Convalescent human IgG, but not IgM, from COVID-19 survivors confers dose-dependent protection against SARS-CoV-2 replication and disease in hamsters.

Introduction: Antibody therapeutic strategies have served an important role during the COVID-19 pandemic, even as their effectiveness has waned with the emergence of escape variants. Here we sought to determine the concentration of convalescent immunoglobulin required to protect against disease from SARS-CoV-2 in a Syrian golden hamster model. Methods: Total IgG and IgM were isolated from plasma of SARS-CoV-2 convalescent donors. Dose titrations of IgG and IgM were infused into hamsters 1 day prior to challenge with SARS-CoV-2 Wuhan-1. Results: The IgM preparation was found to have ~25-fold greater neutralization potency than IgG. IgG infusion protected hamsters from disease in a dose-dependent manner, with detectable serum neutralizing titers correlating with protection. Despite a higher in vitro neutralizing potency, IgM failed to protect against disease when transferred into hamsters. Discussion: This study adds to the growing body of literature that demonstrates neutralizing IgG antibodies are important for protection from SARS-CoV-2 disease, and confirms that polyclonal IgG in sera can be an effective preventative strategy if the neutralizing titers are sufficiently high. In the context of new variants, against which existing vaccines or monoclonal antibodies have reduced efficacy, sera from individuals who have recovered from infection with the emerging variant may potentially remain an efficacious tool.

A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50 µg) SpFN vaccine, given twice 28 days apart, induced a Th1-biased CD4 T cell helper response and elicited neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates following high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates supports the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations.

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV-like Sarbecovirus vaccine development.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of other coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2 in mice. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, along with high magnitude neutralizing titers against major VoC. These results provide design strategies for pan-coronavirus vaccine development. HIGHLIGHTS: Iterative structure-based design of four Spike-domain Ferritin nanoparticle classes of immunogensSpFN-ALFQ and RFN-ALFQ immunization elicits potent neutralizing activity against SARS-CoV-2, variants of concern, and SARS-CoV-1Passively transferred IgG from immunized C57BL/6 mice protects K18-hACE2 mice from lethal SARS-CoV-2 challenge.

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques.

Emergence of novel variants of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean neutralizing antibody titers of 14,000-21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within four days in 7 of 8 animals receiving 50 µg RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only ∼2-fold relative to USA-WA1. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-like betacoronavirus vaccine development. SIGNIFICANCE STATEMENT: The emergence of SARS-CoV-2 variants of concern (VOC) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 Spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation, efficacious vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses exceeding those of current vaccines in terms of breadth and potency and protects against high-dose respiratory tract challenge. Neutralization activity against the B.1.351 VOC within two-fold of wild-type virus and against SARS-CoV-1 indicate exceptional breadth. Our results support consideration of RFN for SARS-like betacoronavirus vaccine development.

Efficacy of a Broadly Neutralizing SARS-CoV-2 Ferritin Nanoparticle Vaccine in Nonhuman Primates.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50 µ g) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFN's potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses. ONE-SENTENCE SUMMARY: A SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

Synthetic Host Defense Peptides Inhibit Venezuelan Equine Encephalitis Virus Replication and the Associated Inflammatory Response.

Venezuelan equine encephalitis virus (VEEV), a New World alphavirus of the Togaviridae family of viruses causes periodic outbreaks of disease in humans and equines. Disease following VEEV infection manifests as a febrile illness with flu-like symptoms, which can progress to encephalitis and cause permanent neurological sequelae in a small number of cases. VEEV is classified as a category B select agent due to ease of aerosolization and high retention of infectivity in the aerosol form. Currently, there are no FDA-approved vaccines or therapeutics available to combat VEEV infection. VEEV infection in vivo is characterized by extensive systemic inflammation that can exacerbate infection by potentially increasing the susceptibility of off-site cells to infection and dissemination of the virus. Hence, a therapeutic targeting both the infection and associated inflammation represents an unmet need. We have previously demonstrated that host defense peptides (HDPs), short peptides that are key components of the innate immune response, exhibit antiviral activity against a multitude of viruses including VEEV. In this study, we designed synthetic peptides derived from indolicidin, a naturally occurring HDP, and tested their efficacy against VEEV. Two candidate synthetic peptides inhibited VEEV replication by approximately 1000-fold and decreased the expression of inflammatory mediators such as IL1α, IL1ß, IFNγ, and TNFα at both the gene and protein expression levels. Furthermore, an increase in expression levels of genes involved in chemotaxis of leukocytes and anti-inflammatory genes such as IL1RN was also observed. Overall, we conclude that our synthetic peptides inhibit VEEV replication and the inflammatory burden associated with VEEV infection.

Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKß Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis.

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKß inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKß and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKß kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKß can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

Efficacy of FDA-Approved Anti-Inflammatory Drugs Against Venezuelan Equine Encephalitis Virus Infection.

