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

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

Intradermal Administration of Influenza Vaccine with Trehalose and Pullulan-Based Dissolving Microneedle Arrays

Most influenza vaccines are administered via intramuscular injection which has several disadvantages that might jeopardize the compliance of vaccinees. Intradermal administration of dissolving-microneedle-arrays (dMNAs) could serve as minimal invasive alternative to needle injections. However, during the production process of dMNAs antigens are subjected to several stresses, which may reduce their potency. Moreover, the needles need to have sufficient mechanical strength to penetrate the skin and subsequently dissolve effectively to release the incorporated antigen. Here, we investigated whether blends of trehalose and pullulan are suitable for the production of stable dMNA fulfilling these criteria. Our results demonstrate that production of trehalose/pullulan-based dMNAs rendered microneedles that were sharp and stiff enough to pierce into ex vivo human skin and subsequently dissolve within 15 min. The mechanical properties of the dMNAs were maintained well even after four weeks of storage at temperatures up to 37°C. In addition, immunization of mice with influenza antigens via both freshly prepared dMNAs and dMNAs after storage (four weeks at 4°C or 37°C) resulted in antibody titers of similar magnitude as found in intramuscularly injected mice and partially protected mice from influenza virus infection. Altogether, our results demonstrate the potential of trehalose/pullulan-based dMNAs as alternative dosage form for influenza vaccination.Drug Delivery Technolog

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

Intranasal Delivery of Influenza Subunit Vaccine Formulated with GEM Particles as an Adjuvant

Nasal administration of influenza vaccine has the potential to facilitate influenza control and prevention. However, when administered intranasally (i.n.), commercially available inactivated vaccines only generate systemic and mucosal immune responses if strong adjuvants are used, which are often associated with safety problems. We describe the successful use of a safe adjuvant Gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis for i.n. vaccination with subunit influenza vaccine in mice. It is shown that simple admixing of the vaccine with the GEM particles results in a strongly enhanced immune response. Already after one booster, the i.n. delivered GEM subunit vaccine resulted in hemagglutination inhibition titers in serum at a level equal to the conventional intramuscular (i.m.) route. Moreover, i.n. immunization with GEM subunit vaccine elicited superior mucosal and Th1 skewed immune responses compared to those induced by i.m. and i.n. administered subunit vaccine alone. In conclusion, GEM particles act as a potent adjuvant for i.n. influenza immunization

Superior Immunogenicity of Inactivated Whole Virus H5N1 Influenza Vaccine is Primarily Controlled by Toll-like Receptor Signalling

In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic

Towards an oral influenza vaccine:Comparison between intragastric and intracolonic delivery of influenza subunit vaccine in a murine model

In this paper we investigated to which part of the gastro-intestinal (GI) tract, the upper or Lower part, an oral influenza vaccine should be targeted to result in an effective immune response in mice. Our study demonstrates that without adjuvant substantial systemic but tow respiratory mucosal immune responses were induced in mice after delivery of influenza subunit vaccine to the upper GI-tract (intragastric) as well as the lower GI-tract (intracolonically). When the vaccine was adjuvanted with Escherichia coli heat-labile enterotoxin (LT) these responses were significantly enhanced. Interestingly, intracolonic administration of vaccine with adjuvant also resulted in enhanced cellular immune responses and the desired Th1-skewing of these responses. Intragastric administration of the adjuvanted vaccine also increased T-helper responses. However, Th1-skewing was absent. In conclusion, the right combination of strong mucosal adjuvant (e.g. LT) and antigen delivery site (e.g. the lower part of the gastro-intestinal tract) might result in effective vaccination via the oral route. (c) 2007 Elsevier Ltd. All rights reserved

Virosomes in vaccine development:Induction of cytotoxic T lymphocyte activity with virosome-encapsulated protein antigens

Virosomes are reconstituted viral envelopes which lack the genetic material but retain the cell entry and membrane fusion characteristics of the virus they are derived from. Thus, influenza virosomes are taken up by cells via receptor-mediated endocytosis, which directs the particles to the endosomal cell compartment. Subsequently, the virosomal membrane fuses with the endosomal membrane induced by the mildly acidic pH within the endosomes. This fusion process establishes continuity between the lumen of the virosome and the cell cytosol. Upon interaction of virosomes with antigen-presenting cells (APCs), protein antigens encapsulated within virosomes will be delivered to the cell cytosol, and thus, into the MHC class I presentation pathway. Indeed, virosome-mediated delivery of antigens in vivo results in efficient priming of a class I MHC-restricted cytotoxic T lymphocyte (CTL) response

Rational design of an influenza subunit vaccine powder with sugar glass technology:preventing conformational changes of haemagglutinin during freezing and freeze-drying

The development of a stable influenza subunit vaccine in the dry state was investigated. The influence of various carbohydrates, buffer types and freezing rates on the integrity of haemagglutinin after freeze-thawing or freeze-drying was investigated with a range of analytical and immunological methods. The use of fast freezing, Hepes buffer and carbohydrates (trehalose, inulin or dextran) as cryo- and lyoprotectants resulted in a significant reduction or even absence of conformational changes of HA as revealed by the used methods. The subunit vaccine in the powder was shown to remain immunogenic in an in vivo study in mice, using reconstituted powder. Moreover, the HA potency of the influenza subunit vaccine powder was stable for at least 26 weeks at room temperature

Incorporation of LpxL1, a detoxified lipopolysaccharide adjuvant, in influenza H5N1 virosomes increases vaccine immunogenicity

The increasing number of human influenza H5N1 infections accentuates the need for the development of H5N1 vaccine candidates to prevent a potential influenza pandemic. The use of adjuvants in such vaccines can contribute significantly to antigen dose-sparing. In this study, we evaluated the capacity of the non toxic Neisseria meningitidis lipopolysaccharide analog LpxL1 to function as an adjuvant for an influenza H5N1 virosomal vaccine. Inactivated influenza H5N1 virus (NIBRG-14) was used to construct virosomes (reconstituted virus envelopes) with LpxL1 incorporated in the virosomal membrane thus combining the influenza hemagglutinin (HA) antigen and the adjuvant in the same particle. Mice were immunized in a one- or two-dose immunization regimen with H5N1 virosomes with or without incorporated LpxL1 After a single immunization, H5N1 virosomes with incorporated LpxL1 induced significantly enhanced H5N1-specific total IgG titers as compared to non-adjuvanted virosomes but hemagglutination inhibition (HI) titers remained low. In the two-dose immunization regimen, LpxL1-modified H5N1 virosomes induced HI titers above 40 which were significantly higher than those obtained with non-adjuvanted virosomes. Incorporation of LpxL1 had little effect on virosome-induced IgG1 levels, but significantly increased IgC2a levels in both the one- and two-close immunization regimen. Compared to non-adjuvanted virosomes, LpxL1-modified virosomes induced similar numbers of IFN gamma-producing T cells but decreased numbers of IL-4-producing T cells irrespective of the number of immunizations. We conclude that LpxL1 incorporated in H5N1 influenza virosomes has the capacity to function as a potent adjuvant particularly Stimulating Th1-type immune reactions. (C) 2008 Elsevier Ltd. All rights reserved