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Immune response to two SARS-CoV-2 mRNA vaccine doses among kidney transplant recipients (KTRs) is limited. We aimed to evaluate humoral and cellular response to a third BNT162b2 dose. In this prospective study, 190 KTRs were evaluated before and â¼3 weeks after the third vaccine dose. The primary outcomes were anti-spike antibody level >4160 AU/ml (neutralization-associated cutoff) and any seropositivity. Univariate and multivariate analyses were conducted to identify variables associated with antibody response. T-cell response was evaluated in a subset of participants. Results were compared to a control group of 56 healthcare workers. Among KTRs, we found a seropositivity rate of 70% (133/190) after the third dose (37%, 70/190, after the second vaccine dose); and 27% (52/190) achieved levels above 4160 AU/ml after the third dose, compared to 93% of controls. Variables associated with antibody response included higher antibody levels after the second dose (odds ratio [OR] 30.8 per log AU/ml, 95% confidence interval [CI]11-86.4, p < 0.001); and discontinuation of antimetabolite prior to vaccination (OR 9.1,95% CI 1.8-46.5, p = 0.008). T-cell response was demonstrated in 13% (7/53). In conclusion, third dose BNT162b2 improved immune response among KTRs, however 30% still remained seronegative. Pre-vaccination temporary immunosuppression reduction improved antibody response.
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COVID-19 , Transplante de Rim , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade , Estudos Prospectivos , SARS-CoV-2 , Transplantados , Vacinas Sintéticas , Vacinas de mRNARESUMO
rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.
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Vacinas contra COVID-19/toxicidade , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , Cricetinae , Feminino , Glicoproteínas de Membrana/genética , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Suínos , Vacinação , Vacinas Sintéticas/toxicidade , Proteínas do Envelope Viral/genéticaRESUMO
The COVID-19 pandemic led to development of mRNA vaccines, which became a leading anti-SARS-CoV-2 immunization platform. Preclinical studies are limited to infection-prone animals such as hamsters and monkeys in which protective efficacy of vaccines cannot be fully appreciated. We recently reported a SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc) mRNA vaccine delivered via lipid nanoparticles (LNPs). BALB/c mice demonstrated specific immunologic responses following RBD-hFc mRNA vaccination. Now, we evaluated the protective effect of this RBD-hFc mRNA vaccine by employing the K18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model. Administration of an RBD-hFc mRNA vaccine to K18-hACE2 mice resulted in robust humoral responses comprising binding and neutralizing antibodies. In correlation with this response, 70% of vaccinated mice withstood a lethal SARS-CoV-2 dose, while all control animals succumbed to infection. To the best of our knowledge, this is the first nonreplicating mRNA vaccine study reporting protection of K18-hACE2 against a lethal SARS-CoV-2 infection.
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COVID-19 , Nanopartículas , Vacinas , Animais , Humanos , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Pandemias , RNA Mensageiro/genética , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
OBJECTIVE: Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. DATA DESCRIPTION: We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.
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Burkholderia pseudomallei , Genoma Bacteriano , Melioidose , Filogenia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/isolamento & purificação , Burkholderia pseudomallei/patogenicidade , Melioidose/microbiologia , Melioidose/epidemiologia , Tailândia/epidemiologia , Humanos , Genoma Bacteriano/genética , Índia , Animais , Israel/epidemiologia , Virulência/genética , Camundongos , Sequenciamento Completo do GenomaRESUMO
Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have emerged as an effective vaccination strategy. Although currently applied toward viral pathogens, data concerning the platform's effectiveness against bacterial pathogens are limited. Here, we developed an effective mRNA-LNP vaccine against a lethal bacterial pathogen by optimizing mRNA payload guanine and cytosine content and antigen design. We designed a nucleoside-modified mRNA-LNP vaccine based on the bacterial F1 capsule antigen, a major protective component of Yersinia pestis, the etiological agent of plague. Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of humankind. Now, the disease is treated effectively with antibiotics; however, in the case of a multiple-antibiotic-resistant strain outbreak, alternative countermeasures are required. Our mRNA-LNP vaccine elicited humoral and cellular immunological responses in C57BL/6 mice and conferred rapid, full protection against lethal Y. pestis infection after a single dose. These data open avenues for urgently needed effective antibacterial vaccines.
