mRNA-LNP vaccine-induced CD8+ T cells protect mice from lethal SARS-CoV-2 infection in the absence of specific antibodies.

The role of CD8+ T cells in SARS-CoV-2 pathogenesis or mRNA-LNP vaccine-induced protection from lethal COVID-19 is unclear. Using mouse-adapted SARS-CoV-2 virus (MA30) in C57BL/6 mice, we show that CD8+ T cells are unnecessary for the intrinsic resistance of female or the susceptibility of male mice to lethal SARS-CoV-2 infection. Also, mice immunized with a di-proline prefusion-stabilized full-length SARS-CoV-2 Spike (S-2P) mRNA-LNP vaccine, which induces Spike-specific antibodies and CD8+ T cells specific for the Spike-derived VNFNFNGL peptide, are protected from SARS-CoV-2 infection-induced lethality and weight loss, while mice vaccinated with mRNA-LNPs encoding only VNFNFNGL are protected from lethality but not weight loss. CD8+ T cell depletion ablates protection in VNFNFNGL but not in S-2P mRNA-LNP-vaccinated mice. Therefore, mRNA-LNP vaccine-induced CD8+ T cells are dispensable when protective antibodies are present but essential for survival in their absence. Hence, vaccine-induced CD8+ T cells may be critical to protect against SARS-CoV-2 variants that mutate epitopes targeted by protective antibodies.

Resistance To Poxvirus Lethality Does Not Require the Necroptosis Proteins RIPK3 or MLKL.

Receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) are proteins that are critical for necroptosis, a mechanism of programmed cell death that is both activated when apoptosis is inhibited and thought to be antiviral. Here, we investigated the role of RIPK3 and MLKL in controlling the Orthopoxvirus ectromelia virus (ECTV), a natural pathogen of the mouse. We found that C57BL/6 (B6) mice deficient in RIPK3 (Ripk3-/-) or MLKL (Mlkl-/-) were as susceptible as wild-type (WT) B6 mice to ECTV lethality after low-dose intraperitoneal infection and were as resistant as WT B6 mice after ECTV infection through the natural footpad route. Additionally, after footpad infection, Mlkl-/- mice, but not Ripk3-/- mice, endured lower viral titers than WT mice in the draining lymph node (dLN) at three days postinfection and in the spleen or in the liver at seven days postinfection. Despite the improved viral control, Mlkl-/- mice did not differ from WT mice in the expression of interferons or interferon-stimulated genes or in the recruitment of natural killer (NK) cells and inflammatory monocytes (iMOs) to the dLN. Additionally, the CD8 T-cell responses in Mlkl-/- and WT mice were similar, even though in the dLNs of Mlkl-/- mice, professional antigen-presenting cells were more heavily infected. Finally, the histopathology in the livers of Mlkl-/- and WT mice at 7 dpi did not differ. Thus, the mechanism of the increased virus control by Mlkl-/- mice remains to be defined. IMPORTANCE The molecules RIPK3 and MLKL are required for necroptotic cell death, which is widely thought of as an antiviral mechanism. Here we show that C57BL/6 (B6) mice deficient in RIPK3 or MLKL are as susceptible as WT B6 mice to ECTV lethality after a low-dose intraperitoneal infection and are as resistant as WT B6 mice after ECTV infection through the natural footpad route. Mice deficient in MLKL are more efficient than WT mice at controlling virus loads in various organs. This improved viral control is not due to enhanced interferon, natural killer cell, or CD8 T-cell responses. Overall, the data indicate that deficiencies in the molecules that are critical to necroptosis do not necessarily result in worse outcomes following viral infection and may improve virus control.

Resistance to lethal ectromelia virus infection requires Type I interferon receptor in natural killer cells and monocytes but not in adaptive immune or parenchymal cells.

