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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

The magnitude of SARS-CoV-2–specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

BACKGROUND: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined. STUDY DESIGN AND METHODS: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay. RESULTS: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization. CONCLUSION: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity.

Clostridium difficile causes nearly 500,000 infections and nearly 30,000 deaths each year in the U.S., which is estimated to cost $4.8 billion. C. difficile infection (CDI) arises from bacteria colonizing the large intestine and releasing two toxins, toxin A (TcdA) and toxin B (TcdB). Generating humoral immunity against C. difficile&rsquo, s toxins provides protection against primary infection and recurrence. Thus, a vaccine may offer the best opportunity for sustained, long-term protection. We developed a novel single-cycle adenovirus (SC-Ad) vaccine against C. difficile expressing the receptor-binding domains from TcdA and TcdB. The single immunization of mice generated sustained toxin-binding antibody responses and protected them from lethal toxin challenge for up to 38 weeks. Immunized Syrian hamsters produced significant toxin-neutralizing antibodies that increased over 36 weeks. Single intramuscular immunization provided complete protection against lethal BI/NAP1/027 spore challenge 45 weeks later. These data suggest that this replicating vaccine may prove useful against CDI in humans.

Most infections occur at mucosal surfaces. Providing a barrier of protection at these surfaces may be a useful strategy to combat the earliest events in infection when there are relatively few pathogens to address. The majority of vaccines are delivered systemically by the intramuscular (IM) route. While IM vaccination can drive mucosal immune responses, mucosal immunization at intranasal (IN) or oral sites can lead to better immune responses at mucosal sites of viral entry. In macaques, IN immunization with replicating single-cycle adenovirus (SC-Ads) and protein boosts generated favorable mucosal immune responses. However, there was an apparent &ldquo, distance effect&rdquo, in generating mucosal immune responses. IN immunization generated antibodies against HIV envelope (env) nearby in the saliva, but weaker responses in samples collected from the distant vaginal samples. To improve on this, we tested here if SC-Ads expressing genetic adjuvants could be used to amplify antibody responses in distant vaginal samples when they are codelivered with SC-Ads expressing clade C HIV env immunogen. SC-Ads env 1157 was coadministered with SC-Ads expressing 4-1BBL, granulocyte macrophage colony-stimulating factor (GMCSF), IL-21, or Clostridoides difficile (C. diff.) toxin fragments by IN or IM routes. These data show that vaginal antibody responses were markedly amplified after a single immunization by the IN or IM routes, with SC-Ad expressing HIV env if this vaccine is complemented with SC-Ads expressing genetic adjuvants. Furthermore, the site and combination of adjuvants appear to &ldquo, tune&rdquo, these antibody responses towards an IgA or IgG isotype bias. Boosting these priming SC-Ad responses with another SC-Ad or with SOSIP native-like env proteins markedly amplifies env antibody levels in vaginal washes. Together, this data may be useful in informing the choice of route of delivery adenovirus and peptide vaccines against HIV-1.

Recent West African Ebola virus (EBOV) epidemics have led to testing different anti-EBOV vaccines, including a replication-defective adenovirus (RD-Ad) vector (ChAd3-EBOV) and an infectious, replication-competent recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (rVSV-EBOV; also known as rVSV-ZEBOV). While RD-Ads elicit protection, when scaled up to human trials, the level of protection may be much lower than that of vaccines containing viruses that can replicate. Although a replication-competent Ad (RC-Ad) vaccine might generate a level of protection approximating that of rVSV, this infectious vector would also risk causing adenovirus disease. We recently described a "single-cycle" adenovirus (SC-Ad) vector that amplifies antigen genes like RC-Ad, but that avoids the risk of adenovirus infection. Here we have tested an SC-Ad6 vector expressing the glycoprotein (GP) from a 2014 EBOV strain in mice, hamsters, and rhesus macaques. We show that SC-Ad6-EBOV GP induces a high level of serum antibodies in all species and mediates significant protection against pseudo-challenge with rVSV-EBOV expressing luciferase in mice and hamsters. These data suggest that SC-Ad6-EBOV GP may be useful during future EBOV outbreaks.

Summary In a recent issue of Cell Host and Microbe, Fiege and colleagues1 report that laboratory mice exposed to pathogens from pet-store mice exhibit impaired humoral immunity to influenza vaccination and display gene expression signatures that more authentically reflect human vaccine responses., In a recent issue of Cell Host and Microbe, Fiege and colleagues1 report that laboratory mice exposed to pathogens from pet-store mice exhibit impaired humoral immunity to influenza vaccination and display gene expression signatures that more authentically reflect human vaccine responses.

