Enhancing the protection of influenza virus vaccines with BECC TLR4 adjuvant in aged mice

Abstract Influenza A virus (IAV) is a leading cause of respiratory disease worldwide often resulting in severe morbidity and mortality. We have previously shown that the Bacterial Enzymatic Combinatorial Chemistry (BECC) adjuvants, BECC438 and BECC470, formulated with an influenza virus hemagglutinin (HA) protein vaccine, offer greater protection from influenza virus challenge in mouse respiratory models using adult mice than standard HA:adjuvant combinations. In this study, we determined that immunization with HA + BECC adjuvants also significantly broadened the epitopes targeted on HA as compared with other adjuvants, resulting in increased titers of antibodies directed against the highly conserved HA stalk domain. Importantly, we demonstrate that BECC470 combined with an influenza virus HA protein antigen in a prime-only immunization regimen was able to achieve complete protection from challenge in a ~ 12-month-old mouse aged model. Together, this demonstrates the heightened protection provided by the BECC470 adjuvant in an influenza virus vaccine model and shows the enhanced immune response, as compared to other adjuvants elicited by the formulation of HA with BECC470

Differences in HHV-6-specific antibody responses and detection of viral DNA between experimental groups.

<p>(A) Comparison of virus-specific IgM and IgG responses between HHV-6A and HHV-6B-intravenously inoculated (iv) marmosets. AUC is calculated from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003138#ppat-1003138-g003" target="_blank">Figure 3</a>. (B) Significantly elevated virus-specific IgM and IgG responses in marmosets inoculated with HHV-6A intravenously compared to marmosets inoculated with HHV-6A intranasally (p = 0.0286, Mann Whitney U test). AUC is calculated from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003138#ppat-1003138-g003" target="_blank">Figures 3</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003138#ppat-1003138-g005" target="_blank">5</a>. (C, D) The number of marmosets testing positive for viral DNA was significantly greater in the HHV-6A intranasal group compared to the HHV-6A intravenous group (p = 0.003, Mann Whitney U test). AUC in (D) is calculated from (C). <i>AUC: Area under the curve.</i></p

Marmosets inoculated intravenously with HHV-6A exhibited clinical symptoms without weight loss.

<p>Percent weight change is on the left y-axis (dashed line). Clinical score is on the right y-axis (solid line). The scoring system is as follows, 0: no clinical signs, 0.5: apathy or altered walking pattern without ataxia, 1: lethargy or tremor, 2: ataxia or optic disease, 2.25: monoparesis, 2.5: paraparesis or sensory loss, 3: paraplegia or hemiplegia. Arrows represent times of HHV-6A intravenous inoculations.</p

Bilateral hyperintense MRI signal in corpus callosum of M04 (red arrows), inoculated with HHV-6A intravenously.

<p>(a) Baseline, acquired before viral inoculation, (b) 183 days post-inoculation, (c) 194 days post-inoculation, (d) post-mortem scan (433 days post-inoculation).</p

Spinal cord pathology in two HHV-6A intravenously inoculated marmosets.

<p>Iba-1 is specific for microglia and macrophages, Luxol Fast Blue (LFB) stains myelin and Bielschowski's stains neurofibrils. Cervical spinal cord pathology of M03 includes (A) microglial/macrophageal aggregates identified by Iba-1, and swollen myelin sheaths identified by (B) LFB and (C) Bielschowski's. Spinal cord pathology of M04 includes microglial/macrophageal aggregates identified by Iba-1 in the (D) thoracic and (E) lumbar spinal cord and (F) myelin abnormalities identified by LFB in the dorsal root ganglia, specifically variations in sheath size and focal neuronal chromatolysis (black arrow), indicative of mild reversible damage.</p