IgGκ Signal Peptide Enhances the Efficacy of an Influenza Vector Vaccine against Respiratory Syncytial Virus Infection in Mice.

Intranasal vaccination using influenza vectors is a promising approach to developing vaccines against respiratory pathogens due to the activation of the mucosa-associated immune response. However, there is no clear evidence of a vector design that could be considered preferable. To find the optimal structure of an influenza vector with a modified NS genomic segment, we constructed four vector expressing identical transgene sequences inherited from the F protein of the respiratory syncytial virus (RSV). Two vectors were designed aiming at transgene accumulation in the cytosol. Another two were supplemented with an IgGκ signal peptide prior to the transgene for its extracellular delivery. Surprisingly, adding the IgGκ substantially enhanced the T-cell immune response to the CD8 epitope of the transgene. Moreover, this strategy allowed us to obtain a better protection of mice from the RSV challenge after a single intranasal immunization. Protection was achieved without antibodies, mediated by a balanced T-cell immune response including the formation of the RSV specific effector CD8+ IFNγ+/IL10+-producing cells and the accumulation of Treg cells preventing immunopathology in the lungs of infected mice. In addition to the presented method for optimizing the influenza vector, our results highlight the possibility of achieving protection against RSV through a respiratory-associated T-cell immune response alone.

Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses.

Respiratory syncytial virus (RSV) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. There is an urgent need to develop a safe and efficacious vaccine against RSV that would induce immunological memory without causing immunopathology following natural RSV infection. We have previously generated two recombinant live attenuated influenza vaccine (LAIV) viruses that encode immunodominant T-cell epitopes of RSV M2 protein in the neuraminidase or NS1 genes. These chimeric vaccines afforded protection against influenza and RSV infection in mice, without causing pulmonary eosinophilia or inflammatory RSV disease. The current study assessed the formation of influenza-specific and RSV-specific CD4 and CD8 T-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory T cell subsets (TRM). The RSV epitopes did not affect influenza-specific CD4 effector memory T cell (Tem) levels in the lungs. The majority of these cells formed by LAIV or LAIV-RSV viruses had CD69+CD103- phenotype. Both LAIV+NA/RSV and LAIV+NS/RSV recombinant viruses induced significant levels of RSV M282 epitope-specific lung-localized CD8 Tem cells expressing both CD69 and CD103 TRM markers. Surprisingly, the CD69+CD103+ influenza-specific CD8 Tem responses were augmented by the addition of RSV epitopes, possibly as a result of the local microenvironment formed by the RSV-specific memory T cells differentiating to TRM in the lungs of mice immunized with LAIV-RSV chimeric viruses. This study provides evidence that LAIV vector-based vaccination can induce robust lung-localized T-cell immunity to the inserted T-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself.

Therapeutic efficacy of arginine-rich exenatide on diabetic neuropathy in rats.

Diabetes mellitus is characterized by metabolic dysregulation associated with a number of health complications. More than 50% of patients with diabetes mellitus suffer from diabetic polyneuropathy, which involves the presence of peripheral nerve dysfunction symptoms. The aim of this study was to evaluate the potential of a new synthetic arginine-rich exendin-4 (Peptide D) in the treatment of complications caused by diabetes, including peripheral neuropathy, in rats. Diabetes was induced by administering streptozotocin (STZ). Three groups of diabetic rats were treated with Peptide D (0.1, 1, and 10 µg/kg). One group of diabetic rats was treated with Byetta® (1 µg/kg) for 80 days. Neuropathic pain development was assessed by tactile allodynia. STZ-treated rats showed an increased level of tactile allodynia unlike naïve animals. A histological study revealed that the diameter of the sciatic nerve fibers in STZ-treated rats was smaller than that of the naïve animals. An IHC study demonstrated decreased expression of myelin basic protein (MBP) in the sciatic nerve of diabetic rats compared to that in the naïve animals. Peptide D reduced the severity of tactile allodynia. This effect was more pronounced in the Peptide D treated groups than in the group treated with Byetta®. Peptide D and Byetta® treatment resulted in increased MBP expression in the sciatic nerve and increased diameter of myelinated nerve fibers. These findings suggest that poly-arginine peptides are promising agents for the treatment of peripheral polyneuropathies.

Sequential Immunization with Universal Live Attenuated Influenza Vaccine Candidates Protects Ferrets against a High-Dose Heterologous Virus Challenge.

