Intranasal administration of unadjuvanted SARS-CoV-2 spike antigen boosts antigen-specific immune responses induced by parenteral protein subunit vaccine prime in mice and hamsters.

With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines. Herein, we evaluated the immunogenicity of heterologous prime-boost regimens in mice and hamsters based on a parenteral vaccination of the antigen in combination with sulfated lactosylarchaeol (SLA) archaeosomes, a liposome adjuvant comprised of a single semisynthetic archaeal lipid, followed by an intranasally administered unadjuvanted SARS-CoV-2 spike antigen. Intranasal administration of unadjuvanted spike to mice and hamsters increased serum spike-specific IgG titers and spike-neutralizing activity compared with nonboosted animals. Spike-specific IgA responses were also detected in the bronchoalveolar lavage fluid in the lungs of mice that received an intranasal boost. In hamsters, the intranasal boost showed high efficacy against SARS-CoV-2 infection by protecting from body weight loss and reducing viral titers in the lungs and nasal turbinate. Overall, our heterologous intramuscular prime-intranasal boost with SLA-adjuvanted and unadjuvanted spike, respectively, demonstrated the potential of protein subunit formulations to promote antigen-specific systemic and mucosal immune responses.

Effectiveness of VSV vectored SARS-CoV-2 spike when administered through intranasal, intramuscular or a combination of both.

A critical feature of the VSV vector platform is the ability to pseudotype the virus with different glycoproteins from other viruses, thus altering cellular tropism of the recombinant virus. The route of administration is critical in triggering local and systemic immune response and protection. Most of the vaccine platforms used at the forefront are administered by intramuscular injection. However, it is not known at what level ACE2 is expressed on the surface of skeletal muscle cells, which will have a significant impact on the efficiency of a VSV-SARS-CoV-2 spike vaccine to mount a protective immune response when administered intramuscularly. In this study, we investigate the immunogenicity and efficacy of a prime-boost immunization regimen administered intranasally (IN), intramuscularly (IM), or combinations of the two. We determined that the prime-boost combinations of IM followed by IN immunization (IM + IN) or IN followed by IN immunization (IN + IN) exhibited strong spike-specific IgG, IgA and T cell response in vaccinated K18 knock-in mice. Hamsters vaccinated with two doses of VSV expressing SARS-CoV-2 spike, both delivered by IN or IM + IN, showed strong protection against SARS-CoV-2 variants of concern Alpha and Delta. This protection was also observed in aged hamsters. Our study underscores the highly crucial role immunization routes have with the VSV vector platform to elicit a strong and protective immune response.

Corrigendum: Effects of in ovo inoculation of multi-strain lactobacilli on cytokine gene expression and antibody-mediated immune responses in chickens.

[This corrects the article DOI: 10.3389/fvets.2020.00105.].

Corrigendum: In ovo and oral administration of probiotic lactobacilli modulate cell- and antibody-mediated immune responses in newly hatched chicks.

[This corrects the article DOI: 10.3389/fimmu.2021.664387.].

Cross protection to SARS-CoV-2 variants in hamsters with naturally-acquired immunity.

Since SARS-CoV-2 was first reported in late 2019, multiple variations of the original virus have emerged. Each variant harbors accumulations of mutations, particularly within the spike glycoprotein, that are associated with increased viral transmissibility and escape immunity. The different mutations in the spike protein of different variants shape the subsequent antibody and T cell responses, such that exposure to different spike proteins can result in reduced or enhanced responses to heterologous variants further down the line. Globally, people have been exposed and re-exposed to multiple variations of the Ancestral strain, including the five variants of concerns. Studies have shown that the protective immune response of an individual is influenced by which strain or combination of strains they are exposed to. The initial exposure to a specific strain may also shape their subsequent immune patterns and response to later infections with a heterologous virus. Most immunological observations were carried out early during the pandemic when the Ancestral strain was circulating. However, SARS-CoV-2 variants exhibit varying patterns of disease severity, waning immunity, immune evasion and sensitivity to therapeutics. Here we investigated the cross-protection in hamsters previously infected with a variant of concern (VOC) and subsequently re-infected with a heterologous variant. We also determined if cross-protection and immunity were dependent on the specific virus to which the hamster was first exposed. We further profiled the host cytokine response induced by each SARS-CoV-2 variants as well as subsequent to re-infection. A comparative analysis of the three VOCs revealed that Alpha variant was the most pathogenic VOC to emerge. We showed that naturally acquired immunity protected hamsters from subsequent re-infection with heterologous SARS-CoV-2 variant, regardless which variant the animal was first exposed to. Our study supports observations that heterologous infection of different SARS-CoV-2 variants do not exacerbate disease in subsequent re-infections. The continual emergence of new SARS-CoV-2 variants mandates a better understanding of cross-protection and immune imprinting in infected individuals. Such information is essential to guide vaccine strategy and public policy to emerging SARS-CoV-2 VOCs and future novel pandemic coronaviruses.

