Marcela Henao Tamayo - Profile on Academia.edu (original) (raw)

Papers by Marcela Henao Tamayo

The Journal of Immunology, 2022

Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2... more Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. In this study, we evaluated immune and endothelial blood cell profiles of patients with coronavirus disease 2019 to determine critical differences between those with mild, moderate, or severe COVID-19 using spectral flow cytometry. We examined a suite of immune phenotypes, including monocytes, T cells, NK cells, B cells, endothelial cells, and neutrophils, alongside surface and intracellular markers of activation. Our results showed progressive lymphopenia and depletion of T cell subsets (CD3 + , CD4 + , and CD8 + ) in patients with severe disease and a significant increase in the CD56 + CD14 + Ki67 + IFN-g + monocyte population in patients with moderate and severe COVID-19 that has not been previously described. Enhanced circulating endothelial cells (CD45 2 CD31 + CD34 + CD146 + ), circulating endothelial progenitors (CD45 2 CD31 + CD34 +/2 CD146 2 ), and neutrophils (CD11b + CD66b + ) were coevaluated for COVID-19 severity. Spearman correlation analysis demonstrated the synergism among age, obesity, and hypertension with upregulated CD56 + monocytes, endothelial cells, and decreased T cells that lead to severe outcomes of SARS-CoV-2 infection. Circulating monocytes and endothelial cells may represent important cellular markers for monitoring postacute sequelae and impacts of SARS-CoV-2 infection during convalescence and for their role in immune host defense in high-risk adults after vaccination.

Vaccine, 2020

Hemicellulases from microbial cells have become biotechnologically important since they hydrolyze... more Hemicellulases from microbial cells have become biotechnologically important since they hydrolyze complex polysaccharides of plant tissues into simple molecules. In paper and pulp industry, role of hemicellulases is well established and recently its applicability in the food and feed technology, coffee extraction, oil drilling and detergent industry also have been found. Hemicellulases are enzymes produced mainly from microorganisms but its production from plants and animals have also been reported. Bacterial hemicellulases are mostly extracellular enzymes that can act in a wide pH and temperature range, though acidic and neutral hemicellulases are more common. In this review we will focus on complex hemicellulases structure and the microbial enzyme complex involved in its complete breakdown, hemicellulases sources, its production conditions and their applications in the commercial sector. However, the major emphasis of the review is on the microbial hemicellulases but reference to plant and animal hemicellulases also has been made to complete the overview.

Infection with Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Di... more Infection with Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of the Spike S1 protein encoding mRNA, incorporated in a lipid nanoparticle, stabilized by polyethylene glycol (PEG). mRNA vaccines are novel in many respects, including cellular uptake, the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences encoding the SARS-CoV-2 Spike S1 protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to induction of virus neutralizing antibodies and cytotoxic T-lymphocytes will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side e...

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of v... more The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX™) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopath...

Scientific Reports, 2020

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; ho... more Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The ...

Minipigs as a neonatal animal model for tuberculosis vaccine efficacy testing

Veterinary Immunology and Immunopathology, 2019

Many vaccines against childhood diseases are administered early after birth, but vaccine developm... more Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48 h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.

Tuberculosis (Edinburgh, Scotland), 2017

In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Curren... more In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Current animal models study TB during adulthood but animal models for infant TB are scarce. Here we propose that minipigs can be used as an animal model to study adult, adolescent and infant TB including natural transmission. In these studies, two-month old minipigs (representing infant age in humans) and six-month old minipigs (representing adolescence in humans) were infected via the aerosol route with hyper-virulent clinical strain W-Beijing Mycobacterium tuberculosis (Mtb) HN878 and were monitored for 11 or 36 weeks post-challenge, respectively. In the same studies, infected and unchallenged animals were housed together. Viable bacteria were recovered from pulmonary and thoracic lymph nodes from both -infected and their initially unchallenged natural contacts. Bacillary load, gross lesions and histopathology revealed similarities to the spectrum of disease observed in human TB. The study d...