The development of universal influenza vaccines has been a priority for more than 20 years. We conducted a preclinical study in ferrets of two sets of live attenuated influenza vaccines (LAIVs) expressing chimeric hemagglutinin (cHA). These vaccines contained the HA stalk domain from H1N1pdm09 virus but had antigenically unrelated globular head domains from avian influenza viruses H5N1, H8N4 and H9N2. The viral nucleoproteins (NPs) in the two sets of universal LAIV candidates were from different sources: one LAIV set contained NP from A/Leningrad/17 master donor virus (MDV), while in the other set this gene was from wild-type (WT) H1N1pdm09 virus, in order to better match the CD8 T-cell epitopes of currently circulating influenza A viruses. To avoid any difference in protective effect of the various anti-neuraminidase (NA) antibodies, all LAIVs were engineered to contain the NA gene of Len/17 MDV. Naïve ferrets were sequentially immunized with three doses of (i) classical LAIVs containing non-chimeric HA and NP from MDV (LAIVs (NP-MDV)); (ii) cHA-based LAIVs containing NP from MDV (cHA LAIVs (NP-MDV)); and (iii) cHA-based LAIVs containing NP from H1N1pdm09 virus (cHA LAIVs (NP-WT)). All vaccination regimens were safe, producing no significant increase in body temperature or weight loss, in comparison with the placebo group. The two groups of cHA-based vaccines induced a broadly reactive HA stalk-directed antibody, while classical LAIVs did not. A high-dose challenge with H1N1pdm09 virus induced significant pathology in the control, non-immunized ferrets, including high virus titers in respiratory tissues, clinical signs of disease and histopathological changes in nasal turbinates and lung tissues. All three vaccination regimens protected animals from clinical manifestations of disease: immunized ferrets did not lose weight or show clinical symptoms, and their fever was significantly lower than in the control group. Further analysis of virological and pathological data revealed the following hierarchy in the cross-protective efficacy of the vaccines: cHA LAIVs (NP-WT) > cHA LAIVs (NP-MDV) > LAIVs (NP-MDV). This ferret study showed that prototype universal cHA-based LAIVs are highly promising candidates for further clinical development.

Rationale for vaccination with trivalent or quadrivalent live attenuated influenza vaccines: Protective vaccine efficacy in the ferret model.

BACKGROUND AND AIM: The majority of seasonal influenza vaccines are trivalent, containing two A virus strains (H1N1 and H3N2) and one B virus strain. The co-circulation of two distinct lineages of B viruses can lead to mismatch between the influenza B virus strain recommended for the trivalent seasonal vaccine and the circulating B virus. This has led some manufacturers to produce quadrivalent influenza vaccines containing one strain from each B lineage in addition to H1N1 and H3N2 strains. However, it is also important to know whether vaccines containing a single influenza B strain can provide cross-protectivity against viruses of the antigenically distinct lineage. The aim of this study was to assess in naïve ferrets the potential cross-protective activity of trivalent live attenuated influenza vaccine (T-LAIV) against challenge with a heterologous wild-type influenza B virus belonging to the genetically different lineage and to compare this activity with effectiveness of quadrivalent LAIV (Q-LAIV) in the ferret model. METHODS AND RESULTS: Ferrets were vaccinated with either one dose of trivalent LAIV containing B/Victoria or B/Yamagata lineage virus, or quadrivalent LAIV (containing both B lineages), or placebo. They were then challenged with B/Victoria or B/Yamagata lineage wild-type virus 28 days after vaccination. The ferrets were monitored for clinical signs and morbidity. Nasal swabs and lung tissue samples were analyzed for the presence of challenge virus. Antibody response to vaccination was assessed by routine hemagglutination inhibition assay. All LAIVs tested were found to be safe and effective against wild-type influenza B viruses based on clinical signs, and virological and histological data. The absence of interference between vaccine strains in trivalent and quadrivalent vaccine formulations was confirmed. Trivalent LAIVs were shown to have the potential to be cross-protective against infection with genetically different influenza B/Victoria and B/Yamagata lineages. CONCLUSIONS: In this ferret model, quadrivalent vaccine provided higher protection to challenge against both B/Victoria and B/Yamagata lineage viruses. However, T-LAIV provided some cross-protection in the case of a mismatch between circulating and vaccine type B strains. Notably, B/Victoria-based T-LAIV was more protective compared to B/Yamagata-based T-LAIV.

Two Live Attenuated Vaccines against Recent Low⁻and Highly Pathogenic H7N9 Influenza Viruses Are Safe and Immunogenic in Ferrets.

Influenza H7N9 virus is a potentially pandemic subtype to which most people are immunologically naïve. To be better prepared for the potential occurrence of an H7N9 pandemic, in 2017 the World Health Organization recommended developing candidate vaccine viruses from two new H7N9 viruses, A/Guangdong/17SF003/2016 (A/GD) and A/Hong Kong/125/2017 (A/HK). This report describes the development of live attenuated influenza vaccine (LAIV) candidates against A/GD and A/HK viruses and study of their safety and immunogenicity in the ferret model in order to choose the most promising one for a phase I clinical trial. The A/HK-based vaccine candidate (A/17/HK) was developed by classical reassortment in eggs. The A/GD-based vaccine candidate (A/17/GD) was generated by reverse genetics. Ferrets were vaccinated with two doses of LAIV or phosphate-buffered saline. Both H7N9 LAIVs tested were safe for ferrets, as shown by absence of clinical signs, and by virological and histological data; they were immunogenic after a single vaccination. These results provide a compelling argument for further testing of these vaccines in volunteers. Since the A/HK virus represents the cluster that has caused the majority of human cases, and because the A/HK-based LAIV candidate was developed by classical reassortment, this is the preferred candidate for a phase I clinical trial.