Multivalent IgM scaffold enhances the therapeutic potential of variant-agnostic ACE2 decoys against SARS-CoV-2.

As immunological selection for escape mutants continues to give rise to future SARS-CoV-2 variants, novel universal therapeutic strategies against ACE2-dependent viruses are needed. Here we present an IgM-based decavalent ACE2 decoy that has variant-agnostic efficacy. In immuno-, pseudovirus, and live virus assays, IgM ACE2 decoy had potency comparable or superior to leading SARS-CoV-2 IgG-based mAb therapeutics evaluated in the clinic, which were variant-sensitive in their potency. We found that increased ACE2 valency translated into increased apparent affinity for spike protein and superior potency in biological assays when decavalent IgM ACE2 was compared to tetravalent, bivalent, and monovalent ACE2 decoys. Furthermore, a single intranasal dose of IgM ACE2 decoy at 1 mg/kg conferred therapeutic benefit against SARS-CoV-2 Delta variant infection in a hamster model. Taken together, this engineered IgM ACE2 decoy represents a SARS-CoV-2 variant-agnostic therapeutic that leverages avidity to drive enhanced target binding, viral neutralization, and in vivo respiratory protection against SARS-CoV-2.

Subcutaneous BCG vaccination protects against streptococcal pneumonia via regulating innate immune responses in the lung.

Bacillus Calmette-Guérin (BCG) still remains the only licensed vaccine for TB and has been shown to provide nonspecific protection against unrelated pathogens. This has been attributed to the ability of BCG to modulate the innate immune system, known as trained innate immunity (TII). Trained innate immunity is associated with innate immune cells being in a hyperresponsive state leading to enhanced host defense against heterologous infections. Both epidemiological evidence and prospective studies demonstrate cutaneous BCG vaccine-induced TII provides enhanced innate protection against heterologous pathogens. Regardless of the extensive progress made thus far, the effect of cutaneous BCG vaccination against heterologous respiratory bacterial infections and the underlying mechanisms still remain unknown. Here, we show that s.c. BCG vaccine-induced TII provides enhanced heterologous innate protection against pulmonary Streptococcus pneumoniae infection. We further demonstrate that this enhanced innate protection is mediated by enhanced neutrophilia in the lung and is independent of centrally trained circulating monocytes. New insight from this study will help design novel effective vaccination strategies against unrelated respiratory bacterial pathogens.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Intranasal immunization with a proteosome-adjuvanted SARS-CoV-2 spike protein-based vaccine is immunogenic and efficacious in mice and hamsters.

With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.

Phenotypic characterization of gamma delta (γδ) T cells in chickens infected with or vaccinated against Marek's disease virus.

Marek's disease (MD) vaccines reduce the incidence of MD but cannot control virus shedding. To develop new vaccines, it is essential to elucidate mechanisms of immunity to Marek's disease virus (MDV) infection. In this regard, gamma delta (γδ) T cells may play a significant role in prevention of viral spread and tumor surveillance. Here we demonstrated that MDV vaccination induced interferon (IFN)-γ+CD8α+ γδ T cells and transforming growth factor (TGF)-ß+ γδ T cells in lungs. γδ T cells from MDV-infected chickens exhibited cytotoxic activity. Importantly, γδ T cells from the vaccinated/challenged group exhibited maximum cytotoxic activity following ex vivo stimulation. These results suggest that MDV vaccines activate effector γδ T cells which may be involved in the development of protective immune responses against MD. Further, it was demonstrated that MDV infection increases the frequency of a subpopulation of γδ T cells expressing membrane-bound TGF-ß in MDV-infected birds.