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Vacina contra a Peste , Peste , Yersinia pestis , Camundongos , Animais , Peste/prevenção & controle , Vacina contra a Peste/genética , Proteínas de Bactérias/genética , Camundongos Endogâmicos C57BL , Yersinia pestis/genética , Antígenos de Bactérias/genéticaRESUMO
Two doses of mRNA SARS-CoV-2 vaccines elicit an attenuated humoral immune response among immunocompromised patients. Our study aimed to assess the immunogenicity of a third dose of the BNT162b2 vaccine among lung transplant recipients (LTRs). We prospectively evaluated the humoral response by measuring anti-spike SARS-CoV-2 and neutralizing antibodies in 139 vaccinated LTRs ~4-6 weeks following the third vaccine dose. The t-cell response was evaluated by IFNγ assay. The primary outcome was the seropositivity rate following the third vaccine dose. Secondary outcomes included: positive neutralizing antibody and cellular immune response rate, adverse events, and COVID-19 infections. Results were compared to a control group of 41 healthcare workers. Among LTRs, 42.4% had a seropositive antibody titer, and 17.2% had a positive t-cell response. Seropositivity was associated with younger age (t = 3.736, p < 0.001), higher GFR (t = 2.355, p = 0.011), and longer duration from transplantation (t = -1.992, p = 0.024). Antibody titer positively correlated with neutralizing antibodies (r = 0.955, p < 0.001). The current study may suggest the enhancement of immunogenicity by using booster doses. Since monoclonal antibodies have limited effectiveness against prevalent sub-variants and LTRs are prone to severe COVID-19 morbidity, vaccination remains crucial for this vulnerable population.
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This study examines the efficacy, bacterial load, and humoral response of extensively delayed ciprofloxacin or doxycycline treatments following airway exposure of mice to Francisella tularensis subsp. holarctica (strain LVS) or to the highly virulent F. tularensis subsp. tularensis (strain SchuS4). A delay in onset of both antibiotic treatments allowed the rescue of all LVS-infected animals. However, for animals infected with SchuS4, only ciprofloxacin was efficacious and prolongation of treatment rescued all animals.
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Ciprofloxacina/uso terapêutico , Doxiciclina/uso terapêutico , Francisella tularensis/efeitos dos fármacos , Francisella tularensis/patogenicidade , Tularemia/tratamento farmacológico , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
mRNA Lipid nanoparticles (LNPs) have recently been propelled onto the center stage of therapeutic platforms due to the success of the SARS-CoV-2 mRNA LNP vaccines (mRNA-1273 and BNT162b2), with billions of mRNA vaccine doses already shipped worldwide. While mRNA vaccines seem like an overnight success to some, they are in fact a result of decades of scientific research. The advantage of mRNA-LNP vaccines lies in the modularity of the platform and the rapid manufacturing capabilities. However, there is a multitude of choices to be made when designing an optimal mRNA-LNP vaccine regarding efficacy, stability and toxicity. Herein, we provide a brief on what we consider to be the most important aspects to cover when designing mRNA-LNPs from what is currently known and how to optimize them. Lastly, we give our perspective on which of these aspects is most crucial and what we believe are the next steps required to advance the field.
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Lipossomos , Nanopartículas , Desenvolvimento de Vacinas , Vacinas de mRNA , Vacina BNT162 , COVID-19 , Humanos , Vacinas SintéticasRESUMO
The progression of the COVID-19 pandemic has led to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about reduced protective T cell immunity and, consequently, more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type Wuhan-1 SARS-CoV-2 ancestral spike protein and the Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from eight healthy volunteers 4-5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or the Omicron SARS-CoV-2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring cells secreting interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4). For all the examined individuals, comparable levels of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg-secreting cells and only limited numbers of IL-10- and IL-4-secreting cells. The data demonstrate stable T cell activity in response to the emerging Omicron variant in the tested individuals; therefore, the protective immunity to the variant following BNT162b2 vaccination is not significantly affected.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacina BNT162/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacina BNT162/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , Citocinas/análise , Citocinas/imunologia , ELISPOT , Feminino , Humanos , Interferon gama/análise , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia , Células Th1/imunologia , Adulto JovemRESUMO
The design of efficient vaccines for long-term protective immunity against pathogens represents an objective of utmost public health priority. In general, live attenuated vaccines are considered to be more effective than inactivated pathogens, yet potentially more reactogenic. Accordingly, inactivation protocols which do not compromise the pathogen's ability to elicit protective immunity are highly beneficial. One of the sentinel mechanisms of the host innate immune system relies on the production of reactive nitrogen intermediates (RNI), which efficiently inactivate pathogens. Peroxynitrite (PN) is a prevalent RNI, assembled spontaneously upon the interaction of nitric oxide (NO) with superoxide. PN exerts its bactericidal effect by via the efficient oxidation of a broad range of biological molecules. Furthermore, the interaction of PN with proteins results in structural/chemical modifications, such as the oxidation of tryptophan, tyrosine, and cysteine residues, as well as the formation of carbonyl, dityrosine, and nitrotyrosine (NT). In addition to their role in innate immunity, these PN-mediated modifications of pathogen components may also augment the antigenicity of pathogen peptides and proteins, hence contributing to specific humoral responses. In the study reported here, a novel approach for vaccine development, consisting of pathogen inactivation by PN, combined with increased immunity of NT-containing peptides, is implemented as a proof-of-concept for vaccination against the intracellular pathogen Francisella tularensis (F. tularensis). In vivo experiments in a murine model of tularemia confirm that PN-inactivated F. tularensis formulations may rapidly stimulate innate and adaptive immune cells, conferring efficient protection against a lethal challenge, superior to that elicited by bacteria inactivated by the widely used formalin treatment.