Antimicrobial Agents and Chemotherapy, 2015

Over the last 10 years,Mycobacterium abscessusgroup strains have emerged as important human patho... more Over the last 10 years,Mycobacterium abscessusgroup strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections withM. abscessusare often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection withM. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an opt...

The open AIDS journal, 2011

Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in ... more Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis. Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijin...

BMC Microbiology, 2015

Background: Macrophages are the primary effector cells responsible for killing Mycobacterium tube... more Background: Macrophages are the primary effector cells responsible for killing Mycobacterium tuberculosis (MTB) through various mechanisms, including apoptosis. However, MTB can evade host immunity to create a favorable environment for intracellular replication. MTB-infected human macrophages produce interleukin-32 (IL-32). IL-32 is a pro-inflammatory cytokine and has several isoforms. We previously found that IL-32γ reduced the burden of MTB in human macrophages, in part, through the induction of caspase-3-dependent apoptosis. However, based on our previous studies, we hypothesized that caspase-3-independent death pathways may also mediate IL-32 control of MTB infection. Herein, we assessed the potential roles of cathepsin-mediated apoptosis, caspase-1-mediated pyroptosis, and apoptosis-inducing factor (AIF) in mediating IL-32γ control of MTB infection in THP-1 cells. Results: Differentiated human THP-1 macrophages were infected with MTB H37Rv alone or in the presence of specific inhibitors to caspase-1, cathepsin B/D, or cathepsin L for up to four days, after which TUNEL-positive cells were quantified; in addition, MTB was quantified by culture as well as by the percentage of THP-1 cells that were infected with green fluorescent protein (GFP)-labeled MTB as determined by microscopy. AIF expression was inhibited using siRNA technology. Inhibition of cathepsin B/D, cathepsin L, or caspase-1 activity significantly abrogated the IL-32γ-mediated reduction in the number of intracellular MTB and of the percentage of GFP-MTB-infected macrophages. Furthermore, inhibition of caspase-1, cathepsin B/D, or cathepsin L in the absence of exogenous IL-32γ resulted in a trend toward an increased proportion of MTB-infected THP-1 cells. Inhibition of AIF activity in the absence of exogenous IL-32γ also increased intracellular burden of MTB. However, since IL-32γ did not induce AIF and because the relative increases in MTB with inhibition of AIF were similar in the presence or absence of IL-32γ, our results indicate that AIF does not mediate the host-protective effect of IL-32γ against MTB. The anti-MTB effects of IL-32γ are mediated through classical caspase-3-dependent apoptosis as well as caspase-3-independent apoptosis.

Tuberculosis, 2009

Enhancing immunity to tuberculosis in animal models after exposure to the infection has proved di... more Enhancing immunity to tuberculosis in animal models after exposure to the infection has proved difficult. In this study we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs of guinea pigs challenged by low dose aerosol infection with Mycobacterium tuberculosis and vaccinated ten days later. On day forty after infection the fusion protein F36 and a pool of Ag85A and ESAT-6 vaccines had significant effects on the bacterial load, showed increased expression of the activation marker CD45+ on CD4+ T cells, and reduced numbers of heterophils. Lung pathology and pathology scores were marginally improved in animals given these vaccines, but lymph node pathology was not influenced. Despite early effects no changes in long term survival were seen. These results suggest that a single postexposure vaccination can initially slow the disease process. However, this effect is transient, but this could be of use in an multidrug resistant/extremely drug resistant outbreak situation because it could potentially slow the infection long enough to complete drug susceptibility testing and initiate effective chemotherapy.

Memory T cell subsets in tuberculosis: What should we be targeting?

