Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains. Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza. The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses

Imatinib in patients with severe COVID-19: a randomised, double-blind, placebo-controlled, clinical trial

Background The major complication of COVID-19 is hypoxaemic respiratory failure from capillary leak and alveolar oedema. Experimental and early clinical data suggest that the tyrosine-kinase inhibitor imatinib reverses pulmonary capillary leak.Methods This randomised, double-blind, placebo-controlled, clinical trial was done at 13 academic and non-academic teaching hospitals in the Netherlands. Hospitalised patients (aged >= 18 years) with COVID-19, as confirmed by an RT-PCR test for SARS-CoV-2, requiring supplemental oxygen to maintain a peripheral oxygen saturation of greater than 94% were eligible. Patients were excluded if they had severe pre-existing pulmonary disease, had pre-existing heart failure, had undergone active treatment of a haematological or non-haematological malignancy in the previous 12 months, had cytopenia, or were receiving concomitant treatment with medication known to strongly interact with imatinib. Patients were randomly assigned (1:1) to receive either oral imatinib, given as a loading dose of 800 mg on day 0 followed by 400 mg daily on days 1-9, or placebo. Randomisation was done with a computer-based clinical data management platform with variable block sizes (containing two, four, or six patients), stratified by study site. The primary outcome was time to discontinuation of mechanical ventilation and supplemental oxygen for more than 48 consecutive hours, while being alive during a 28-day period. Secondary outcomes included safety, mortality at 28 days, and the need for invasive mechanical ventilation. All efficacy and safety analyses were done in all randomised patients who had received at least one dose of study medication (modified intention-to-treat population). This study is registered with the EU Clinical Trials Register (EudraCT 2020-001236-10).Findings Between March 31, 2020, and Jan 4, 2021, 805 patients were screened, of whom 400 were eligible and randomly assigned to the imatinib group (n=204) or the placebo group (n=196). A total of 385 (96%) patients (median age 64 years [IQR 56-73]) received at least one dose of study medication and were included in the modified intention-to-treat population. Time to discontinuation of ventilation and supplemental oxygen for more than 48 h was not significantly different between the two groups (unadjusted hazard ratio [HR] 0.95 [95% CI 0.76-1.20]). At day 28, 15 (8%) of 197 patients had died in the imatinib group compared with 27 (14%) of 188 patients in the placebo group (unadjusted HR 0.51 [0.27-0.95]). After adjusting for baseline imbalances between the two groups (sex, obesity, diabetes, and cardiovascular disease) the HR for mortality was 0.52 (95% CI 0.26-1.05). The HR for mechanical ventilation in the imatinib group compared with the placebo group was 1.07 (0.63-1.80; p=0.81). The median duration of invasive mechanical ventilation was 7 days (IQR 3-13) in the imatinib group compared with 12 days (6-20) in the placebo group (p=0.0080). 91 (46%) of 197 patients in the imatinib group and 82 (44%) of 188 patients in the placebo group had at least one grade 3 or higher adverse event. The safety evaluation revealed no imatinib-associated adverse events.Interpretation The study failed to meet its primary outcome, as imatinib did not reduce the time to discontinuation of ventilation and supplemental oxygen for more than 48 consecutive hours in patients with COVID-19 requiring supplemental oxygen. The observed effects on survival (although attenuated after adjustment for baseline imbalances) and duration of mechanical ventilation suggest that imatinib might confer clinical benefit in hospitalised patients with COVID-19, but further studies are required to validate these findings. Copyright (C) 2021 Elsevier Ltd. All rights reserved.Pathogenesis and treatment of chronic pulmonary disease

Functional Constraints of Influenza A Virus Epitopes Limit Escape from Cytotoxic T Lymphocytes

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes

Vaccination against Seasonal Influenza A/H3N2 Virus Reduces the Induction of Heterosubtypic Immunity against Influenza A/H5N1 Virus Infection in Ferrets ▿ †

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza

Induction of serum antibodies against IAV HK/68 (H3N2) by vaccination.

