Immunization with V987H-stabilized Spike glycoprotein protects K18-hACE2 mice and golden Syrian hamsters upon SARS-CoV-2 infection.

Safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are crucial to fight against the coronavirus disease 2019 pandemic. Most vaccines are based on a mutated version of the Spike glycoprotein [K986P/V987P (S-2P)] with improved stability, yield and immunogenicity. However, S-2P is still produced at low levels. Here, we describe the V987H mutation that increases by two-fold the production of the recombinant Spike and the exposure of the receptor binding domain (RBD). S-V987H immunogenicity is similar to S-2P in mice and golden Syrian hamsters (GSH), and superior to a monomeric RBD. S-V987H immunization confer full protection against severe disease in K18-hACE2 mice and GSH upon SARS-CoV-2 challenge (D614G or B.1.351 variants). Furthermore, S-V987H immunized K18-hACE2 mice show a faster tissue viral clearance than RBD- or S-2P-vaccinated animals challenged with D614G, B.1.351 or Omicron BQ1.1 variants. Thus, S-V987H protein might be considered for future SARS-CoV-2 vaccines development.

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.

A monoclonal antibody targeting a large surface of the receptor binding motif shows pan-neutralizing SARS-CoV-2 activity.

Here we report the characterization of 17T2, a SARS-CoV-2 pan-neutralizing human monoclonal antibody isolated from a COVID-19 convalescent individual infected during the first pandemic wave. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA+ memory B cell, with a broad neutralizing activity against former and new SARS-CoV-2 variants, including XBB.1.16 and BA.2.86 Omicron subvariants. Consistently, 17T2 demonstrates in vivo prophylactic and therapeutic activity against Omicron BA.1.1 infection in K18-hACE2 mice. Cryo-electron microscopy reconstruction shows that 17T2 binds the BA.1 spike with the RBD in "up" position and blocks the receptor binding motif, as other structurally similar antibodies do, including S2E12. Yet, unlike S2E12, 17T2 retains its neutralizing activity against all variants tested, probably due to a larger RBD contact area. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 as a potential candidate for future clinical interventions.

Immunisation efficacy of a stabilised SARS-CoV-2 spike glycoprotein in two geriatric animal models.

Age is associated with reduced efficacy of vaccines and linked to higher risk of severe COVID-19. Here we determined the impact of ageing on the efficacy of a SARS-CoV-2 vaccine based on a stabilised Spike glycoprotein (S-29) that had previously shown high efficacy in young animals. Thirteen to 18-month-old golden Syrian hamsters (GSH) and 22-23-month-old K18-hCAE2 mice were immunised twice with S-29 protein in AddaVaxTM adjuvant. GSH were intranasally inoculated with SARS-CoV-2 either two weeks or four months after the booster dose, while all K18-hACE2 mice were intranasally inoculated two weeks after the second immunisation. Body weight and clinical signs were recorded daily post-inoculation. Lesions and viral load were investigated in different target tissues. Immunisation induced seroconversion and production of neutralising antibodies; however, animals were only partially protected from weight loss. We observed a significant reduction in the amount of viral RNA and a faster viral protein clearance in the tissues of immunized animals. Infectious particles showed a faster decay in vaccinated animals while tissue lesion development was not altered. In GSH, the shortest interval between immunisation and inoculation reduced RNA levels in the lungs, while the longest interval was equally effective in reducing RNA in nasal turbinates; viral nucleoprotein amount decreased in both tissues. In mice, immunisation was able to improve the survival of infected animals. Despite the high protection shown in young animals, S-29 efficacy was reduced in the geriatric population. Our research highlights the importance of testing vaccine efficacy in older animals as part of preclinical vaccine evaluation.

Exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endangered Iberian lynx (Lynx pardinus).

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging zoonotic virus of public and animal health concern, of which felids have been suggested as potential reservoirs. Although SARS-CoV-2 exposure has been detected in domestic and wild captive animals belonging to Felidae family, surveillance has not been carried out in free-ranging wild felids so far. The aim of the present study was to assess SARS-CoV-2 exposure in the Iberian lynx (Lynx pardinus), the most endangered felid in the world. Between 2019 and 2022, we conducted a seroepidemiological study of SARS-CoV-2 in 276 free-ranging and captive Iberian lynxes. Our results evidenced limited (0.4%; 95%CI: 0.0-1.1) but not negligible exposure to this emerging virus in this endangered felid species, increasing the SARS-CoV-2 host range. The circulation of this virus in wildlife evidences the need of integrated European wildlife monitoring.

Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant.

Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production and immunogenicity of the recombinant S trimer, suggesting that these two parameters are related. Nevertheless, S-2P still shows some molecular instability and it is produced with low yield. Here we described a novel set of mutations identified by molecular modeling and located in the S2 region of the S-2P that increase its production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development.

Middle East respiratory coronavirus (MERS-CoV) internalized by llama alveolar macrophages does not result in virus replication or induction of pro-inflammatory cytokines.

Severe Middle East respiratory syndrome (MERS) is characterized by massive infiltration of immune cells in lungs. MERS-coronavirus (MERS-CoV) replicates in vitro in human macrophages, inducing high pro-inflammatory responses. In contrast, camelids, the main reservoir for MERS-CoV, are asymptomatic carriers. Although limited infiltration of leukocytes has been observed in the lower respiratory tract of camelids, their role during infection remains unknown. Here we studied whether llama alveolar macrophages (LAMs) are susceptible to MERS-CoV infection and can elicit pro-inflammatory responses. MERS-CoV did not replicate in LAMs; however, they effectively capture and degrade viral particles. Moreover, transcriptomic analyses showed that LAMs do not induce pro-inflammatory cytokines upon MERS-CoV sensing.

Transmission of severe acute respiratory syndrome coronavirus 2 from humans to animals: is there a risk of novel reservoirs?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic virus able to infect humans and multiple nonhuman animal species. Most natural infections in companion, captive zoo, livestock, and wildlife species have been related to a reverse transmission, raising concern about potential generation of animal reservoirs due to human-animal interactions. To date, American mink and white-tailed deer are the only species that led to extensive intraspecies transmission of SARS-CoV-2 after reverse zoonosis, leading to an efficient spread of the virus and subsequent animal-to-human transmission. Viral host adaptations increase the probability of new SARS-CoV-2 variants' emergence that could cause a major global health impact. Therefore, applying the One Health approach is crucial to prevent and overcome future threats for human, animal, and environmental fields.

Quantification of camelid cytokine mRNA expression in PBMCs by microfluidic qPCR technology.

Camelids are economically and socially important in several parts of the world and might carry pathogens with epizootic or zoonotic potential. However, biological research in these species is limited due to lack of reagents. Here, we developed RT-qPCR assays to quantify a panel of camelid innate and adaptive immune response genes, which can be monitored in a single run. The assays were validated with PHA, PMA-ionomycin, and Poly I:C-stimulated PBMCs from alpaca, dromedary camel and llama, including normalization by multiple reference genes. Further, comparative gene expression analyses for the different camelid species were performed by a unique microfluidic qPCR assay. Compared to unstimulated controls, PHA and PMA-ionomycin stimulation elicited robust Th1 and Th2 responses in PBMCs from camelid species. Additional activation of type I and type III IFN signalling pathways was described exclusively in PHA-stimulated dromedary lymphocytes, in contrast to those from alpaca and llama. We also found that PolyI:C stimulation induced robust antiviral response genes in alpaca PBMCs. The proposed methodology should be useful for the measurement of immune responses to infection or vaccination in camelid species.

Animal models to study the neurological manifestations of the post-COVID-19 condition.

More than 40% of individuals infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have experienced persistent or relapsing multi-systemic symptoms months after the onset of coronavirus disease 2019 (COVID-19). This post-COVID-19 condition (PCC) has debilitating effects on the daily life of patients and encompasses a broad spectrum of neurological and neuropsychiatric symptoms including olfactory and gustative impairment, difficulty with concentration and short-term memory, sleep disorders and depression. Animal models have been instrumental to understand acute COVID-19 and validate prophylactic and therapeutic interventions. Similarly, studies post-viral clearance in hamsters, mice and nonhuman primates inoculated with SARS-CoV-2 have been useful to unveil some of the aspects of PCC. Transcriptomic alterations in the central nervous system, persistent activation of immune cells and impaired hippocampal neurogenesis seem to have a critical role in the neurological manifestations observed in animal models infected with SARS-CoV-2. Interestingly, the proinflammatory transcriptomic profile observed in the central nervous system of SARS-CoV-2-inoculated mice partially overlaps with the pathological changes that affect microglia in humans during Alzheimer's disease and aging, suggesting shared mechanisms between these conditions. None of the currently available animal models fully replicates PCC in humans; therefore, multiple models, together with the fine-tuning of experimental conditions, will probably be needed to understand the mechanisms of PCC neurological symptoms. Moreover, given that the intrinsic characteristics of the new variants of concern and the immunological status of individuals might influence PCC manifestations, more studies are needed to explore the role of these factors and their combinations in PCC, adding further complexity to the design of experimental models.