In ovo co-administration of vitamins (A and D) and probiotic lactobacilli modulates immune responses in broiler chickens.

There is evidence that probiotic lactobacilli, in addition to essential vitamins, such as vitamin A and D, have immunomodulatory properties that enhance immune response of neonatal chickens against infections. The present study evaluated the effects of in ovo administration of retinoic acid (RA), 25-Hydroxyvitamin D3 (VitD), and a lactobacilli cocktail on cytokine gene expression, antibody responses and spleen cell subsets in chickens. RA (90 µmol/egg) and VitD (0.6 µg/egg) were administered in ovo, either alone or in combination with lactobacilli (107 CFU/egg), at embryonic d 18. On d 5 and 10 posthatch, gene expression and cellular composition were analyzed in the bursa of Fabricius and spleen. Birds were immunized on d 14 and 21 posthatch with 2 T-dependent antigens, sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), to assess their antibody responses. Sera were collected from the immunized chickens on d 14, 21, 28, and 35 posthatch. The results demonstrated that lactobacilli treatment increased the number of monocyte/macrophages (KUL01+) and CD3+CD4+ T cells in the spleen, and enhanced serum anti-KLH IgM and IgY on d 14 postprimary immunization (P < 0.05). RA significantly increased serum IgY and IgM titers to KLH and enhanced the expression of interferon (IFN)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-12, IL-13, and transforming growth factor-ß (TGF-ß) in the bursa of Fabricius (P < 0.05). The percentage of CD3+CD8+ T cells, and monocyte/macrophages (KUL01+) was elevated in the spleen as well (P < 0.05). These findings reveal that prehatch administration of RA improves immunocompetency of neonatal chickens by increasing the production of cytokines that regulate innate immunity and through enhancing antibody-mediated response against T-dependent antigens.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

The Regulatory Microenvironment in Feathers of Chickens Infected with Very Virulent Marek's Disease Virus.

Vaccines against Marek's disease can protect chickens against clinical disease; however, infected chickens continue to propagate the Marek's disease virus (MDV) in feather follicles and can shed the virus into the environment. Therefore, the present study investigated if MDV could induce an immunoregulatory microenvironment in feathers of chickens and whether vaccines can overcome the immune evasive mechanisms of MDV. The results showed an abundance of CD4+CD25+ and CD4+ transforming growth factor-beta (TGF-ß)+ T regulatory cells in the feathers of MDV-infected chickens at 21 days post-infection. In contrast, vaccinated chickens had a lower number of regulatory T cells. Furthermore, the expression of TGF-ß and programmed cell death receptor (PD)-1 increased considerably in the feathers of Marek's disease virus-infected chickens. The results of the present study raise the possibility of an immunoregulatory environment in the feather pulp of MDV-infected chickens, which may in turn favor replication of infectious MDV in this tissue. Exploring the evasive strategies employed by MDV will facilitate the development of control measures to prevent viral replication and transmission.

In Ovo and Oral Administration of Probiotic Lactobacilli Modulate Cell- and Antibody-Mediated Immune Responses in Newly Hatched Chicks.

