and thus aid with the manufacture of CSL TIVs indicated for use in the paediatric population.

Correlation of polymerase replication fidelity with genetic evolution of influenza A/Fujian/411/02(H3N2) viruses†

Influenza virus evolves continuously through mutations presumed to result from evolutionary pressure driving viral replication. This study examined the relationship between the genetic evolution and replication fidelity of influenza viruses. Analysis of influenza sequences from National Centre for Biotechnology Information (NCBI) database revealed a gradual decrease in the rate of genetic evolution of A/Fujian/411/02(H3N2)‐like variants after the emergence and predominance of the A/H3N2 Fujian strain in 2002. This decrease may be related to an increase in replication fidelity, which was investigated by assessing mutation frequencies of reassortant viruses carrying the PB1 segment of Fujian variants isolated between 2003 and 2005 in a sequencing‐based plaque assay. The data revealed a threefold decrease in substitution per site of the reassortant viruses carrying the Fujian PB1 segments isolated in 2004–2005 compared with those circulating in 2003. The decrease in mutation frequency paralleled a decrease in genetic evolution of the Fujian variants from the NCBI database. This correlation implicates changes in the polymerase replication fidelity as contributing to altered genetic evolution of influenza viruses. J. Med. Virol. 83:510–516, 2011.

Molecular Characterisation of the Haemagglutinin Glycan-Binding Specificity of Egg-Adapted Vaccine Strains of the Pandemic 2009 H1N1 Swine Influenza A Virus

The haemagglutinin (HA) glycan binding selectivity of H1N1 influenza viruses is an important determinant for the host range of the virus and egg-adaption during vaccine production. This study integrates glycan binding data with structure-recognition models to examine the impact of the K123N, D225G and Q226R mutations (as seen in the HA of vaccine strains of the pandemic 2009 H1N1 swine influenza A virus). The glycan-binding selectivity of three A/California/07/09 vaccine production strains, and purified recombinant A/California/07/09 HAs harboring these mutations was examined via a solid-phase ELISA assay. Wild-type A/California/07/09 recombinant HA bound specifically to α2,6-linked sialyl-glycans, with no affinity for the α2,3-linked sialyl-glycans in the array. In contrast, the vaccine virus strains and recombinant HA harboring the Q226R HA mutation displayed a comparable pattern of highly specific binding to α2,3-linked sialyl-glycans, with a negligible affinity for α2,6-linked sialyl-glycans. The D225G A/California/07/09 recombinant HA displayed an enhanced binding affinity for both α2,6- and α2,3-linked sialyl-glycans in the array. Notably its α2,6-glycan affinity was generally higher compared to its α2,3-glycan affinity, which may explain why the double mutant was not naturally selected during egg-adaption of the virus. The K123N mutation which introduces a glycosylation site proximal to the receptor binding site, did not impact the α2,3/α2,6 glycan selectivity, however, it lowered the overall glycan binding affinity of the HA; suggesting glycosylation may interfere with receptor binding. Docking models and ‘per residues’ scoring were employed to provide a structure-recognition rational for the experimental glycan binding data. Collectively, the glycan binding data inform future vaccine design strategies to introduce the D225G or Q226R amino acid substitutions into recombinant H1N1 viruses.

Investigation into alternative testing methodologies for characterization of influenza virus vaccine

The objective of this study was to explore various testing methodologies suitable for characterizing sedimented or agglomerated material. To model this, bioCSLs split influenza virus vaccine, Fluvax® was utilized. The investigation was conducted on 5 dispensed lots of commercially manufactured vaccine, formulated for the 2013 Southern Hemisphere season. Vaccine syringes were initially inspected by visual tests; the material was then aseptically pooled for characterization assessment by microscopy and several agglomeration assays. All syringes passed bioCSLs description test where any fine or large sized particles of sediment observed in the vaccine were resuspended upon shaking; inverted light microscopy verified that the sediment morphology was consistent with influenza vaccine. Electron microscopic examination of pooled vaccine material demonstrated the presence of typical influenza structures including split virus, virosomes, whole virus particles and agglomerates. An optical density turbidity assay revealed relatively high protein recoveries in the vaccine supernatant post-centrifugation treatment, thus indicative of a well-dispersed vaccine formulation. This was corroborated by particle sizing analysis using dynamic light scattering which generated reproducible volume particle size distributions of a polydisperse nature. Ultraviolet-visible absorbance profiles further confirmed the presence of some agglomerated material. Data from all methods demonstrated consistent results between all batches of vaccine. Therefore, this investigation revealed the suitability and usefulness of the various methodologies in characterizing the appearance of agglomerated vaccine material. It is suggested that such methods may be applicable and beneficial for the development of a wider spectrum of heterogeneous and agglomerated formulations to provide safe, efficacious and superior quality biopharmaceutical products.

Reverse engineering the antigenic architecture of the haemagglutinin from influenza H5N1 clade 1 and 2.2 viruses with fine epitope mapping using monoclonal antibodies

The induction of neutralising antibodies to the viral surface glycoprotein, haemagglutinin (HA) is considered the cornerstone of current seasonal and pandemic influenza vaccines. Mapping of neutralising epitopes using monoclonal antibodies (mAbs) helps define mechanisms of antigenic drift, neutralising escape and facilitates pre-pandemic vaccine design. In the present study we reverse engineered the antigenic structure of the HAs of two highly pathogenic H5N1 vaccine strains representative of currently circulating clade 1 and 2.2 H5N1 viruses. The HA sequence of the A/Vietnam/1194/04 clade 1 virus was progressively mutated into the HA sequence of the clade 2.2 virus, A/Bar-headed Goose/Qinghai/1A/05. Fine mapping of clade-specific neutralising epitopes was performed by examining the cross-reactivity of mAbs raised against the native HA of each parent virus. The reactivity across all clade specific mAbs centred around a constellation of mutations at positions 140, 145, 171 and 172, all of which are proximal to the receptor binding site on the membrane distal globular head of the HA. Overlapping cross-reactivity of these antigenic sites suggests that these amino acid positions relate to the antigenic evolution of the H5 clade 1 and 2.2 viruses. This finding may prove useful for the design of vaccines with broader neutralising cross-reactivity against the different H5 HA sublineages currently in circulation. These findings provide important information about the amino acid changes involved in the cross-clade evolution of H5N1 viruses and their potential for human to human transmission; and facilitates a greater understanding of the pandemic potential of H5N1 isolates.