Type I interferons (IFN-I) are antiviral cytokines that signal through the ubiquitous IFN-I receptor (IFNAR). Following footpad infection with ectromelia virus (ECTV), a mouse-specific pathogen, C57BL/6 (B6) mice survive without disease, while B6 mice broadly deficient in IFNAR succumb rapidly. We now show that for survival to ECTV, only hematopoietic cells require IFNAR expression. Survival to ECTV specifically requires IFNAR in both natural killer (NK) cells and monocytes. However, intrinsic IFNAR signaling is not essential for adaptive immune cell responses or to directly protect non-hematopoietic cells such as hepatocytes, which are principal ECTV targets. Mechanistically, IFNAR-deficient NK cells have reduced cytolytic function, while lack of IFNAR in monocytes dampens IFN-I production and hastens virus dissemination. Thus, during a pathogenic viral infection, IFN-I coordinates innate immunity by stimulating monocytes in a positive feedback loop and by inducing NK cell cytolytic function.

Mechanisms of Antiviral Cytotoxic CD4 T Cell Differentiation.

Cytotoxic CD4 T lymphocytes (CD4-CTL) are important in antiviral immunity. For example, we have previously shown that in mice, CD4-CTL are important to control ectromelia virus (ECTV) infection. How viral infections induce CD4-CTL responses remains incompletely understood. We demonstrate here that not only ECTV but also vaccinia virus and lymphocytic choriomeningitis virus induce CD4-CTL, though the response to ECTV is stronger. Using ECTV, we also demonstrate that in contrast to CD8-CTL, CD4-CTL differentiation requires constant virus replication and ceases once the virus is controlled. We also show that major histocompatibility complex class II molecules on CD11c+ cells are required for CD4-CTL differentiation and for mousepox resistance. Transcriptional analysis indicated that antiviral CD4-CTL and noncytolytic T helper 1 (Th1) CD4 T cells have similar transcriptional profiles, suggesting that CD4-CTL are terminally differentiated classical Th1 cells. Interestingly, CD4-CTL and classical Th1 cells expressed similar mRNA levels of the transcription factors ThPOK and GATA-3, necessary for CD4 T cell linage commitment, and Runx3, required for CD8 T cell development and effector function. However, at the protein level, CD4-CTL had higher levels of the three transcription factors, suggesting that further posttranscriptional regulation is required for CD4-CTL differentiation. Finally, CRISPR/Cas9-mediated deletion of Runx3 in CD4 T cells inhibited CD4-CTL but not classical Th1 cell differentiation in response to ECTV infection. These results further our understanding of the mechanisms of CD4-CTL differentiation during viral infection and the role of posttranscriptionally regulated Runx3 in this process. IMPORTANCE While it is well established that cytotoxic CD4 T cells (CD4-CTLs) directly contribute to viral clearance, it remains unclear how CD4-CTL are induced. We now show that CD4-CTLs require sustained antigen presentation and are induced by CD11c-expressing antigen-presenting cells. Moreover, we show that CD4-CTLs are derived from the terminal differentiation of classical T helper 1 (Th1) subset of CD4 cells. Compared to Th1 cells, CD4-CTLs upregulate protein levels of the transcription factors ThPOK, Runx3, and GATA-3 posttranscriptionally. Deletion of Runx3 in differentiated CD4 T cells prevents induction of CD4-CTLs but not classical Th1 cells. These results advance our knowledge of how CD4-CTLs are induced during viral infection.

Chronic Lymphocytic Choriomeningitis Infection Causes Susceptibility to Mousepox and Impairs Natural Killer Cell Maturation and Function.

Chronic viral infections. like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus or those of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), result in immune dysfunction that predisposes the host to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV), and that this resistance requires natural killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox but that most of those that recovered from acute infection with the LCMV Armstrong (Arm) strain survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm infection have a reduced frequency and a reduced number of NK cells. However, at steady state, NK cells in mice that have recovered from Arm infection mature normally and, in response to ECTV, get activated, become more mature, proliferate, and increase their cytotoxicity in vivo Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated, and following ECTV infection, they do not mature, proliferate, or increase their cytotoxicity. Given the well-established importance of NK cells in resistance to mousepox, these data suggest that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.IMPORTANCE Infection of adult mice with the clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, we show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV; the agent of mousepox) and that natural killer (NK) cells in CL13-infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues why humans chronically infected with certain pathogens are less resistant to viral diseases.

Loss of Resistance to Mousepox during Chronic Lymphocytic Choriomeningitis Virus Infection Is Associated with Impaired T-Cell Responses and Can Be Rescued by Immunization.

