Induction of Heterosubtypic Cross-Protection against Influenza by a Whole Inactivated Virus Vaccine:The Role of Viral Membrane Fusion Activity

BACKGROUND: The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge. CONCLUSION/SIGNIFICANCE: The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity and full immunogenicity of the vaccine

A PBMC-Based System to Assess Human T Cell Responses to Influenza Vaccine Candidates In Vitro

Vaccine development is an expensive and time-consuming process that heavily relies on animal models. Yet, vaccine candidates that have previously succeeded in animal experiments often fail in clinical trials questioning the predictive value of animal models. Alternative assay systems that can add to the screening and evaluation of functional characteristics of vaccines in a human context before embarking on costly clinical trials are therefore urgently needed. In this study, we have established an in vitro system consisting of long-term cultures of unfractionated peripheral blood mononuclear cells (PBMCs) from healthy volunteers to assess (recall) T cell responses to vaccine candidates. We observed that different types of influenza vaccines (whole inactivated virus (WIV), split, and peptide vaccines) were all able to stimulate CD4 and CD8 T cell responses but to different extents in line with their reported in vivo properties. In-depth analyses of different T cell subsets revealed that the tested vaccines evoked mainly recall responses as indicated by the fact that the vast majority of the responding T cells had a memory phenotype. Furthermore, we observed vaccine-induced activation of T follicular helper cells, which are associated with the induction of humoral immune responses. Our results demonstrate the suitability of the established PBMC-based system for the in vitro evaluation of memory T cell responses to vaccines and the comparison of vaccine candidates in a human immune cell context. As such, it can help to bridge the gap between animal experiments and clinical trials and assist in the selection of promising vaccine candidates, at least for recall antigens

Assessing the Immunomodulatory Effect of Size on the Uptake and Immunogenicity of Influenza- and Hepatitis B Subunit Vaccines In Vitro

Viral subunit vaccines are a safer and more tolerable alternative to whole inactivated virus vaccines. However, they often come with limited efficacy, necessitating the use of adjuvants. Using free and particle-bound viral antigens, we assessed whether size affects the uptake of those antigens by human monocyte-derived dendritic cells (Mo-DCs) and whether differences in uptake affect their capacity to stimulate cytokine production by T cells. To this end, influenza antigens and hepatitis B surface antigen (HBsAg) were covalently conjugated to polystyrene particles of 500 nm and 3 μm. Cellular uptake of the antigens, either unconjugated or conjugated, and their capacity to stimulate T cells within a population of human peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry. Conjugation of both antigens to particles significantly increased their uptake by Mo-DCs. Moreover, both the 500 nm and 3 μm influenza conjugates induced significantly higher numbers of cytokine-producing CD4+ T cells and induced increased production of the pro-inflammatory cytokines IFNγ and TNFα. In contrast, conjugation of HBsAg to particles did not notably affect the T cell response. In conclusion, conjugation of antigen to 500 nm and 3 μm particles leads to increased antigen uptake by human Mo-DCs, although the capacity of such conjugates to induce T cell stimulation likely depends on the immunological status of the PBMC donor

Heterosubtypic cross-protection induced by whole inactivated influenza virus vaccine in mice:Influence of the route of vaccine administration

Background: Development of influenza vaccines capable of inducing broad protection against different virus subtypes is necessary given the ever-changing viral genetic landscape. Previously, we showed that vaccination with whole inactivated virus (WIV) induces heterosubtypic protection against lethal virus infection in mice. Whole inactivated virus-induced cross-protection was found to be mediated primarily by flu-specific CD8+ T cells. Objectives: As it has been demonstrated that the route of vaccine administration strongly influences both the quantity and quality of vaccine-induced immunity, in this study, we determined which route of WIV administration induces optimal heterosubtypic cross-protection. Methods: We compared the magnitude of the immune response and heterosubtypic protection against lethal A/PR/8/34 (H1N1) infection after subcutaneous (SC), intramuscular (IM), and intranasal (IN) vaccination with A/NIBRG-14 (H5N1) WIV. Results: Subcutaneous and IM administration was superior to IN administration of influenza WIV in terms of flu-specific CD8+ T-cell induction and protection of mice against lethal heterosubtypic challenge. Surprisingly, despite the very low flu-specific CD8+ T-cell responses detected in IN-vaccinated mice, these animals were partially protected, most likely due to cross-reactive IgA antibodies. Conclusion: The results of this study show that the magnitude of WIV-induced flu-specific CD8+ T-cell activity depends on the applied vaccination route. We conclude that parenteral administration of WIV vaccine, in particular IM injection, is superior to IN vaccine delivery for the induction of heterosubtypic cross-protection and generally appears to elicit stronger immune responses than mucosal vaccination with WIV

Innate responses induced by whole inactivated virus or subunit influenza vaccines in cultured dendritic cells correlate with immune responses in vivo