There is some evidence that lactobacilli can strengthen the immune system of chickens. This study evaluated the effects of in ovo and oral administration of a lactobacilli cocktail on cytokine gene expression, antibody-mediated immune responses, and spleen cellularity in chickens. Lactobacilli were administered either in ovo at embryonic day 18, orally at days 1, 7, 14, 21, and 28 post-hatches, or a combination of both in ovo and post-hatch inoculation. On day 5 and 10 post-hatch, spleen and bursa of Fabricius were collected for gene expression and cell composition analysis. On days 14 and 21 post-hatch, birds were immunized with sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), and sera were collected on days 7, 14, and 21 post-primary immunization. Birds that received lactobacilli (107 CFU) via in ovo followed by weekly oral administration showed a greater immune response by enhancing antibody responses, increasing the percentage of CD4+ and CD4+CD25+ T cells in the spleen and upregulating the expression of interferon (IFN)-α, IFN-ß, interleukin (IL)-8, IL-13, and IL-18 in the spleen and expression of IFN-γ, IL-2, IL-6, IL-8, IL-12, and IL-18 in the bursa. These findings suggest that pre-and post-hatch administration of lactobacilli can modulate the immune response in newly hatched chickens.

Effects of administration of probiotic lactobacilli on immunity conferred by the herpesvirus of turkeys vaccine against challenge with a very virulent Marek's disease virus in chickens.

Several vaccines have been used to control Marek's disease (MD) in chickens. However, the emergence of new strains of Marek's disease virus (MDV) imposes a threat to vaccine efficacy. Therefore, the current study was carried out to investigate whether concurrent administration of probiotics with the herpesvirus of turkeys (HVT) vaccine enhances its protective efficacy against MDV infection. In this regard, a cocktail comprised of four Lactobacillus species was administered with HVT to chicken embryos at embryonic day 18 (ED18) and/or from day 1 to day 4 post-hatch. The results revealed that the administration of a probiotic Lactobacillus with HVT at ED18 followed by oral gavage with the same lactobacilli cocktail to newly hatched chicks for the first 4 days post-hatch increased the expression of major histocompatibility complex (MHC) II on macrophages and B cells in spleen and decreased the number of CD4+CD25+ T regulatory cells in the spleen. Subsequently, chicks were infected with MDV. The chickens that received in ovo HVT and lactobacilli or HVT had higher expression of IFN-α at 21dpi in the spleen compared to the chickens that were challenged with MDV. Also, the expression of IFN-ß in cecal tonsils at 10dpi was higher in the groups that received in ovo HVT and lactobacilli and oral lactobacilli compared to the group that received in ovo HVT alone. Moreover, the expression of tumor growth factor (TGF)-ß4 at 4 days post-infection was reduced in the group that received both HVT and probiotics at ED18. Additionally, concurrent probiotics administration reduced tumor incidence by half when compared to HVT vaccine alone indicating enhancing effect of lactobacilli with HVT vaccine on host immune responses. In conclusion, these findings suggest the potential use of probiotic lactobacilli as adjuvants with the HVT vaccine against MDV infection in chickens.

Gut microbiota is associated with protection against Marek's disease virus infection in chickens.

Marek's Disease Virus (MDV) infects chickens via respiratory route and causes lymphomas in internal organs including gastrointestinal tract. MDV infection causes a shift in the gut microbiota composition. However, interactions between the gut microbiota and immune responses against MDV infection are not well understood. Therefore, the current study was performed to understand the effect of the gut microbiota on Marek's disease (MD) pathogenesis. The findings showed that depletion of gut microbiota increased the severity of MD in infected chickens. In addition, an increase in the transcription of interferon (IFN)-α, IFN-ß and IFN-γ in the bursa of Fabricius at 4 days post-infection (dpi) was observed in the gut microbiota depleted chickens. The observations in this study shed more light on the association between the gut microbiota and MDV infection in chickens. More research is needed to explore the mechanisms of involvement of the gut microbiota in immunity against MD in chickens.

Effects of in ovo Inoculation of Multi-Strain Lactobacilli on Cytokine Gene Expression and Antibody-Mediated Immune Responses in Chickens.