It is well established that chronic viral infections can cause immune suppression, resulting in increased susceptibility to other infectious diseases. However, the effects of chronic viral infection on T-cell responses and vaccination against highly pathogenic viruses are not well understood. We have recently shown that C57BL/6 (B6) mice lose their natural resistance to wild-type (WT) ectromelia virus (ECTV) when chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13). Here we compared the T-cell response to ECTV in previously immunologically naive mice that were chronically infected with CL13 or that were convalescent from acute infection with the Armstrong (Arm) strain of LCMV. Our results show that mice that were chronically infected with CL13 but not those that had recovered from Arm infection have highly defective ECTV-specific CD8+ and CD4+ T-cell responses to WT ECTV. These defects are at least partly due to the chronic infection environment. In contrast to mice infected with WT ECTV, mice chronically infected with CL13 survived without signs of disease when infected with ECTV-Δ036, a mutant ECTV strain that is highly attenuated. Strikingly, mice chronically infected with CL13 mounted a strong CD8+ T-cell response to ECTV-Δ036 and survived without signs of disease after a subsequent challenge with WT ECTV. Our work suggests that enhanced susceptibility to acute viral infections in chronically infected individuals can be partly due to poor T-cell responses but that sufficient T-cell function can be recovered and resistance to acute infection can be restored by immunization with highly attenuated vaccines.IMPORTANCE Chronic viral infections may result in immunosuppression and enhanced susceptibility to infections with other pathogens. For example, we have recently shown that mice chronically infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) are highly susceptible to mousepox, a disease that is caused by ectromelia virus and that is the mouse homolog of human smallpox. Here we show chronic CL13 infection severely disrupts the expansion, proliferation, activation, and cytotoxicity of T cells in response due at least in part to the suppressive effects of the chronic infection milieu. Notably, despite this profound immunodeficiency, mice chronically infected with CL13 could be protected by vaccination with a highly attenuated variant of ECTV. These results demonstrate that protective vaccination of immunosuppressed individuals is possible, provided that proper immunization tools are used.

Recombinogenic, genotoxic, and cytotoxic effects of azathioprine using in vivo assays.

Azathioprine (Aza) is a purine antimetabolite immunosuppressant that is widely employed for immunosuppressive therapy in post-transplant recipients or patients with autoimmune diseases. Chronic use of immunosuppressants might produce several side effects, including a high rate of neoplasms in these patients. Considering that genotoxic effects are associated with an increased risk of developing cancer, the aim of this study was to examine the recombinogenic, genotoxic, and cytotoxic effects of Aza using Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster, as well as comet and micronucleus assays in mouse bone marrow cells. Further, the adverse effects of Aza were determined in mouse hepatic and renal tissues using histopathological analysis. Data demonstrated that Aza induced significant increased genotoxicity in D. melanogaster and mouse bone marrow cells at all concentrations tested. Homologous recombination was the predominant genotoxic event noted for the first time to be initiated by Aza in SMART. In histopathological analysis, Aza did not show any marked toxic activity in mouse hepatic and renal tissues. Therefore, the high rate of neoplasms reported in patients with long-term use of Aza may be attributed, at least partially, to the genotoxic action of this drug.

Cutting Edge: Protection by Antiviral Memory CD8 T Cells Requires Rapidly Produced Antigen in Large Amounts.

Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Inhibition kinetics of digestive proteases for Anticarsia gemmatalis.

Anticarsia gemmatalis Hübner, 1818 (Lepidoptera) is a major pest of soybean in the Brazil. It is known that the reduction of proteolytic activity by the ingestion of protease inhibitors reduces digestion and larval development of the insects. Control via inhibition of the digestive enzymes necessitates deeper knowledge of the enzyme kinetics and the characterization of the inhibition kinetics of these proteases, for better understanding of the active centers and action mechanisms of this enzyme. Trypsin-like proteases found in the gut of Anticarsia gemmatalis were purified in a p-aminobenzamidine agarose column. Kinetic characterization showed KM 0.503 mM for the L-BApNA substrate; Vmax= 46.650 nM s-1; Vmax/[E]= 9.256 nM s-1 mg L-1 and Vmax/[E]/KM= 18.402 nM s-1 mg L-1 mM. The Ki values for the inhibitors benzamidine, berenil, SKTI and SBBI were 11.2 µM, 32.4 µM, 0.25 nM and 1.4 nM, respectively, and all revealed linear competitive inhibition. The SKTI showed the greatest inhibition, which makes it a promising subject for future research to manufacture peptide mimetic inhibitors.