Tuberculosis, 2014

The purpose of vaccination is to establish a stable population of long lived memory T cells. In t... more The purpose of vaccination is to establish a stable population of long lived memory T cells. In the context of tuberculosis, the BCG vaccine has been widely used for well over 60 years, but during that time its weaknesses, particularly its ineffectiveness in adults, has been increasingly recognized. In this commentary we review what is known about memory T cells, both in general and in the context of their role in expressing specific acquired resistance to tuberculosis. Current knowledge indicates that both effector memory and central memory can be generated, depending on the experimental conditions, but both in animal models and in clinical studies it is clear that effector memory T cells are the predominant subset. These issues are of importance, given the concerted effort to make new TB vaccines, not all of which may work in precisely the same manner. At the present time whether a TB vaccine would work better if it targeted one specific T cell subset rather than another is as yet completely unknown, and this is now further complicated by new evidence that suggests other subsets such as IL-17 secreting CD4 T cells and cells with stem cell-like qualities may also play important roles.

A mouse model of tuberculosis reinfection

Tuberculosis, 2012

Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis wh... more Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis who live in endemic areas are four times more likely to develop secondary disease, often as not caused by exogenous reinfection. In a mouse model described here, we show that mice infected with the virulent W-Beijing Mycobacterium tuberculosis strain HN878, then given chemotherapy to clear the infection, were resistant to re-challenge with the same organism thereafter. This resistance, which was mediated by rapid expression of CD4 T cells expressing markers consistent with both central and effector memory immunity, was only transient however. After 20-30 days of the reinfection the numbers of these cells steadily declined, the bacterial load in the lungs surged up, and the lung tissues became increasingly consolidated. No evidence was found for a regulatory T cell response in these mice, but many T cells harvested from the lungs showed evidence of increased PD-1 expression, indicating exhaustion. These data indicate that the memory T cell response to reinfection may not be as stable and long lived as previously thought, a finding with obvious implications for vaccine development.

Vaccine, 2012

Bacillus-Calmette-Guerin (BCG), the only human tuberculosis vaccine, primes a partially protectiv... more Bacillus-Calmette-Guerin (BCG), the only human tuberculosis vaccine, primes a partially protective immune response against M. tuberculosis infection in humans and animals. In guinea pigs, BCG vaccination slows the progression of disease and reduces the severity of necrotic granulomas, which harbor a population of drug-tolerant bacilli. The objective of this study was to determine if reducing disease severity by BCG vaccination of guinea pigs prior to M. tuberculosis challenge enhanced the efficacy of combination drug therapy. At 20 days of infection, treatment of vaccinated and non-vaccinated animals with rifampin, isoniazid, and pyrizinamide (RHZ) was initiated for 4 or 8 weeks. On days 50, 80 and 190 of infection (10 weeks after drug were withdrawn), treatment efficacy was evaluated by quantifying clinical condition, bacterial loads, lesion severity, and dynamic changes in peripheral blood and lung leukocyte numbers by flow cytometry. In a separate, long-term survival study, treatment efficacy was evaluated by determining disease reactivation frequency post-mortem. BCG vaccination alone delayed pulmonary and extra-pulmonary disease progression, but failed to prevent dissemination of bacilli and the formation of necrotic granulomas. Drug therapy either alone or in combination with BCG, was more effective at lessening clinical disease and lesion severity compared to control animals or those receiving BCG alone. Fewer residual lesions in BCG vaccinated and drug treated animals, equated to a reduced frequency of reactivation disease and improvement in survival even out to 500 days of infection. The combining of BCG vaccination and drug therapy was more effective at resolving granulomas such that fewer animals had evidence of residual infection and thus less reactivation disease.

Vaccine, 2007

A mutant of Mycobacterium tuberculosis lacking the 19-kilodalton lipoprotein Rv3763 is highly att... more A mutant of Mycobacterium tuberculosis lacking the 19-kilodalton lipoprotein Rv3763 is highly attenuated in vivo but retains potent vaccinogenic properties

Tuberculosis, 2011

Predictive biomarkers illustrating the effectiveness of chemotherapeutic regimens for tuberculosi... more Predictive biomarkers illustrating the effectiveness of chemotherapeutic regimens for tuberculosis still remain elusive. To date, most are predicated on assays using sputum or serum; as a result, if not predictive, treatment failure in patients may not be evident for some time. We report here the results of a simple screening study in which T cell surface markers were examined in mice infected with Mycobacterium tuberculosis and then treated with drugs. These studies identified certain markers, the exhaustion markers PD-1 and TIM-3, as well as the marker KLRG-1, particularly on CD8 T cells, that changed in concert with reduction of the bacterial load in the lungs. While there is no guarantee these changes would also be seen on T cells in the blood, this approach should be further investigated.