<p>Serum antibody levels were determined before and at the indicated time points after vaccination of mice with PBS (groups 1, 3 and 4; ○), subunit vaccine with alum (groups 2 and 5; ▴), subunit vaccine only (group 6; ▪) and alum only (group 7; ×) by HI assay (A) and VN assay (B).</p

Outcome of infection with IAV IND/05 (H5N1).

<p>Mice were inoculated with IAV IND/05 (groups 2 (▴), 3 (○), 4 (▿), 5 (⋄), 6 (▪) and 7 (×)) or PBS (group 1 (). (A) Body weight after infection was determined daily and expressed as the percentage of the original body weight before infection. (B) Survival rates after infection with IAV IND/05. The proportion of mice from the indicated groups that survived infection is shown in a Kaplan-Meier plot. Moribund animals were euthanized when they reached pre-fixed criteria regarding weight loss (>20%) and disease severity score, which was used to determine mortality rates. (C) Lung virus titers measured on 7 days p.i. in mice from the indicated groups. Horizontal bars represent the average of 2–6 mice. The difference in virus titers between mice of group 2 and group 3 was statistically significant (p = 0.025). N.S.: not significant. (D) Virus-specific CD8+ T cell responses on day 7 p.i.. The frequency of CD3+ CD8+ splenocytes specific for peptide NP_366–374 and PA_224–233 derived from IAV IND/05 was determined by intracellular IFN-γ staining. The horizontal bars represent the average frequency of IFN-γ+ cells in the CD8+ T cell population of 2–7 mice in the indicated groups. Differences between group 2 and group 3 were statistically significant for both peptides.</p

Outcome of infection with IAV HK/68 (H3N2).

<p>Mice were inoculated with IAV HK/68 (groups 2 (▴), 3 (○), 6 (▪) and 7 (×)) or PBS (groups 1 (), 4 (▿) and 5 (⋄)). (A) Body weight after infection was determined daily and expressed as the percentage of the original body weight before infection. (B) Lung virus titers measured on day 4 p.i. in mice from the indicated experimental groups. Horizontal bars represent the average titers of five mice. The dotted line represents the cut-off value for obtaining a positive result. *This mouse from group 6 had before infection an HI antibody titer of 40. (C) Vaccination prevented the induction of iBALT after infection. Twenty-eight days post infection with IAV HK/68 iBALT was detected in mice from group 3, but not in mice from group 2. Lung tissue sections were stained with HE. (D) Virus-specific CD8+ T cell responses detected 28 days post infection. Splenocytes of mice from the indicated experimental groups were tested for the presence of CD8+ T cells that bound the H2-Db NP_HK Tetramer. Horizontal bars represent the average of 2–4 mice. The difference in %CD8+ Tm+ T cells between groups 2 and 3 was statistically significant (<i>P</i> = 0.030).</p

Experimental groups and design of the study.

<p>Mice were divided over seven groups and were either vaccinated twice with subunit vaccine with or without adjuvant (Alum), PBS, or adjuvant only as indicated. Four weeks after the second vaccination, mice were infected with IAV HK/68 (H3N2) or mock-infected. Twenty-nine days after the infection with IAV HK/68, mice were challenged with IAV IND/05 (H5N1).</p

Histopathological analysis and immunohistochemistry of the lungs of mice infected with IAV IND/05.

<p>Mouse lung sections were stained for influenza A virus nucleoprotein. Cytoplasm of infected cells stain red, the nuclei of infected cells stain deep red. In the groups without a history of productive A/H3N2 infection, including group 2 (A,B), infection with IAV IND/05 led to severe histopathological changes and to viral antigen expression in cells of the bronchiolar walls and in the alveoli (group 4: E,F and group 5: G,H). In mice of groups 3 (C,D) and 7 (I,J) that had experienced a productive infection with IAV HK/68 only moderate histopathological changes were observed and virus infected cells were detected sporadically (see insert in panel D). For more information please see text.</p