Parkia platycephala lectin enhances the antibiotic activity against multi-resistant bacterial strains and inhibits the development of Haemonchus contortus.

Lectins have been studied in the past few years as an alternative to inhibit the development of pathogenic bacteria and gastrointestinal nematodes of small ruminants. The development of new antibacterial and anthelmintic compounds is necessary owing to the increase in drug resistance among important pathogens. Therefore, this study aimed to evaluate the capacity of a glucose/mannose-binding lectin from Parkia platycephala seeds (PPL) to inhibit the development of Haemonchus contortus and to modulate antibiotic activity against multi-resistant bacterial strains, thereby confirming its efficacy when used in combination with gentamicin. PPL at the concentration of 1.2 mg/mL did not show inhibitory activity on H. contortus in the egg hatch test or the exsheathment assay. However, it did show significant inhibition of H. contortus larval development with an IC50 of 0.31 mg/mL. The minimum inhibitory concentration (MIC) obtained for PPL against all tested bacterial strains was not clinically relevant (MIC ≥ 1024 µg/mL). However, when PPL was combined with gentamicin, a significant increase in antibiotic activity was observed against S. aureus and E.coli multi-resistant strains. The inhibition of hemagglutinating activity by gentamicin (MIC = 50 mM) revealed that it may be interacting with the carbohydrate-binding site of PPL. It is this interaction between the antibiotic and lectin carbohydrate-binding site that may be responsible for the enhanced activity of gentamicin against multi-resistant strains. It can be concluded that PPL showed selective anthelmintic effect, inhibiting the development of H. contortus larvae and that it increased the effect of the antibiotic gentamicin against multi-resistant bacterial strains, thus constituting a potential therapeutic resource against resistant bacterial strains and H. contortus.

Molecular identification of three species of Oncideres (Coleoptera: Cerambycidae) using RAPD markers.

Twig girdlers have a short adult life and a long larval stage (up to one year in some species). This fact, together with lack of morphological traits to identify the species from the larval stage, poses obstacles for the taxonomic identification and characterization of the many twig girdlers found in the wild. To solve this matter, Random Amplified Polymorphic DNA (RAPD) molecular markers have been applied to identify some insect species and to determine species boundaries. The aim of this study was to identify three species of Oncideres using the RAPD technique. Adults and larvae of O. saga, O. ocularis and O. ulcerosa were collected in Minas Gerais, Brazil. Two RAPD primers were used (OPA-05 and OPB-13), which produced different electrophoretic profiles that were used to construct a UPGMA phenogram. Three groups identified in the analyses accurately separated the three species, based on individuals in two different stages of development (adults and larvae): I: O. saga, II: O. ulcerosa, and III: O. ocularis. Molecular markers, such as RAPD, are valuable tools that help taxonomists in species identification.

Protease inhibitory, insecticidal and deterrent effects of the trypsin-inhibitor benzamidine on the velvetbean caterpillar in soybean.

The recognition of protease inhibitors with insecticidal activity is important as a basis for the development of mimetic peptides with potential use as biorational insecticides. We sprayed benzamidine on soybean plants and assessed whether this potent synthetic trypsin-inhibitor has protease inhibitory, insecticidal and deterrent effects on the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae). Activity of trypsin inhibition in soybean leaves was increased and total proteolytic activity in the midgut extract from larvae fed on these leaves was reduced by benzamidine. Different concentrations of benzamidine sprayed on the plant caused approximately 50 % of larval mortality, and larval choice and moth preference and oviposition were all negatively affected. Low concentrations of benzamidine increased mortality and hindered insect choice and oviposition as well as higher doses. Since many synthetic protease inhibitors are usually expensive, small doses of benzamidine may be effective to protect soybean against A. gemmatalis attack. Our results highlight the potential of synthetic protease inhibitors for insecticidal and deterrent purposes in insect pest management.

