Circulating precursor CCR7(lo)PD-1(hi) 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.

Cross-reactive influenza-specific antibody-dependent cellular cytotoxicity in intravenous immunoglobulin as a potential therapeutic against emerging influenza viruses.

BACKGROUND: Intravenous immunoglobulin (IVIG) is a purified pool of human antibodies from thousands of donors that is used to prevent or treat primary immune deficiency, several infectious diseases, and autoimmune diseases. The antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against heterologous influenza strains may be present in IVIG preparations. METHODS: We tested 8 IVIG preparations prior to the 2009 H1N1 swine-origin influenza pandemic and 10 IVIG preparations made after 2010 for their ability to mediate influenza-specific ADCC. RESULTS: ADCC mediating antibodies to A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) were detected in IVIG preparations prior to the 2009-H1N1 pandemic. The HA-specific ADCC targeted both the HA1 and HA2 regions of A(H1N1)pdm09 HA and was capable of recognizing a broad range of HA proteins including those from recent avian influenza strains A(H5N1) and A(H7N9). The low but detectable ADCC recognition of A(H7N9) was likely due to rare individuals in the population contributing cross-reactive antibodies to IVIG. CONCLUSIONS: IVIG preparations contain broadly cross-reactive ADCC mediating antibodies. IVIG may provide at least some level of protection for individuals at high risk of severe influenza disease, especially during influenza pandemics prior to the development of effective vaccines.

An ELISA-based assay for determining haemagglutinin potency in egg, cell, or recombinant protein derived influenza vaccines.

Background: The current compendial assay for haemagglutinin antigen potency in influenza vaccine is the single radial immunodiffusion (SRID) which is time consuming and can lead to delays in release of vaccine. We previously described an alternate capture and detection enzyme linked immunoassay (ELISA) that utilizes sub-type specific, sub-clade cross-reactive monoclonal antibodies (mAbs) that are haemagglutination inhibiting (HAI) and correlate with SRID. The aim of this study is to determine the applicability of ELISA across current platforms for quantitation of seasonal quadrivalent vaccine. Methods: A single mAb capture and detection ELISA was employed to quantitate hemagglutinin (HA) derived from different vaccine platforms and host organisms and compared to SRID and a polyclonal antibody based ELISA. Results: We selected mAbs that displayed appropriate characteristics for a stability indicating potency assay which reacted to avian, insect and mammalian derived HA. Qualification of the homologous mAb assay against egg and cell derived HA demonstrated performance similar to that of the SRID however, superiority in sensitivity and specificity against strains from both influenza B/Victoria and B/Yamagata lineages. Analysis of drifted strains across multiple seasons demonstrated continued utility of this approach, reducing the need to develop reagents each season. With modification of the assay, we were able to accurately measure HA from different platforms and process stages using a single calibrated reference standard. We demonstrated the accuracy of ELISA when testing vaccine formulations containing selected adjuvants at standard and higher concentrations. Accelerated stability analysis indicated a strong correlation in the rate of degradation between the homologous mAb ELISA and SRID but not with ELISA utilizing polyclonal antisera. Further, we demonstrated specificity was restricted to the trimeric and oligomeric forms of HA but not monomeric HA. Conclusion: We believe this homologous mAb ELISA is a suitable replacement for the SRID compendial assay for HA antigen quantitation and stability assessment. Identification of suitable mAbs that are applicable across multiple vaccine platforms with extended sub-type reactivity across a number of influenza seasons, indicate that this assay has broad applicability, leading to earlier availability of seasonal and pandemic vaccines without frequent replacement of polyclonal antisera that is required with SRID.

Potency Testing of Venoms and Antivenoms in Embryonated Eggs: An Ethical Alternative to Animal Testing.

Venoms are complex mixtures of biologically active molecules that impact multiple physiological systems. Manufacture of antivenoms (AVs) therefore requires potency testing using in vivo models to ensure AV efficacy. As part of ongoing research to replace small animals as the standard model for AV potency testing, we developed an alternate in vivo method using the embryonated egg model (EEM). In this model, the survival of chicken embryos envenomated in ovo is determined prior to 50% gestation, when they are recognized as animals by animal welfare legislation. Embryos were found to be susceptible to a range of snake, spider, and marine venoms. This included funnel-web spider venom for which the only other vertebrate, non-primate animal model is newborn mice. Neutralization of venom with standard AV allowed correlation of AV potency results from the EEM to results from animal assays. Our findings indicate that the EEM provides an alternative, insensate in vivo model for the assessment of AV potency. The EEM may enable reduction or replacement of the use of small animals, as longer-term research that enables the elimination of animal use in potency testing continues.

Intravenous Immunoglobulin Protects Against Severe Pandemic Influenza Infection.

Influenza is a highly contagious, acute, febrile respiratory infection that can have fatal consequences particularly in individuals with chronic illnesses. Sporadic reports suggest that intravenous immunoglobulin (IVIg) may be efficacious in the influenza setting. We investigated the potential of human IVIg to ameliorate influenza infection in ferrets exposed to either the pandemic H1N1/09 virus (pH1N1) or highly pathogenic avian influenza (H5N1). IVIg administered at the time of influenza virus exposure led to a significant reduction in lung viral load following pH1N1 challenge. In the lethal H5N1 model, the majority of animals given IVIg survived challenge in a dose dependent manner. Protection was also afforded by purified F(ab')2 but not Fc fragments derived from IVIg, supporting a specific antibody-mediated mechanism of protection. We conclude that pre-pandemic IVIg can modulate serious influenza infection-associated mortality and morbidity. IVIg could be useful prophylactically in the event of a pandemic to protect vulnerable population groups and in the critical care setting as a first stage intervention.

Development of an enzyme-linked immunoassay for the quantitation of influenza haemagglutinin: an alternative method to single radial immunodiffusion.

BACKGROUND: The current method used to measure haemagglutinin (HA) content for influenza vaccine formulation, single radial immunodiffusion (SRID), is lengthy and relies on the availability of matched standardised homologous reagents. The 2009 influenza pandemic highlighted the need to develop alternate assays that are able to rapidly quantitate HA antigen for vaccine formulation. OBJECTIVES: The aim of this work was to develop an enzyme-linked immunoassay (EIA) for the rapid quantitation of H1, H3, H5 and B influenza HA antigens. METHODS: Monoclonal antibodies (mAbs) selected for haemagglutination inhibition (HAI) activity were conjugated with horseradish peroxidase and used to establish a capture-detection EIA for the quantitation of HA antigen. Results were compared with the appropriate reference SRID assays to investigate assay performance and utility. RESULTS: Quantitation of HA antigen by EIA correlated well with current reference SRID assays. EIA results showed equivalent precision and exhibited a similar capacity to detect HA antigen in virus samples that had been used in either stability or splitting studies, or subjected to physical or chemical stresses. EIA exhibited greater sensitivity than SRID and has the potential to be used in high-throughput applications. CONCLUSIONS: We demonstrated the utility of EIA as a suitable alternative to SRID for HA antigen quantitation and stability assessment. This approach would lead to earlier availability of both seasonal and pandemic vaccines, because of the extended cross-reactivity of reagents.

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.

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.