Tuberculosis, 2011

There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to ... more There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to the W-Beijing genotype of newly emerging strains are often of very high virulence when tested in small animal models, including the mouse and guinea pig. In this report we provide further evidence to support this contention, and show that two W-Beijing strains are of very high virulence when introduced by low dose aerosol into out-bred guinea pigs. In addition to severe lung pathology, each of these infections was associated with large influxes of activated CD4 and CD8 T cells into the lungs. Large influxes of macrophages were also observed, but the fraction of these showing evidence of activation by Class-II expression was relatively low. A progressive increase in neutrophils was also seen, with highest levels accumulating in the lungs of the W-Beijing infected animals. In the case of these two infections mRNA levels for TH1 cytokines was elevated early, but these then declined, and were replaced by increasing levels of message encoding for Foxp3, IL-10, and TGFβ. These observations support the hypothesis that W-Beijing strains are potent inducers of regulatory T cells, and that this event may enhance survival and transmission of these bacilli.

PLoS ONE, 2011

Supporting Information S1 (A) Partial sequencing of the hsp65 and rpoB genes of M. massiliense CI... more Supporting Information S1 (A) Partial sequencing of the hsp65 and rpoB genes of M. massiliense CIP 108297 and CRM-0019. The corresponding sequences of M. abscessus ATCC 19977 are included in the alignments as a reference. PCR amplification were performed as described by Ringuet et al. (1999) and Kirshner et al. (1993) (see references below).

Nature Medicine, 2011

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 re... more We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Desx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4 + memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4 + cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.

Journal of Leukocyte Biology, 2005

In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosi... more In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosis representing a range of virulence for their capacity to grow in bone marrow-derived macrophages. The rate of growth of each of the isolates in macrophages reflected their known virulence, but the most virulent isolates strongly induced production of the cytokine tumor necrosis factor ␣. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains after several days in culture. Staining of cell monolayers for DNA fragmentation indicative of apoptosis showed that this was minimal and only evident to any degree in macrophages infected with the most virulent strains. In contrast, electron microscopy revealed damage of macrophages consistent with cell necrosis. These results suggest that rapid intracellular growth rate and induction of necrotic cell death within host macrophages are virulence factors of M. tuberculosis in the early stages of bacterial infection. They further imply that infected cell apoptosis, regarded as a defense mechanism or cross-priming mechanism, plays a minimal role.

The Journal of Immunology, 2022

Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2... more Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. In this study, we evaluated immune and endothelial blood cell profiles of patients with coronavirus disease 2019 to determine critical differences between those with mild, moderate, or severe COVID-19 using spectral flow cytometry. We examined a suite of immune phenotypes, including monocytes, T cells, NK cells, B cells, endothelial cells, and neutrophils, alongside surface and intracellular markers of activation. Our results showed progressive lymphopenia and depletion of T cell subsets (CD3 + , CD4 + , and CD8 + ) in patients with severe disease and a significant increase in the CD56 + CD14 + Ki67 + IFN-g + monocyte population in patients with moderate and severe COVID-19 that has not been previously described. Enhanced circulating endothelial cells (CD45 2 CD31 + CD34 + CD146 + ), circulating endothelial progenitors (CD45 2 CD31 + CD34 +/2 CD146 2 ), and neutrophils (CD11b + CD66b + ) were coevaluated for COVID-19 severity. Spearman correlation analysis demonstrated the synergism among age, obesity, and hypertension with upregulated CD56 + monocytes, endothelial cells, and decreased T cells that lead to severe outcomes of SARS-CoV-2 infection. Circulating monocytes and endothelial cells may represent important cellular markers for monitoring postacute sequelae and impacts of SARS-CoV-2 infection during convalescence and for their role in immune host defense in high-risk adults after vaccination.