Venezuelan equine encephalitis virus (VEEV) is a category B select agent pathogen that can be aerosolized. Infections in murine models and humans can advance to an encephalitic phenotype which may result in long-term neurological complications or death. No specific FDA-approved treatments or vaccines are available for the treatment or prevention of VEEV infection. Neurotropic viral infections have two damaging components: neuronal death caused by viral replication, and damage from the subsequent inflammatory response. Reducing the level of inflammation may lessen neurological tissue damage that often arises following VEEV infection. In this study, three commercially available anti-inflammatory drugs, Celecoxib, Rolipram, and Tofacitinib, were evaluated for antiviral activity in an astrocyte and a microglial model of VEEV infection. The inhibitors were tested against the vaccine strain VEEV TC-83, as well as the wild-type VEEV Trinidad donkey strain. Celecoxib, Tofacitinib, and Rolipram significantly decreased viral titers both after pre-treatment and post-treatment of infected cells. VEEV Trinidad Donkey (TrD) titers were reduced 6.45-fold in cells treated with 50 µM of Celecoxib, 2.45-fold when treated with 50 µM of Tofacitinib, and 1.81-fold when treated with 50 µM of Rolipram. Celecoxib was also shown to decrease inflammatory gene expression in the context of TC-83 infection. Overall, Celecoxib demonstrated potency as a countermeasure strategy that slowed VEEV infection and infection-induced inflammation in an in vitro model.

Human Antimicrobial Peptides as Therapeutics for Viral Infections.

Successful in vivo infection following pathogen entry requires the evasion and subversion of multiple immunological barriers. Antimicrobial peptides (AMPs) are one of the first immune pathways upregulated during infection by multiple pathogens, in multiple organs in vivo. In humans, there are many classes of AMPs exhibiting broad antimicrobial activities, with defensins and the human cathelicidin LL-37 being the best studied examples. Whereas historically the efficacy and therapeutic potential of AMPs against bacterial infection has been the primary focus of research, recent studies have begun to elucidate the antiviral properties of AMPs as well as their role in regulation of inflammation and chemoattraction. AMPs as therapeutic tools seem especially promising against emerging infectious viral pathogens for which no approved vaccines or treatments are currently available, such as dengue virus (DENV) and Zika virus (ZIKV). In this review, we summarize recent studies elucidating the efficacy and diverse mechanisms of action of various classes of AMPs against multiple viral pathogens, as well as the potential use of human AMPs in novel antiviral therapeutic strategies.

Human cathelicidin peptide LL-37 as a therapeutic antiviral targeting Venezuelan equine encephalitis virus infections.

Venezuelan equine encephalitis virus (VEEV), a new world alphavirus belonging to the Togaviridae family, causes periodic disease outbreaks in humans and equines with high associated mortality and morbidity. VEEV is highly infectious via the aerosol route and so has been developed as a biological weapon (Hawley and Eitzen, 2001). Despite its current classification as a category B select agent, there are no FDA approved vaccines or therapeutics to counter VEEV infections. Here we utilize a naturally occurring host defense peptide, LL-37, as a therapeutic strategy to inhibit VEEV multiplication in infected cells. LL-37 has previously demonstrated activity against several viruses by directly interacting with viral particles and indirectly by establishing an antiviral state in the host cell. We show that LL-37 exhibited potent antiviral activity against VEEV by inhibiting viral replication. Genomic RNA copies of the TC-83 strain of VEEV and viral titers were significantly reduced compared to non-treated controls. LL-37 also inhibited the virulent Trinidad Donkey (TrD) strain of VEEV. Entry assays revealed a robust reduction of viral RNA copies at the early stages of TC-83 infection. Pre-incubation of cells with LL-37 and TC-83 resulted in a strong inhibitory response, indicating that LL-37 impacts early stages of the infectious process. Confocal and electron microscopy images confirmed the aggregation of viral particles, which potentially accounts for entry prevention and hence reduced viral infection. LL-37 treatment also modulated type I interferon (IFN) expression in infected cells. LL-37 treatment dramatically increased IFNß1 expression in treated cells in a time-dependent manner. Our results establish LL-37 as a relevant and novel potential therapeutic strategy for the treatment of VEEV infections.

Host-based processes as therapeutic targets for Rift Valley fever virus.

Rift Valley fever virus (RVFV) is an enveloped, segmented, negative sense RNA virus that replicates within the host's cytoplasm. To facilitate its replication, RVFV must utilize host cell processes and as such, these processes may serve as potential therapeutic targets. This review summarizes key host cell processes impacted by RVFV infection. Specifically the influence of RVFV on host transcriptional regulation, post-transcriptional regulation, protein half-life and availability, host signal transduction, trafficking and secretory pathways, cytoskeletal modulation, and mitochondrial processes and oxidative stress are discussed. Therapeutics targeted towards host processes that are essential for RVFV to thrive as well as their efficacy and importance to viral pathogenesis are highlighted.