Breakthrough infections by SARS-CoV-2 variants boost cross-reactive hybrid immune responses in mRNA-vaccinated Golden Syrian hamsters.

Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.

Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different in vitro and in vivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced in vitro and in vivo by the decrease of proinflammatory cytokines.

Ally, adversary, or arbitrator? The context-dependent role of eosinophils in vaccination for respiratory viruses and subsequent breakthrough infections.

Eosinophils are a critical type of immune cell and central players in type 2 immunity. Existing literature suggests that eosinophils also can play a role in host antiviral responses, typically type 1 immune events, against multiple respiratory viruses, both directly through release of antiviral mediators and indirectly through activation of other effector cell types. One way to prime host immune responses toward effective antiviral responses is through vaccination, where typically a type 1-skewed immunity is desirable in the context of intracellular pathogens like respiratory viruses. In the realm of breakthrough respiratory viral infection in vaccinated hosts, an event in which virus can still establish productive infection despite preexisting immunity, eosinophils are most prominently known for their link to vaccine-associated enhanced respiratory disease upon natural respiratory syncytial virus infection. This was observed in a pediatric cohort during the 1960s following vaccination with formalin-inactivated respiratory syncytial virus. More recent research has unveiled additional roles of the eosinophil in respiratory viral infection and breakthrough infection. The specific contribution of eosinophils to the quality of vaccine responses, vaccine efficacy, and antiviral responses to infection in vaccinated hosts remains largely unexplored, especially regarding their potential roles in protection. On the basis of current findings, we will speculate upon the suggested function of eosinophils and consider the many potential ways by which eosinophils may exert protective and pathological effects in breakthrough infections. We will also discuss how to balance vaccine efficacy with eosinophil-related risks, as well as the use of eosinophils and their products as potential biomarkers of vaccine efficacy or adverse events.

Induction of protective immune responses at respiratory mucosal sites.

Many pathogens enter the host through mucosal sites. Thus, interfering with pathogen entry through local neutralization at mucosal sites therefore is an effective strategy for preventing disease. Mucosally administered vaccines have the potential to induce protective immune responses at mucosal sites. This manuscript delves into some of the latest developments in mucosal vaccination, particularly focusing on advancements in adjuvant technologies and the role of these adjuvants in enhancing vaccine efficacy against respiratory pathogens. It highlights the anatomical and immunological complexities of the respiratory mucosal immune system, emphasizing the significance of mucosal secretory IgA and tissue-resident memory T cells in local immune responses. We further discuss the differences between immune responses induced through traditional parenteral vaccination approaches vs. mucosal administration strategies, and explore the protective advantages offered by immunization through mucosal routes.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in QIV vaccinated mice.

Adjuvants can enhance vaccine effectiveness of currently licensed influenza vaccines. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus derived defective interfering (SDI) RNA, a RIG-I agonist, and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), TLR7/8 adjuvant. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating the direct delivery of a RIG-I agonist to the cytosol. We have previously tested SDI and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated in a licensed vaccine setting (quadrivalent influenza vaccine or QIV) against H1N1 influenza virus, showing robust induction of antibody titres and T cell responses. Depending on the adjuvant combination and LNP lipid composition (K-Ac7-Dsa or S-Ac7-Dog lipids), humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with protection during viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was examined against challenge with the vaccine-matching strain of H1N1 influenza A virus. Groups that received either LNP formulated with SDI, IMDQ-PEG-Chol or both showed very low levels of viral replication in their lungs at five days post virus infection. LNP ionizable lipid composition as well as loading (empty versus SDI) also skewed host responses to infection, as reflected in the cytokine and chemokine levels in lungs of vaccinated animals upon infection. These studies show the potential of LNPs as adjuvant delivery vehicles for licensed vaccines and illustrate the importance of LNP composition for subsequent host responses to infection, an important point of consideration for vaccine safety.

Enhanced mucosal B- and T-cell responses against SARS-CoV-2 after heterologous intramuscular mRNA prime/intranasal protein boost vaccination with a combination adjuvant.