Vaccine, 2020

Hemicellulases from microbial cells have become biotechnologically important since they hydrolyze... more Hemicellulases from microbial cells have become biotechnologically important since they hydrolyze complex polysaccharides of plant tissues into simple molecules. In paper and pulp industry, role of hemicellulases is well established and recently its applicability in the food and feed technology, coffee extraction, oil drilling and detergent industry also have been found. Hemicellulases are enzymes produced mainly from microorganisms but its production from plants and animals have also been reported. Bacterial hemicellulases are mostly extracellular enzymes that can act in a wide pH and temperature range, though acidic and neutral hemicellulases are more common. In this review we will focus on complex hemicellulases structure and the microbial enzyme complex involved in its complete breakdown, hemicellulases sources, its production conditions and their applications in the commercial sector. However, the major emphasis of the review is on the microbial hemicellulases but reference to plant and animal hemicellulases also has been made to complete the overview.

Infection with Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Di... more Infection with Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of the Spike S1 protein encoding mRNA, incorporated in a lipid nanoparticle, stabilized by polyethylene glycol (PEG). mRNA vaccines are novel in many respects, including cellular uptake, the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences encoding the SARS-CoV-2 Spike S1 protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to induction of virus neutralizing antibodies and cytotoxic T-lymphocytes will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side e...

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of v... more The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX™) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopath...

Scientific Reports, 2020

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; ho... more Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The ...

Minipigs as a neonatal animal model for tuberculosis vaccine efficacy testing

Veterinary Immunology and Immunopathology, 2019

Many vaccines against childhood diseases are administered early after birth, but vaccine developm... more Many vaccines against childhood diseases are administered early after birth, but vaccine development studies frequently test efficacy in adult rather than in neonatal animal models. In countries with endemic tuberculosis (TB), Bacillus Calmette-Guerin (BCG) is administered as part of the neonatal vaccine regimen because it prevents against the disseminated form of TB in children, although it has variable efficacy against pulmonary TB. Several promising new vaccines against TB are currently being tested in adult animal models. Here we evaluated neonatal piglets as an animal model to test vaccine efficacy. For this purpose, minipigs were vaccinated or not with BCG 48 h after birth and their immune response followed longitudinally until adolescence. We characterized the memory and activation phenotype of T cells, cytokine profile, and monocyte activation in response to BCG stimulation from 4 weeks of age into adolescence- age of 24 weeks. Immunological responses in vaccinated and non-vaccinated animals were further monitored upon infection with a low dose exposure to Mycobacterium tuberculosis strain HN878 via the aerosol route. Comparing the immunological response elicited by BCG vaccination in minipigs vs similar studies in infants, suggest that minipigs have the potential to serve as an effective neonatal animal model for vaccine development.

Tuberculosis (Edinburgh, Scotland), 2017

In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Curren... more In endemic countries more than 20% of tuberculosis (TB) cases are in infants and children. Current animal models study TB during adulthood but animal models for infant TB are scarce. Here we propose that minipigs can be used as an animal model to study adult, adolescent and infant TB including natural transmission. In these studies, two-month old minipigs (representing infant age in humans) and six-month old minipigs (representing adolescence in humans) were infected via the aerosol route with hyper-virulent clinical strain W-Beijing Mycobacterium tuberculosis (Mtb) HN878 and were monitored for 11 or 36 weeks post-challenge, respectively. In the same studies, infected and unchallenged animals were housed together. Viable bacteria were recovered from pulmonary and thoracic lymph nodes from both -infected and their initially unchallenged natural contacts. Bacillary load, gross lesions and histopathology revealed similarities to the spectrum of disease observed in human TB. The study d...