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Both humoral and cellular anamnestic responses are significant for protective immunity against SARS-CoV-2. In the current study, the responses in elderly people before and after a fourth vaccine dose of BNT162b2 were compared to those of individuals immunized with three vaccine doses. Although a boost effect was observed, the high response following the third administration questions the necessity of an early fourth boost.
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The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.
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Enzima de Conversão de Angiotensina 2 , COVID-19 , Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fator de Transcrição GATA6/genética , Humanos , Peptidil Dipeptidase A/metabolismo , Provírus/genética , SARS-CoV-2/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple, and rapid platform for immunization, and therefore have been employed in recent studies toward the development of a SARS-CoV-2 vaccine. Herein, we present the design of an mRNA vaccine, based on lipid nanoparticles (LNPs)-encapsulated SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc). Several ionizable lipids have been evaluated in vivo in a luciferase (luc) mRNA reporter assay, and two leading LNPs formulations have been chosen for the subsequent RBD-hFc mRNA vaccine strategy. Intramuscular administration of LNP RBD-hFc mRNA elicited robust humoral response, a high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. The data in the current study demonstrate the potential of these lipids as promising candidates for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular.
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COVID-19 , Nanopartículas , Vacinas , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Humanos , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20-30% loss of weight at day 7 and full recovery at days 11-13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.
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We previously demonstrated that the HtrA (High Temperature Requirement A) protease/chaperone active in the quality control of protein synthesis, represents an important virulence determinant of Bacillus anthracis. Virulence attenuation of htrA-disrupted Bacillus anthracis strains was attributed to susceptibility of ΔhtrA strains to stress insults, as evidenced by affected growth under various stress conditions. Here, we report a comparative RNA-seq transcriptomic study generating a database of differentially expressed genes in the B. anthracishtrA-disrupted and wild type parental strains under oxidative stress. The study demonstrates that, apart from protease and chaperone activities, HtrA exerts a regulatory role influencing expression of more than 1000 genes under stress. Functional analysis of groups or individual genes exhibiting strain-specific modulation, evidenced (i) massive downregulation in the ΔhtrA and upregulation in the WT strains of various transcriptional regulators, (ii) downregulation of translation processes in the WT strain, and (iii) downregulation of metal ion binding functions and upregulation of sporulation-associated functions in the ΔhtrA strain. These modulated functions are extensively discussed. Fifteen genes uniquely upregulated in the wild type strain were further interrogated for their modulation in response to other stress regimens. Overexpression of one of these genes, encoding for MazG (a nucleoside triphosphate pyrophosphohydrolase involved in various stress responses in other bacteria), in the ΔhtrA strain resulted in partial alleviation of the H2O2-sensitive phenotype.
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The high-temperature requirement chaperone/protease (HtrA) is involved in the stress response of the anthrax-causing pathogen Bacillus anthracis Resilience to oxidative stress is essential for the manifestation of B. anthracis pathogenicity. Here, we announce transcriptome data sets detailing global gene expression in B. anthracis wild-type and htrA-disrupted strains following H2O2-induced oxidative stress.
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Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting the need for effective new treatments. It has been demonstrated that binding of antibody-coated bacteria to the Fc receptor located on phagocytic cells is a key process needed for efficient protection against Ft. Yet, Ft utilizes the same receptor to enter the phagocytic cells in order to escape the immune system. To address the question whether an anti-Ft LPS antibody lacking the ability to bind the Fc receptor may inhibit the entry of Ft into host cells, a soluble scFv (TL1-scFv) was constructed from an anti Ft-LPS antibody (TL1) that was isolated from an immune single-chain (scFv) phage-display library. Bacterial uptake was assessed upon infection of macrophages with Ft live attenuated strain (LVS) in the presence of either TL1 or TL1-scFv. While incubation of LVS in the presence of TL1 greatly enhanced bacterial uptake, LVS uptake was significantly inhibited in the presence of TL1-scFv. These results prompt further experiments probing the therapeutic efficacy of TL1-scFv, alone or in combination with antibiotic treatment.
