Optimizing immunogenicity and product presentation of a SARS-CoV-2 subunit vaccine composition: effects of delivery route, heterologous regimens with self-amplifying RNA vaccines, and lyophilization.

Introduction: Dozens of vaccines have been approved or authorized internationally in response to the ongoing SARS-CoV-2 pandemic, covering a range of modalities and routes of delivery. For example, mucosal delivery of vaccines via the intranasal (i.n.

Exploring yeast glucans for vaccine enhancement: Sustainable strategies for overcoming adjuvant challenges in a SARS-CoV-2 model.

Vaccine adjuvants are important for enhancing vaccine efficacy, and although aluminium salts (Alum) are the most used, their limited ability to induce specific immune responses has spurred the search for new adjuvants. However, many adjuvants fail during product development due to manufacturability, supply, stability, or safety concerns. This work hypothesizes that protein-free yeast glucans can be used as vaccine adjuvants due to their known immunostimulatory activity and high abundancy.

Modulation of type I interferon responses potently inhibits SARS-CoV-2 replication and inflammation in rhesus macaques.

Type I interferons (IFN-I) are critical mediators of innate control of viral infections but also drive the recruitment of inflammatory cells to sites of infection, a key feature of severe coronavirus disease 2019. Here, IFN-I signaling was modulated in rhesus macaques (RMs) before and during acute SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection using a mutated IFN-α2 (IFN-modulator; IFNmod), which has previously been shown to reduce the binding and signaling of endogenous IFN-I. IFNmod treatment in uninfected RMs was observed to induce a modest up-regulation of only antiviral IFN-stimulated genes (ISGs); however, in SARS-CoV-2-infected RMs, IFNmod reduced both antiviral and inflammatory ISGs.

A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability.

mRNA vaccines were the first to be authorized for use against SARS-CoV-2 and have since demonstrated high efficacy against serious illness and death. However, limitations in these vaccines have been recognized due to their requirement for cold storage, short durability of protection, and lack of access in low-resource regions. We have developed an easily-manufactured, potent self-amplifying RNA (saRNA) vaccine against SARS-CoV-2 that is stable at room temperature.

Heterologous saRNA Prime, DNA Dual-Antigen Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies.

We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (AAHI-SC2) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression and an N antigen modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the AAHI-SC2 vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to confer protease resistance.

A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery.

Current RNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at -20°C or -70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA.

Live-attenuated RNA hybrid vaccine technology provides single-dose protection against Chikungunya virus.

We present a live-attenuated RNA hybrid vaccine technology that uses an RNA vaccine delivery vehicle to deliver in vitro-transcribed, full-length, live-attenuated viral genomes to the site of vaccination. This technology allows ready manufacturing in a cell-free environment, regardless of viral attenuation level, and it promises to avoid many safety and manufacturing challenges of traditional live-attenuated vaccines. We demonstrate this technology through development and testing of a live-attenuated RNA hybrid vaccine against Chikungunya virus (CHIKV), comprised of an in vitro-transcribed, highly attenuated CHIKV genome delivered by a highly stable nanostructured lipid carrier (NLC) formulation as an intramuscular injection.

Polymorphisms in STING Affect Human Innate Immune Responses to Poxviruses.

We conducted a large genome-wide association study (GWAS) of the immune responses to primary smallpox vaccination in a combined cohort of 1,653 subjects. We did not observe any polymorphisms associated with standard vaccine response outcomes (e.g.

Intramuscular Delivery of Replicon RNA Encoding ZIKV-117 Human Monoclonal Antibody Protects against Zika Virus Infection.

Monoclonal antibody (mAb) therapeutics are an effective modality for the treatment of infectious, autoimmune, and cancer-related diseases. However, the discovery, development, and manufacturing processes are complex, resource-consuming activities that preclude the rapid deployment of mAbs in outbreaks of emerging infectious diseases. Given recent advances in nucleic acid delivery technology, it is now possible to deliver exogenous mRNA encoding mAbs for expression following intravenous (i.

Sex Differences in Older Adults' Immune Responses to Seasonal Influenza Vaccination.