Antimicrobial Agents and Chemotherapy, 2015

Over the last 10 years,Mycobacterium abscessusgroup strains have emerged as important human patho... more Over the last 10 years,Mycobacterium abscessusgroup strains have emerged as important human pathogens, which are associated with significantly higher fatality rates than any other rapidly growing mycobacteria. These opportunistic pathogens are widespread in the environment and can cause a wide range of clinical diseases, including skin, soft tissue, central nervous system, and disseminated infections; by far, the most difficult to treat is the pulmonary form. Infections withM. abscessusare often multidrug-resistant (MDR) and require prolonged treatment with various regimens and, many times, result in high mortality despite maximal therapy. We report here the evaluation of diverse mouse infection models for their ability to produce a progressive high level of infection withM. abscessus. The nude (nu/nu), SCID (severe combined immunodeficiency), gamma interferon knockout (GKO), and granulocyte-macrophage colony-stimulating factor (GMCSF) knockout mice fulfilled the criteria for an opt...

The open AIDS journal, 2011

Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in ... more Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis. Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijin...

BMC Microbiology, 2015

Background: Macrophages are the primary effector cells responsible for killing Mycobacterium tube... more Background: Macrophages are the primary effector cells responsible for killing Mycobacterium tuberculosis (MTB) through various mechanisms, including apoptosis. However, MTB can evade host immunity to create a favorable environment for intracellular replication. MTB-infected human macrophages produce interleukin-32 (IL-32). IL-32 is a pro-inflammatory cytokine and has several isoforms. We previously found that IL-32γ reduced the burden of MTB in human macrophages, in part, through the induction of caspase-3-dependent apoptosis. However, based on our previous studies, we hypothesized that caspase-3-independent death pathways may also mediate IL-32 control of MTB infection. Herein, we assessed the potential roles of cathepsin-mediated apoptosis, caspase-1-mediated pyroptosis, and apoptosis-inducing factor (AIF) in mediating IL-32γ control of MTB infection in THP-1 cells. Results: Differentiated human THP-1 macrophages were infected with MTB H37Rv alone or in the presence of specific inhibitors to caspase-1, cathepsin B/D, or cathepsin L for up to four days, after which TUNEL-positive cells were quantified; in addition, MTB was quantified by culture as well as by the percentage of THP-1 cells that were infected with green fluorescent protein (GFP)-labeled MTB as determined by microscopy. AIF expression was inhibited using siRNA technology. Inhibition of cathepsin B/D, cathepsin L, or caspase-1 activity significantly abrogated the IL-32γ-mediated reduction in the number of intracellular MTB and of the percentage of GFP-MTB-infected macrophages. Furthermore, inhibition of caspase-1, cathepsin B/D, or cathepsin L in the absence of exogenous IL-32γ resulted in a trend toward an increased proportion of MTB-infected THP-1 cells. Inhibition of AIF activity in the absence of exogenous IL-32γ also increased intracellular burden of MTB. However, since IL-32γ did not induce AIF and because the relative increases in MTB with inhibition of AIF were similar in the presence or absence of IL-32γ, our results indicate that AIF does not mediate the host-protective effect of IL-32γ against MTB. The anti-MTB effects of IL-32γ are mediated through classical caspase-3-dependent apoptosis as well as caspase-3-independent apoptosis.

Tuberculosis, 2009

Enhancing immunity to tuberculosis in animal models after exposure to the infection has proved di... more Enhancing immunity to tuberculosis in animal models after exposure to the infection has proved difficult. In this study we used a newly described flow cytometric technique to monitor changes in cell populations accumulating in the lungs of guinea pigs challenged by low dose aerosol infection with Mycobacterium tuberculosis and vaccinated ten days later. On day forty after infection the fusion protein F36 and a pool of Ag85A and ESAT-6 vaccines had significant effects on the bacterial load, showed increased expression of the activation marker CD45+ on CD4+ T cells, and reduced numbers of heterophils. Lung pathology and pathology scores were marginally improved in animals given these vaccines, but lymph node pathology was not influenced. Despite early effects no changes in long term survival were seen. These results suggest that a single postexposure vaccination can initially slow the disease process. However, this effect is transient, but this could be of use in an multidrug resistant/extremely drug resistant outbreak situation because it could potentially slow the infection long enough to complete drug susceptibility testing and initiate effective chemotherapy.