Does mechanical damage on soybean induces the production of flavonoids?

The response of plants to grazing includes the production of chemical defense compounds such as proteases inhibitors and secondary metabolites as flavonoids, which makes them less palatable to feeding and negatively affecting the physiology of insects. The aim of this study was to evaluate the phytochemical response of soybean cultivars (Glycine max (L.) Merrill) resistant (IAC-17, IAC-24) and susceptible (IAC-P1) to insects after mechanical damage. These cultivars were mechanically injured, and after 24 hours samples of these plants were analyzed by HPLC to identify and quantify flavonoids. The flavonoids daidzein, quercetin, and rutin were quantified, with the highest concentration of daidzin in soybean cultivars after mechanical damage. Rutin was biosynthesized by IAC-24. The cultivars IAC-PL1, IAC-17, and IAC-24 did not show a flavonoid response to mechanical damage. The soybean cultivars are not dependent on mechanical damage to produce flavonoids.

A pro-survival role for the intracellular granzyme B inhibitor Serpinb9 in natural killer cells during poxvirus infection.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.

Innate and adaptive immune responses that control lymph-borne viruses in the draining lymph node.

The interstitial fluids in tissues are constantly drained into the lymph nodes (LNs) as lymph through afferent lymphatic vessels and from LNs into the blood through efferent lymphatics. LNs are strategically positioned and have the appropriate cellular composition to serve as sites of adaptive immune initiation against invading pathogens. However, for lymph-borne viruses, which disseminate from the entry site to other tissues through the lymphatic system, immune cells in the draining LN (dLN) also play critical roles in curbing systemic viral dissemination during primary and secondary infections. Lymph-borne viruses in tissues can be transported to dLNs as free virions in the lymph or within infected cells. Regardless of the entry mechanism, infected myeloid antigen-presenting cells, including various subtypes of dendritic cells, inflammatory monocytes, and macrophages, play a critical role in initiating the innate immune response within the dLN. This innate immune response involves cellular crosstalk between infected and bystander innate immune cells that ultimately produce type I interferons (IFN-Is) and other cytokines and recruit inflammatory monocytes and natural killer (NK) cells. IFN-I and NK cell cytotoxicity can restrict systemic viral spread during primary infections and prevent serious disease. Additionally, the memory CD8+ T-cells that reside or rapidly migrate to the dLN can contribute to disease prevention during secondary viral infections. This review explores the intricate innate immune responses orchestrated within dLNs that contain primary viral infections and the role of memory CD8+ T-cells following secondary infection or CD8+ T-cell vaccination.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

Design and preclinical evaluation of a universal SARS-CoV-2 mRNA vaccine.

Because of the rapid mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an effective vaccine against SARS-CoV-2 variants is needed to prevent coronavirus disease 2019 (COVID-19). T cells, in addition to neutralizing antibodies, are an important component of naturally acquired protective immunity, and a number of studies have shown that T cells induced by natural infection or vaccination contribute significantly to protection against several viral infections including SARS-CoV-2. However, it has never been tested whether a T cell-inducing vaccine can provide significant protection against SARS-CoV-2 infection in the absence of preexisting antibodies. In this study, we designed and evaluated lipid nanoparticle (LNP) formulated mRNA vaccines that induce only T cell responses or both T cell and neutralizing antibody responses by using two mRNAs. One mRNA encodes SARS-CoV-2 Omicron Spike protein in prefusion conformation for induction of neutralizing antibodies. The other mRNA encodes over one hundred T cell epitopes (multi-T cell epitope or MTE) derived from non-Spike but conserved regions of the SARS-CoV-2. We show immunization with MTE mRNA alone protected mice from lethal challenge with the SARS-CoV-2 Delta variant or a mouse-adapted virus MA30. Immunization with both mRNAs induced the best protection with the lowest viral titer in the lung. These results demonstrate that induction of T cell responses, in the absence of preexisting antibodies, is sufficient to confer protection against severe disease, and that a vaccine containing mRNAs encoding both the Spike and MTE could be further developed as a universal SARS-CoV-2 vaccine.

The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses.

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.