Sex differences in immune responses to influenza vaccine may impact efficacy across populations. In a cohort of 138 older adults (50-74 years old), we measured influenza A/H1N1 antibody titers, B-cell ELISPOT response, PBMC transcriptomics, and PBMC cell compositions at 0, 3, and 28 days post-immunization with the 2010/11 seasonal inactivated influenza vaccine. We identified higher B-cell ELISPOT responses in females than males.

Transcriptomic signatures of cellular and humoral immune responses in older adults after seasonal influenza vaccination identified by data-driven clustering.

PBMC transcriptomes after influenza vaccination contain valuable information about factors affecting vaccine responses. However, distilling meaningful knowledge out of these complex datasets is often difficult and requires advanced data mining algorithms. We investigated the use of the data-driven Weighted Gene Correlation Network Analysis (WGCNA) gene clustering method to identify vaccine response-related genes in PBMC transcriptomic datasets collected from 138 healthy older adults (ages 50-74) before and after 2010-2011 seasonal trivalent influenza vaccination.

Polymorphisms in the Wilms Tumor Gene Are Associated With Interindividual Variations in Rubella Virus-Specific Cellular Immunity After Measles-Mumps-Rubella II Vaccination.

Rubella vaccination induces widely variable immune responses in vaccine recipients. While rubella vaccination is effective at inducing immunity to rubella infection in most subjects, up to 5% of individuals do not achieve or maintain long-term protective immunity. To expand upon our previous work identifying genetic polymorphisms that are associated with these interindividual differences in humoral immunity to rubella virus, we performed a genome-wide association study in a large cohort of 1843 subjects to discover single-nucleotide polymorphisms (SNPs) associated with rubella virus-specific cellular immune responses.

Genetically defined race, but not sex, is associated with higher humoral and cellular immune responses to measles vaccination.

In addition to host genetic and environmental factors, variations in immune responses to vaccination are influenced by demographic variables, such as race and sex. The influence of genetic race and sex on measles vaccine responses is not well understood, yet important for the development of much-needed improved measles vaccines with lower failure rates. We assessed associations between genetically defined race and sex with measles humoral and cellular immunity after measles vaccination in three independent and geographically distinct cohorts totaling 2872 healthy racially diverse children, older adolescents, and young adults.

Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells.

The virus/host interaction is a complex interplay between pro- and anti-viral factors that ultimately determines the spread or halt of virus infections in tissues. This interplay develops over multiple rounds of infection. The purpose of this study was to determine how cellular-level processes combine to impact the spatial spread of infection.

Defending against smallpox: a focus on vaccines.

Smallpox has shaped human history, from the earliest human civilizations well into the 20th century. With high mortality rates, rapid transmission, and serious long-term effects on survivors, smallpox was a much-feared disease. The eradication of smallpox represents an unprecedented medical victory for the lasting benefit of human health and prosperity.

Neucode Labels for Multiplexed, Absolute Protein Quantification.

We describe a new method to accomplish multiplexed, absolute protein quantification in a targeted fashion. The approach draws upon the recently developed neutron encoding (NeuCode) metabolic labeling strategy and parallel reaction monitoring (PRM). Since PRM scanning relies upon high-resolution tandem mass spectra for targeted protein quantification, incorporation of multiple NeuCode labeled peptides permits high levels of multiplexing that can be accessed from high-resolution tandem mass spectra.

Kinetic Differences and Synergistic Antiviral Effects Between Type I and Type III Interferon Signaling Indicate Pathway Independence.

The spread of acute respiratory viral infections is controlled by type I and III interferon (IFN) signaling. While the mechanisms of type I IFN signaling have been studied in detail, features that distinguish type III IFN signaling remain poorly understood. Type III IFNs play an essential role in limiting infections of intestinal and respiratory epithelial surfaces; however, type III IFNs have been shown to activate similar genes to type I IFNs, raising the question of how these IFNs differ and their signals interact.

Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR.

A quantitative infection assay for human type I, II, and III interferon antiviral activities.

Background: Upon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread. These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system.

Results: We have developed an antiviral assay based on replication inhibition of an engineered fluorescent vesicular stomatitis virus reporter strain on A549 human lung epithelial cells.