Memory T cell subsets in tuberculosis: What should we be targeting?

Tuberculosis, 2014

The purpose of vaccination is to establish a stable population of long lived memory T cells. In t... more The purpose of vaccination is to establish a stable population of long lived memory T cells. In the context of tuberculosis, the BCG vaccine has been widely used for well over 60 years, but during that time its weaknesses, particularly its ineffectiveness in adults, has been increasingly recognized. In this commentary we review what is known about memory T cells, both in general and in the context of their role in expressing specific acquired resistance to tuberculosis. Current knowledge indicates that both effector memory and central memory can be generated, depending on the experimental conditions, but both in animal models and in clinical studies it is clear that effector memory T cells are the predominant subset. These issues are of importance, given the concerted effort to make new TB vaccines, not all of which may work in precisely the same manner. At the present time whether a TB vaccine would work better if it targeted one specific T cell subset rather than another is as yet completely unknown, and this is now further complicated by new evidence that suggests other subsets such as IL-17 secreting CD4 T cells and cells with stem cell-like qualities may also play important roles.

A mouse model of tuberculosis reinfection

Tuberculosis, 2012

Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis wh... more Recent clinical observations shows that individuals treated with chemotherapy for tuberculosis who live in endemic areas are four times more likely to develop secondary disease, often as not caused by exogenous reinfection. In a mouse model described here, we show that mice infected with the virulent W-Beijing Mycobacterium tuberculosis strain HN878, then given chemotherapy to clear the infection, were resistant to re-challenge with the same organism thereafter. This resistance, which was mediated by rapid expression of CD4 T cells expressing markers consistent with both central and effector memory immunity, was only transient however. After 20-30 days of the reinfection the numbers of these cells steadily declined, the bacterial load in the lungs surged up, and the lung tissues became increasingly consolidated. No evidence was found for a regulatory T cell response in these mice, but many T cells harvested from the lungs showed evidence of increased PD-1 expression, indicating exhaustion. These data indicate that the memory T cell response to reinfection may not be as stable and long lived as previously thought, a finding with obvious implications for vaccine development.

Vaccine, 2012

Bacillus-Calmette-Guerin (BCG), the only human tuberculosis vaccine, primes a partially protectiv... more Bacillus-Calmette-Guerin (BCG), the only human tuberculosis vaccine, primes a partially protective immune response against M. tuberculosis infection in humans and animals. In guinea pigs, BCG vaccination slows the progression of disease and reduces the severity of necrotic granulomas, which harbor a population of drug-tolerant bacilli. The objective of this study was to determine if reducing disease severity by BCG vaccination of guinea pigs prior to M. tuberculosis challenge enhanced the efficacy of combination drug therapy. At 20 days of infection, treatment of vaccinated and non-vaccinated animals with rifampin, isoniazid, and pyrizinamide (RHZ) was initiated for 4 or 8 weeks. On days 50, 80 and 190 of infection (10 weeks after drug were withdrawn), treatment efficacy was evaluated by quantifying clinical condition, bacterial loads, lesion severity, and dynamic changes in peripheral blood and lung leukocyte numbers by flow cytometry. In a separate, long-term survival study, treatment efficacy was evaluated by determining disease reactivation frequency post-mortem. BCG vaccination alone delayed pulmonary and extra-pulmonary disease progression, but failed to prevent dissemination of bacilli and the formation of necrotic granulomas. Drug therapy either alone or in combination with BCG, was more effective at lessening clinical disease and lesion severity compared to control animals or those receiving BCG alone. Fewer residual lesions in BCG vaccinated and drug treated animals, equated to a reduced frequency of reactivation disease and improvement in survival even out to 500 days of infection. The combining of BCG vaccination and drug therapy was more effective at resolving granulomas such that fewer animals had evidence of residual infection and thus less reactivation disease.