Current COVID-19 mRNA vaccines delivered intramuscularly (IM) induce effective systemic immunity, but with suboptimal immunity at mucosal sites, limiting their ability to impart sterilizing immunity. There is strong interest in rerouting immune responses induced in the periphery by parenteral vaccination to the portal entry site of respiratory viruses, such as SARS-CoV-2, by mucosal vaccination. We previously demonstrated the combination adjuvant, NE/IVT, consisting of a nanoemulsion (NE) and an RNA-based RIG-I agonist (IVT) induces potent systemic and mucosal immune responses in protein-based SARS-CoV-2 vaccines administered intranasally (IN). Herein, we demonstrate priming IM with mRNA followed by heterologous IN boosting with NE/IVT adjuvanted recombinant antigen induces strong mucosal and systemic antibody responses and enhances antigen-specific T cell responses in mucosa-draining lymph nodes compared to IM/IM and IN/IN prime/boost regimens. While all regimens induced cross-neutralizing antibodies against divergent variants and sterilizing immunity in the lungs of challenged mice, mucosal vaccination, either as homologous prime/boost or heterologous IN boost after IM mRNA prime was required to impart sterilizing immunity in the upper respiratory tract. Our data demonstrate the benefit of hybrid regimens whereby strong immune responses primed via IM vaccination are rerouted by IN vaccination to mucosal sites to provide optimal protection to SARS-CoV-2.

Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity.

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

Portable UV-C Device to Treat High Flow of Infectious Aerosols Generated during Clinical Respiratory Care.

Respiratory interventions including noninvasive ventilation, continuous positive airway pressure and high-flow nasal oxygen generated infectious aerosols may increase risk of airborne disease (SARS-CoV-2, influenza virus) transmission to healthcare workers. We developed/tested a prototype portable UV-C254 device to sterilize high flows of viral-contaminated air from a simulated patient source at airflow rates of up to 100 l/m. Our device consisted of a central quartz tube surrounded 6 high-output UV-C254 lamps, within a larger cylinder allowing recirculation past the UV-C254 lamps a second time before exiting the device. Testing was with nebulized A/PR/8/34 (H1N1) influenza virus. RNA extraction and qRT-PCR showed virus transited through the prototype. Turning on varying numbers of lamps controlled the dose of UVC. Viability experiments at low, medium and high (100 l/min) flows of contaminated gas were conducted with 6, 4, 2 and 1 lamp activated (single-pass and recirculation were tested). Our data show 5-log reduction in particle forming units from a single lamp (single- pass and recirculated conditions) at high and low flows. UVC dose at 100 l/m was calculated at 11.6 mJ/cm2 single pass and 104 mJ/cm2 recirculated. The protype device shows high efficacy in killing nebulized influenza virus in a high flow of contaminated air.

Intranasal SARS-CoV-2 Omicron variant vaccines elicit humoral and cellular mucosal immunity in female mice.

BACKGROUND: In order to prevent the emergence and spread of future variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing vaccines capable of stopping transmission is crucial. The SARS-CoV-2 vaccine NDV-HXP-S can be administered live intranasally (IN) and thus induce protective immunity in the upper respiratory tract. The vaccine is based on Newcastle disease virus (NDV) expressing a stabilised SARS-CoV-2 spike protein. NDV-HXP-S can be produced as influenza virus vaccine at low cost in embryonated chicken eggs. METHODS: The NDV-HXP-S vaccine was genetically engineered to match the Omicron variants of concern (VOC) BA.1 and BA.5 and tested as an IN two or three dose vaccination regimen in female mice. Furthermore, female mice intramuscularly (IM) vaccinated with mRNA-lipid nanoparticles (LNPs) were IN boosted with NDV-HXP-S. Systemic humoral immunity, memory T cell responses in the lungs and spleens as well as immunoglobulin A (IgA) responses in distinct mucosal tissues were characterised. FINDINGS: NDV-HXP-S Omicron variant vaccines elicited high mucosal IgA and serum IgG titers against respective SARS-CoV-2 VOC in female mice following IN administration and protected against challenge from matched variants. Additionally, antigen-specific memory B cells and local T cell responses in the lungs were induced. Host immunity against the NDV vector did not interfere with boosting. Intramuscular vaccination with mRNA-LNPs was enhanced by IN NDV-HXP-S boosting resulting in improvement of serum neutralization titers and induction of mucosal immunity. INTERPRETATION: We demonstrate that NDV-HXP-S Omicron variant vaccines utilised for primary immunizations or boosting efficiently elicit humoral and cellular immunity. The described induction of systemic and mucosal immunity has the potential to reduce infection and transmission. FUNDING: This work was partially funded by the NIAIDCenters of Excellence for Influenza Research and Response (CEIRR) and by the NIAID Collaborative Vaccine Innovation Centers and by institutional funding from the Icahn School of Medicine at Mount Sinai. See under Acknowledgements for details.

The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection.

SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.