Most human immunodeficiency virus type 1 (HIV-1) infections begin at mucosal surfaces. Providing a barrier of protection at these may assist in combating the earliest events in infection. Systemic immunization by intramuscular (i.m.) injection can drive mucosal immune responses, but there are data suggesting that mucosal immunization can better educate these mucosal immune responses. To test this, rhesus macaques were immunized with replicating single-cycle adenovirus (SC-Ad) vaccines expressing clade B HIV-1 gp160 by the intranasal (i.n.) and i.m. routes to compare mucosal and systemic routes of vaccination. SC-Ad vaccines generated significant circulating antibody titers against Env after a single i.m. immunization. Switching the route of second immunization with the same SC-Ad serotype allowed a significant boost in these antibody levels. When these animals were boosted with envelope protein, envelope-binding antibodies were amplified 100-fold, but qualitatively different immune responses were generated. Animals immunized by only the i.m. route had high peripheral T follicular helper (pTfh) cell counts in blood but low Tfh cell counts in lymph nodes. Conversely, animals immunized by the i.n. route had high Tfh cell counts in lymph nodes but low pTfh cell counts in the blood. Animals immunized by only the i.m. route had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity, whereas animals immunized by the mucosal i.n. route had higher ADCC antibody activity. When these Env-immunized animals were challenged rectally with simian-human immunodeficiency virus (SHIV) strain SF162P3 (SHIV(SF162P3)), they all became infected. However, mucosally SC-Ad-immunized animals had lower viral loads in their gastrointestinal tracts. These data suggest that there may be benefits in educating the immune system at mucosal sites during HIV vaccination. IMPORTANCE HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle makes a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

HIV-1 infections occur during sexual contact at mucosal surfaces. Vaccines need to provide mucosal barrier protection and stimulate systemic immune responses to control HIV spread. Most vaccines are delivered by systemic immunization via intramuscular (IM) injection route. While this can drive systemic and mucosal immune responses, there are data show that mucosal immunization may be superior at driving responses at mucosal barriers. To explore this question, we immunized mice with replicating single-cycle adenovirus (SC Ad) vaccines expressing clade B HIV-1 envelope (Env) by intramuscular (IM), intranasal (IN), or intravaginal (IVAG) routes to compare vaccine responses. SC-Ads generated significant antibodies against Env after only a single immunization by the IN route, but not the other routes. These animals were boosted by the same route or by the mucosal IVAG routes. IM and IN primed animals generated strong antibody responses regardless of the boosting route. In contrast, IVAG primed animals failed to generate robust antibodies whether they were boosted by the IVAG or IM routes. These data suggest there may be benefits in first educating the immune system at mucosal sites during HIV vaccination. IN and IM prime-boost were then compared in Syrian hamsters which support SC-Ad DNA replication. In this case, IN immunization again was the only route that generated significant Env antibodies after a single immunization. Following a boost by IN or IM routes, IN primed animals had significantly higher antibody responses than the IM primed animals. Env antibodies could still be detected one year after immunization, but only in animals that received at least one mucosal IN immunization. These data suggest that there is merit in vaccination by mucosal routes.

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction.

We previously selected muscle binding peptides 12.51 and 12.52 from "context-specific" phage display libraries for introduction into adenovirus (Ad) vectors. In this work, these peptides were inserted into the hypervariable region (HVR) 5 loop of the Ad5 hexon protein to display 720 peptides per virions. HVR-12.51 and 12.52 increased transduction of C2C12 cells up to 20-fold when compared to unmodified Ad5. 12.51 increased in vivo muscle transduction 2 to 7-fold over unmodified Ad after intramuscular injection in mice and hamsters. 12.52 did not increase muscle transduction. Notably, insertion of 12.51 into the hexon reduced liver transduction 80-fold when compared to unmodified Ad5 after intravenous injection. Increased muscle transduction in mice translated into increased immune responses after gene-based vaccination. These data suggest there are merits to retargeting and detargeting benefits to modifying the hexons of Ads with peptide ligands.

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro . SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as “needle-free” mucosal vaccines. IMPORTANCE Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors.

Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [C KO ]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The C KO phenotype was dominant: DI-RNAs derived from vac2 with a C KO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection., Author Summary We show in transgenic mouse and human cell culture models that expression of a viral RNA-dependent RNA polymerase (RdRP) profoundly reconfigures mammalian innate antiviral immunity by exposing the normally stringently-sequestered viral RNA copying process to MDA5 detection. In RdRP-transgenic mouse tissues, the mRNAs for critical interferon-stimulated genes (ISGs) are highly elevated, up to 300-fold. Many antiviral ISGs are similarly induced, up to 560-fold, when we express the RdRP in human monocyte and lung epithelial cell lines. The elevated basal ISG profile protects RdRP mice and human cells from viral infection. RdRP mice are robustly healthy with normal longevity despite life-long, constitutive MDA5-mediated innate immune system activation. This work suggests a plausible approach to augmenting innate immunity to block viral pathogenesis.