Vaccine development involves time-consuming and expensive evaluation of candidate vaccines in animal models. As mediators of both innate and adaptive immune responses dendritic cells (DCs) are considered to be highly important for vaccine performance. Here we evaluated how far the response of DCs to a vaccine in vitro is in line with the immune response the vaccine evokes in vivo. To this end, we investigated the response of murine bone marrow-derived DCs to whole inactivated virus (WIV) and subunit (SU) influenza vaccine preparations. These vaccine preparations were chosen because they differ in the immune response they evoke in mice with WIV being superior to SU vaccine through induction of higher virus-neutralizing antibody titers and a more favorable Th1-skewed response phenotype. Stimulation of DCs with WIV, but not SU vaccine, resulted in a cytokine response that was comparable to that of DCs stimulated with live virus. Similarly, the gene expression profiles of DCs treated with WIV or live virus were similar and differed from that of SU vaccine-treated DCs. More specifically, exposure of DCs to WIV resulted in differential expression of genes in known antiviral pathways, whereas SU vaccine did not. The stronger antiviral and more Th1-related response of DCs to WIV as compared to SU vaccine correlates well with the superior immune response found in mice. These results indicate that in vitro stimulation of DCs with novel vaccine candidates combined with the assessment of multiple parameters, including gene signatures, may be a valuable tool for the selection of vaccine candidates

mRNA-1273 COVID-19 vaccination in patients receiving chemotherapy, immunotherapy, or chemoimmunotherapy for solid tumours:a prospective, multicentre, non-inferiority trial

BACKGROUND: Patients with cancer have an increased risk of complications from SARS-CoV-2 infection. Vaccination to prevent COVID-19 is recommended, but data on the immunogenicity and safety of COVID-19 vaccines for patients with solid tumours receiving systemic cancer treatment are scarce. Therefore, we aimed to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine as part of the Vaccination Against COVID in Cancer (VOICE) trial. METHODS: This prospective, multicentre, non-inferiority trial was done across three centres in the Netherlands. Individuals aged 18 years or older with a life expectancy of more than 12 months were enrolled into four cohorts: individuals without cancer (cohort A [control cohort]), and patients with solid tumours, regardless of stage and histology, treated with immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Participants received two mRNA-1273 vaccinations of 100 μg in 0·5 mL intramuscularly, 28 days apart. The primary endpoint, analysed per protocol (excluding patients with a positive baseline sample [>10 binding antibody units (BAU)/mL], indicating previous SARS-CoV-2 infection), was defined as the SARS-CoV-2 spike S1-specific IgG serum antibody response (ie, SARS-CoV-2-binding antibody concentration of >10 BAU/mL) 28 days after the second vaccination. For the primary endpoint analysis, a non-inferiority design with a margin of 10% was used. We also assessed adverse events in all patients who received at least one vaccination, and recorded solicited adverse events in participants who received at least one vaccination but excluding those who already had seroconversion (>10 BAU/mL) at baseline. This study is ongoing and is registered with ClinicalTrials.gov, NCT04715438. FINDINGS: Between Feb 17 and March 12, 2021, 791 participants were enrolled and followed up for a median of 122 days (IQR 118 to 128). A SARS-CoV-2-binding antibody response was found in 240 (100%; 95% CI 98 to 100) of 240 evaluable participants in cohort A, 130 (99%; 96 to >99) of 131 evaluable patients in cohort B, 223 (97%; 94 to 99) of 229 evaluable patients in cohort C, and 143 (100%; 97 to 100) of 143 evaluable patients in cohort D. The SARS-CoV-2-binding antibody response in each patient cohort was non-inferior compared with cohort A. No new safety signals were observed. Grade 3 or worse serious adverse events occurred in no participants in cohort A, three (2%) of 137 patients in cohort B, six (2%) of 244 patients in cohort C, and one (1%) of 163 patients in cohort D, with four events (two of fever, and one each of diarrhoea and febrile neutropenia) potentially related to the vaccination. There were no vaccine-related deaths. INTERPRETATION: Most patients with cancer develop, while receiving chemotherapy, immunotherapy, or both for a solid tumour, an adequate antibody response to vaccination with the mRNA-1273 COVID-19 vaccine. The vaccine is also safe in these patients. The minority of patients with an inadequate response after two vaccinations might benefit from a third vaccination. FUNDING: ZonMw, The Netherlands Organisation for Health Research and Development

Enhanced antiviral activity of human surfactant protein d by site-specific engineering of the carbohydrate recognition domain

Innate immunity is critical in the early containment of influenza A virus (IAV) infection and surfactant protein D (SP-D) plays a crucial role in innate defense against IAV in the lungs. Multivalent lectin-mediated interactions of SP-D with IAVs result in viral aggregation, reduced epithelial infection, and enhanced IAV clearance by phagocytic cells. Previous studies showed that porcine SP-D (pSP-D) exhibits distinct antiviral activity against IAV as compared to human SP-D (hSP-D), mainly due to key residues in the lectin domain of pSP-D that contribute to its profound neutralizing activity. These observations provided the basis for the design of a full-length recombinant mutant form of hSP-D, designated as “improved SP-D” (iSP-D). Inspired by pSP-D, the lectin domain of iSP-D has 5 amino acids replaced (Asp324Asn, Asp330Asn, Val251Glu, Lys287Gln, Glu289Lys) and 3 amino acids inserted (326Gly-Ser-Ser). Characterization of iSP-D revealed no major differences in protein assembly and saccharide binding selectivity as compared to hSP-D. However, hemagglutination inhibition measurements showed that iSP-D expressed strongly enhanced activity compared to hSP-D against 31 different IAV strains tested, including (pandemic) IAVs that were resistant for neutralization by hSP-D. Furthermore, iSP-D showed increased viral aggregation and enhanced protection of MDCK cells against infection by IAV. Importantly, prophylactic or therapeutic application of iSP-D decreased weight loss and reduced viral lung titers in a murine model of IAV infection using a clinical isolate of H1N1pdm09 virus. These studies demonstrate the potential of iSP-D as a novel human-based antiviral inhalation drug that may provide immediate protection against or recovery from respiratory (pandemic) IAV infections in humans