Enhanced antiviral immunity and dampened inflammation in llama lymph nodes upon MERS-CoV sensing: bridging innate and adaptive cellular immune responses in camelid reservoirs.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection can cause fatal pulmonary inflammatory disease in humans. Contrarily, camelids and bats are the main reservoir hosts, tolerant for MERS-CoV replication without suffering clinical disease. Here, we isolated cervical lymph node (LN) cells from MERS-CoV convalescent llamas and pulsed them with two different viral strains (clades B and C). Viral replication was not supported in LN, but a cellular immune response was mounted. Reminiscent Th1 responses (IFN-γ, IL-2, IL-12) were elicited upon MERS-CoV sensing, accompanied by a marked and transient peak of antiviral responses (type I IFNs, IFN-λ3, ISGs, PRRs and TFs). Importantly, expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-8) or inflammasome components (NLRP3, CASP1, PYCARD) was dampened. The role of IFN-λ3 to counterbalance inflammatory processes and bridge innate and adaptive immune responses in camelid species is discussed. Our findings shed light into key mechanisms on how reservoir species control MERS-CoV in the absence of clinical disease.

Preclinical evaluation of a COVID-19 vaccine candidate based on a recombinant RBD fusion heterodimer of SARS-CoV-2.

Current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease, and a decrease in mortality rates. However, SARS-CoV-2 variants are continuously evolving, and development of new accessible COVID-19 vaccines is essential to mitigate the pandemic. Here, we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine (PHH-1V) consisting of an RBD fusion heterodimer comprising the B.1.351 and B.1.1.7 SARS-CoV-2 variants formulated in SQBA adjuvant, an oil-in-water emulsion. A prime-boost immunisation with PHH-1V in BALB/c and K18-hACE2 mice induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralizing activity against several variants, and also showed a good tolerability profile. Significantly, RBD fusion heterodimer vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice, but also reducing Beta, Delta and Omicron infection in lower respiratory airways. These findings demonstrate the feasibility of this recombinant vaccine strategy.

Extended Viral Shedding of MERS-CoV Clade B Virus in Llamas Compared with African Clade C Strain.

Middle East respiratory syndrome coronavirus (MERS-CoV) clade B viruses are found in camelids and humans in the Middle East, but clade C viruses are not. We provide experimental evidence for extended shedding of MERS-CoV clade B viruses in llamas, which might explain why they outcompete clade C strains in the Arabian Peninsula.

Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model.

Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture.

Agreement and differential use of laboratory methods for the detection and quantification of SARS-CoV-2 in experimentally infected animals.

Rodents are widely used for the development of COVID-19-like animal models, the virological outcome being determined through several laboratory methods reported in the literature. Our objective was to assess the agreement between methods performed on different sample types from 342 rodents experimentally infected with SARS-CoV-2 (289 golden Syrian hamsters and 53 K18-hACE2 mice). Our results showed moderate agreement between methods detecting active viral replication, and that increasing viral loads determined by either RT-qPCR or infectious viral titration corresponded to increasing immunohistochemical scores. The percentage of agreement between methods decreased over experimental time points, and we observed poor agreement between RT-qPCR results and viral titration from oropharyngeal swabs. In conclusion, RT-qPCR and viral titration on tissue homogenates are the most reliable techniques to determine the presence and replication of SARS-CoV-2 in the early and peak phases of infection, and immunohistochemistry is valuable to evaluate viral distribution patterns in the infected tissues.

Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals.

Mass vaccination campaigns reduced COVID-19 incidence and severity. Here, we evaluated the immune responses developed in SARS-CoV-2-uninfected patients with predominantly antibody-deficiencies (PAD) after three mRNA-1273 vaccine doses. PAD patients were classified based on their immunodeficiency: unclassified primary antibody-deficiency (unPAD, n = 9), common variable immunodeficiency (CVID, n = 12), combined immunodeficiency (CID, n = 1), and thymoma with immunodeficiency (TID, n = 1). unPAD patients and healthy controls (HCs, n = 10) developed similar vaccine-induced humoral responses after two doses. However, CVID patients showed reduced binding and neutralizing titers compared to HCs. Of interest, these PAD groups showed lower levels of Spike-specific IFN-γ-producing cells. CVID individuals also presented diminished CD8+T cells. CID and TID patients developed cellular but not humoral responses. Although the third vaccine dose boosted humoral responses in most PAD patients, it had limited effect on expanding cellular immunity. Vaccine-induced immune responses in PAD individuals are heterogeneous, and should be immunomonitored to define a personalized therapeutic strategies.

Heterologous Systemic Prime-Intranasal Boosting Using a Spore SARS-CoV-2 Vaccine Confers Mucosal Immunity and Cross-Reactive Antibodies in Mice as well as Protection in Hamsters.