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Anticorpos Antibacterianos/farmacologia , Francisella tularensis/imunologia , Lipopolissacarídeos/imunologia , Fagocitose/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Tularemia/tratamento farmacológico , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Antibacterianos/uso terapêutico , Vacinas Bacterianas/administração & dosagem , Modelos Animais de Doenças , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose/imunologia , Coelhos , Anticorpos de Cadeia Única/sangue , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/uso terapêutico , Tularemia/sangue , Tularemia/imunologia , Tularemia/microbiologia , Vacinas Atenuadas/administração & dosagemRESUMO
Anthrax is a lethal disease caused by the Gram-positive spore-producing bacterium Bacillus anthracis. We previously demonstrated that disruption of htrA gene, encoding the chaperone/protease HtrABA (High Temperature Requirement A of B. anthracis) results in significant virulence attenuation, despite unaffected ability of ΔhtrA strains (in which the htrA gene was deleted) to synthesize the key anthrax virulence factors: the exotoxins and capsule. B. anthracis ΔhtrA strains exhibited increased sensitivity to stress regimens as well as silencing of the secreted starvation-associated Neutral Protease A (NprA) and down-modulation of the bacterial S-layer. The virulence attenuation associated with disruption of the htrA gene was suggested to reflect the susceptibility of ΔhtrA mutated strains to stress insults encountered in the host indicating that HtrABA represents an important B. anthracis pathogenesis determinant. As all HtrA serine proteases, HtrABA exhibits a protease catalytic domain and a PDZ domain. In the present study we interrogated the relative impact of the proteolytic activity (mediated by the protease domain) and the PDZ domain (presumably necessary for the chaperone activity and/or interaction with substrates) on manifestation of phenotypic characteristics mediated by HtrABA. By inspecting the phenotype exhibited by ΔhtrA strains trans-complemented with either a wild-type, truncated (ΔPDZ), or non-proteolytic form (mutated in the catalytic serine residue) of HtrABA, as well as strains exhibiting modified chromosomal alleles, it is shown that (i) the proteolytic activity of HtrABA is essential for its N-terminal autolysis and subsequent release into the extracellular milieu, while the PDZ domain was dispensable for this process, (ii) the PDZ domain appeared to be dispensable for most of the functions related to stress resilience as well as involvement of HtrABA in assembly of the bacterial S-layer, (iii) conversely, the proteolytic activity but not the PDZ domain, appeared to be dispensable for the role of HtrABA in mediating up-regulation of the extracellular protease NprA under starvation stress, and finally (iv) in a murine model of anthrax, the HtrABA PDZ domain, was dispensable for manifestation of B. anthracis virulence. The unexpected dispensability of the PDZ domain may represent a unique characteristic of HtrABA amongst bacterial serine proteases of the HtrA family.
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We report here the draft genome sequence of Burkholderia pseudomallei MAA2018. This highly virulent strain was isolated in 2018 from the first melioidosis case in Israel associated with recreational travel to Goa, India.
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Diagnostic identification of pathogens is usually accomplished by isolation of the pathogen or its substances, and should correlate with the time and site of infection. Alternatively, immunoassays such as enzyme-linked immunosorbent assay (ELISA) tests for quantification of serum antibodies are expedient and are usually employed for retrospective diagnostic of a particular infective agent. Here, the potential of cell-based immunoassays for early pathogen detection was evaluated by quantification of specific, antigen-activated, low-frequency IFNγ-secreting cells in mouse spleens following infection with various pathogens. Using enzyme-linked immunospot (ELISPOT) assays, specific responses were observed within 3-6 days following infection with F. tularensis, B. anthracis, Y. pestis, or Influenza virus. Blood samples collected from F. tularensis-infected mice revealed the presence of IFNγ-producing activated cells within one week post infection. When non-human primates were infected with B. anthracis, cellular response was observed in peripheral blood samples as early as five days post infection, 3-5 days earlier than serum antibodies. Finally, the expression pattern of genes in splenocytes of F. tularensis-infected mice was inspected by a transcriptomic approach, enabling the identification of potential host targets for the future development of genetic-based cellular immunoassays. Altogether, the data demonstrate the potential of cell-based immunoassays for early pathogen detection.