Vaccine, 2007

A mutant of Mycobacterium tuberculosis lacking the 19-kilodalton lipoprotein Rv3763 is highly att... more A mutant of Mycobacterium tuberculosis lacking the 19-kilodalton lipoprotein Rv3763 is highly attenuated in vivo but retains potent vaccinogenic properties

Tuberculosis, 2011

Predictive biomarkers illustrating the effectiveness of chemotherapeutic regimens for tuberculosi... more Predictive biomarkers illustrating the effectiveness of chemotherapeutic regimens for tuberculosis still remain elusive. To date, most are predicated on assays using sputum or serum; as a result, if not predictive, treatment failure in patients may not be evident for some time. We report here the results of a simple screening study in which T cell surface markers were examined in mice infected with Mycobacterium tuberculosis and then treated with drugs. These studies identified certain markers, the exhaustion markers PD-1 and TIM-3, as well as the marker KLRG-1, particularly on CD8 T cells, that changed in concert with reduction of the bacterial load in the lungs. While there is no guarantee these changes would also be seen on T cells in the blood, this approach should be further investigated.

Tuberculosis, 2011

There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to ... more There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to the W-Beijing genotype of newly emerging strains are often of very high virulence when tested in small animal models, including the mouse and guinea pig. In this report we provide further evidence to support this contention, and show that two W-Beijing strains are of very high virulence when introduced by low dose aerosol into out-bred guinea pigs. In addition to severe lung pathology, each of these infections was associated with large influxes of activated CD4 and CD8 T cells into the lungs. Large influxes of macrophages were also observed, but the fraction of these showing evidence of activation by Class-II expression was relatively low. A progressive increase in neutrophils was also seen, with highest levels accumulating in the lungs of the W-Beijing infected animals. In the case of these two infections mRNA levels for TH1 cytokines was elevated early, but these then declined, and were replaced by increasing levels of message encoding for Foxp3, IL-10, and TGFβ. These observations support the hypothesis that W-Beijing strains are potent inducers of regulatory T cells, and that this event may enhance survival and transmission of these bacilli.

PLoS ONE, 2011

Supporting Information S1 (A) Partial sequencing of the hsp65 and rpoB genes of M. massiliense CI... more Supporting Information S1 (A) Partial sequencing of the hsp65 and rpoB genes of M. massiliense CIP 108297 and CRM-0019. The corresponding sequences of M. abscessus ATCC 19977 are included in the alignments as a reference. PCR amplification were performed as described by Ringuet et al. (1999) and Kirshner et al. (1993) (see references below).

Nature Medicine, 2011

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 re... more We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Desx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4 + memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4 + cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.

Journal of Leukocyte Biology, 2005

In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosi... more In this study, we investigated the ability of four clinical isolates of Mycobacterium tuberculosis representing a range of virulence for their capacity to grow in bone marrow-derived macrophages. The rate of growth of each of the isolates in macrophages reflected their known virulence, but the most virulent isolates strongly induced production of the cytokine tumor necrosis factor ␣. A key difference, however, was the degree of cell cytotoxicity observed with the more virulent strains after several days in culture. Staining of cell monolayers for DNA fragmentation indicative of apoptosis showed that this was minimal and only evident to any degree in macrophages infected with the most virulent strains. In contrast, electron microscopy revealed damage of macrophages consistent with cell necrosis. These results suggest that rapid intracellular growth rate and induction of necrotic cell death within host macrophages are virulence factors of M. tuberculosis in the early stages of bacterial infection. They further imply that infected cell apoptosis, regarded as a defense mechanism or cross-priming mechanism, plays a minimal role.