Background: Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are administered systemically and typically result in poor immunogenicity at the mucosa. As a result, vaccination is unable to reduce viral shedding and transmission, ultimately failing to prevent infection. One possible solution is that of boosting a systemic vaccine via the nasal route resulting in mucosal immunity. Here, we have evaluated the potential of bacterial spores as an intranasal boost. Method: Spores engineered to express SARS-CoV-2 antigens were administered as an intranasal boost following a prime with either recombinant Spike protein or the Oxford AZD1222 vaccine. Results: In mice, intranasal boosting following a prime of either Spike or vaccine produced antigen-specific sIgA at the mucosa together with the increased production of Th1 and Th2 cytokines. In a hamster model of infection, the clinical and virological outcomes resulting from a SARS-CoV-2 challenge were ameliorated. Wuhan-specific sIgA were shown to cross-react with Omicron antigens, suggesting that this strategy might offer protection against SARS-CoV-2 variants of concern. Conclusions: Despite being a genetically modified organism, the spore vaccine platform is attractive since it offers biological containment, the rapid and cost-efficient production of vaccines together with heat stability. As such, employed in a heterologous systemic prime-mucosal boost regimen, spore vaccines might have utility for current and future emerging diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and humoral responses against different variants of concern in domestic pet animals and stray cats from North-Eastern Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic in humans, is able to infect several domestic, captive and wildlife animal species. Since reverse zoonotic transmission to pets has been demonstrated, it is crucial to determine their role in the epidemiology of the disease to prevent further spillover events and major spread of SARS-CoV-2. In the present study, we determined the presence of virus and the seroprevalence to SARS-CoV-2, as well as the levels of neutralizing antibodies (nAbs) against several variants of concern (VOCs) in pets (cats, dogs and ferrets) and stray cats from North-Eastern of Spain. We confirmed that cats and dogs can be infected by different VOCs of SARS-CoV-2 and, together with ferrets, are able to develop nAbs against the ancestral (B.1), Alpha (B.1.1.7), Beta (B.1.315), Delta (B.1.617.2) and Omicron (BA.1) variants, with lower titres against the latest in dogs and cats, but not in ferrets. Although the prevalence of active SARS-CoV-2 infection measured as direct viral RNA detection was low (0.3%), presence of nAbs in pets living in COVID-19-positive households was relatively high (close to 25% in cats, 10% in dogs and 40% in ferrets). It is essential to continue monitoring SARS-CoV-2 infections in these animals due to their frequent contact with human populations, and we cannot discard the probability of a higher animal susceptibility to new potential SARS-CoV-2 VOCs.

Evaluation of alpaca tracheal explants as an ex vivo model for the study of Middle East respiratory syndrome coronavirus (MERS-CoV) infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to public health. Here, we established an ex vivo alpaca tracheal explant (ATE) model using an air-liquid interface culture system to gain insights into MERS-CoV infection in the camelid lower respiratory tract. ATE can be infected by MERS-CoV, being 103 TCID50/mL the minimum viral dosage required to establish a productive infection. IFNs and antiviral ISGs were not induced in ATE cultures in response to MERS-CoV infection, strongly suggesting that ISGs expression observed in vivo is rather a consequence of the IFN induction occurring in the nasal mucosa of camelids.

Susceptibility of Domestic Goat (Capra aegagrus hircus) to Experimental Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) B.1.351/Beta Variant.

A wide range of animal species are susceptible to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Natural and/or experimental infections have been reported in pet, zoo, farmed and wild animals. Interestingly, some SARS-CoV-2 variants, such as B.1.1.7/Alpha, B.1.351/Beta, and B.1.1.529/Omicron, were demonstrated to infect some animal species not susceptible to classical viral variants. The present study aimed to elucidate if goats (Capra aegagrus hircus) are susceptible to the B.1.351/Beta variant. First, an in silico approach was used to predict the affinity between the receptor-binding domain of the spike protein of SARS-CoV-2 B.1.351/Beta variant and angiotensin-converting enzyme 2 from goats. Moreover, we performed an experimental inoculation with this variant in domestic goat and showed evidence of infection. SARS-CoV-2 was detected in nasal swabs and tissues by RT-qPCR and/or immunohistochemistry, and seroneutralisation was confirmed via ELISA and live virus neutralisation assays. However, the viral amount and tissue distribution suggest a low susceptibility of goats to the B.1.351/Beta variant. Therefore, although monitoring livestock is advisable, it is unlikely that goats play a role as SARS-CoV-2 reservoir species, and they are not useful surrogates to study SARS-CoV-2 infection in farmed animals.