The metabolic hormone leptin promotes the function of TFH cells and supports vaccine responses.

Follicular helper T (TFH) cells control antibody responses by supporting antibody affinity maturation and memory formation. Inadequate TFH function has been found in individuals with ineffective responses to vaccines, but the mechanism underlying TFH regulation in vaccination is not understood. Here, we report that lower serum levels of the metabolic hormone leptin associate with reduced vaccine responses to influenza or hepatitis B virus vaccines in healthy populations. Leptin promotes mouse and human TFH differentiation and IL-21 production via STAT3 and mTOR pathways. Leptin receptor deficiency impairs TFH generation and antibody responses in immunisation and infection. Similarly, leptin deficiency induced by fasting reduces influenza vaccination-mediated protection for the subsequent infection challenge, which is mostly rescued by leptin replacement. Our results identify leptin as a regulator of TFH cell differentiation and function and indicate low levels of leptin as a risk factor for vaccine failure.

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.

Cold adaptation generates mutations associated with the growth of influenza B vaccine viruses.

Seasonal inactivated influenza vaccines are usually trivalent or quadrivalent and are prepared from accredited seed viruses. Yields of influenza A seed viruses can be enhanced by gene reassortment with high-yielding donor strains, but similar approaches for influenza B seed viruses have been largely unsuccessful. For vaccine manufacture influenza B seed viruses are usually adapted for high-growth by serial passage. Influenza B antigen yields so obtained are often unpredictable and selection of influenza B seed viruses by this method can be a rate-limiting step in seasonal influenza vaccine manufacture. We recently have shown that selection of stable cold-adapted mutants from seasonal epidemic influenza B viruses is associated with improved growth. In this study, specific mutations were identified that were responsible for growth enhancement as a consequence of adaptation to growth at lower temperatures. Molecular analysis revealed that the following mutations in the HA, NP and NA genes are required for enhanced viral growth: G156/N160 in the HA, E253, G375 in the NP and T146 in the NA genes. These results demonstrate that the growth of seasonal influenza B viruses can be optimized or improved significantly by specific gene modifications.

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.

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.

Rapid generation of pandemic influenza virus vaccine candidate strains using synthetic DNA.

BACKGROUND: Vaccination is considered the most effective means of reducing influenza burden. The emergence of H5N1 and pandemic spread of novel H1N1/2009 viruses reinforces the need to have strategies in place to rapidly develop seed viruses for vaccine manufacture. METHODS: Candidate pandemic vaccine strains consisting of the circulating strain haemagglutinin (HA) and neuraminidase (NA) in an A/PR/8/34 backbone were generated using alternative synthetic DNA approaches, including site-directed mutagenesis of DNA encoding related virus strains, and rapid generation of virus using synthetic DNA cloned into plasmid vectors. RESULTS: Firstly, synthetic A/Bar Headed Goose/Qinghai/1A/2005 (H5N1) virus was generated from an A/Vietnam/1194/2004 template using site-directed mutagenesis. Secondly, A/Whooper Swan/Mongolia/244/2005 (H5N1) and A/California/04/09 (H1N1) viruses were generated using synthetic DNA encoding the viral HA and NA genes. Replication and antigenicity of the synthetic viruses were comparable to that of the corresponding non-synthetic viruses. CONCLUSIONS: In the event of an influenza pandemic, the use of these approaches may significantly reduce the time required to generate and distribute the vaccine seed virus and vaccine manufacture. These approaches also offer the advantage of not needing to handle wild-type virus, potentially diminishing biocontainment requirements.

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.

The development of vaccine viruses against pandemic A(H1N1) influenza.

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

The utility of ISCOMATRIX adjuvant for dose reduction of antigen for vaccines requiring antibody responses.

The capacity of an adjuvant to reduce the amount of antigen required in vaccines would be beneficial in a variety of settings, including situations where antigen is difficult or expensive to manufacture, or in situations where demand exceeds production capacity, such as pandemic influenza. The ability to reduce antigen dose would also be a significant advantage in combination vaccines, and vaccines that by necessity must contain multiple antigens to accommodate variability between strains or genotypes. ISCOMATRIX adjuvant was compared to aluminium hydroxide adjuvant (Al(OH3)) for induction of antibody responses and dose sparing of a recombinant HIV gp120 vaccine. Neutralising antibody responses were significantly greater, at the same protein dose, when the gp120 protein was formulated with ISCOMATRIX adjuvant compared to Al(OH3). Moreover, strong responses were achieved with up to 100-fold lower doses of gp120 using ISCOMATRIX adjuvant. Therefore, ISCOMATRIX adjuvant has the potential to substantially reduce the dose of antigen required in human vaccines, without compromising the immune response.