This study was conducted to investigate the effects of various doses of a multi-strain lactobacilli mixture (Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus crispatus, and Lactobacillus johnsonii) on the innate and adaptive immune responses in broiler chickens. At embryonic day eighteen, 200 eggs were injected with PBS, or three different doses of a multi-strain lactobacilli mixture (1 × 105, 1 × 106, and 1 × 107 CFU/egg, P1, P2, and P3 respectively) along with a group of negative control. On days 5 and 10 post-hatch, cecal tonsil, bursa of fabricius, and spleen were collected for gene expression and cellular analysis. On days 14 and 21 post-hatch, birds were immunized intramuscularly with both sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH). Serum samples were collected on days 0, 7, 14, and 21 after primary immunization. The results demonstrated that lactobacilli inoculation increased the splenic expression of cytokines, including interferon (IFN) - α, IFN-ß, IFN-γ, interleukin (IL)-8, and IL-12 on day 5 post-hatch compared to the control group (PBS). However, in cecal tonsils, lactobacilli treatment downregulated the expression of IL-6 on day 5 post-hatch and IL-2 and IL-8 on day 10 post-hatch. No significant differences were observed in the expression of cytokine genes in the bursa except for IL-13 which was upregulated in lactobacilli-treated groups P2 and P3 on days 5 and 10 post-hatch. Flow cytometry analysis showed that the percentage of KUL01, CD4+ and CD8+ splenocytes was not affected by treatments. In addition, no significant differences were observed for antibody titers against SRBC. However, lactobacilli treatment (P1, P2, and P3) was found to increase IgM titers on day 21 post-primary immunization compared to controls. Furthermore, in ovo injection of the highest dose of probiotics (1 × 107, P3) increased serum IgG titers against KLH on day 7 post-primary immunization. In conclusion, this study demonstrated that that in ovo administration of lactobacilli can improve antibody-mediated immune responses and differentially modulate cytokine expression in mucosal and systemic lymphoid tissues of chickens.

Innate immune memory of tissue-resident macrophages and trained innate immunity: Re-vamping vaccine concept and strategies.

In the past few years, our understanding of immunological memory has evolved remarkably due to a growing body of new knowledge in innate immune memory and immunity. Immunological memory now encompasses both innate and adaptive immune memory. The hypo-reactive and hyper-reactive types of innate immune memory lead to a suppressed and enhanced innate immune protective outcome, respectively. The latter is also named trained innate immunity (TII). The emerging information on innate immune memory has not only shed new light on the mechanisms of host defense but is also revolutionizing our long-held view of vaccination and vaccine strategies. Our current review will examine recent progress and knowledge gaps in innate immune memory with a focus on tissue-resident Mϕs, particularly lung Mϕs, and their relationship to local antimicrobial innate immunity. We will also discuss the impact of innate immune memory and TII on our understanding of vaccine concept and strategies and the significance of respiratory mucosal route of vaccination against respiratory pathogens.

Characterization of innate responses induced by in ovo administration of encapsulated and free forms of ligands of Toll-like receptor 4 and 21 in chicken embryos.

Toll-like receptors (TLRs) are a family of innate receptors that recognize pathogen-associated molecular patterns, including double-stranded RNA, CpG DNA and lipopolysaccharide (LPS). After interaction with their ligands, TLRs initiate innate responses that are manifested by activating cells and inducing expression of cytokines that help mediate adaptive immune responses. TLR ligands (TLR-Ls) have the potential to be used prophylactically (alone) or as vaccine adjuvants to promote host immunity. Encapsulating TLR-Ls in nanoparticles, such as Poly (d,l-lactic-co-glycolic acid), may prolong responses through sustained release of the ligands. PLGA nanoparticles protect encapsulated TLR-Ls from degradation and extend the half-life of these ligands by reducing their rapid removal from the body. In this study, encapsulated and free forms of LPS and CpG ODN were administered to embryonation day 18 (ED18) chicken embryos. Spleen, lungs and bursa of Fabricius were collected at 6, 18 and 48hour post-stimulation (hps) and cytokine gene expressions were evaluated using quantitative real-time PCR. Results indicate that both the free and encapsulated forms of LPS and CpG ODN induced innate immune responses in ED18 chicken embryos. Innate responses induced in embryos seem similar to those reported in mature chickens. Significant upregulation of cytokine genes generally occurred by 48hps. Further studies are needed to evaluate long term immunomodulatory effects of encapsulated TLR-Ls and their ability to mediate protection against pathogens of young chicks.