CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.
25 Related JoVE Articles!
Determining the Phagocytic Activity of Clinical Antibody Samples
Institutions: Ragon Institute of MGH, MIT, and Harvard, Dartmouth College.
Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6
. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7
. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 μm fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple FcγRs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of FcγRs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9
Immunology, Issue 57, Phagocytosis, Antibody, ADCC, Effector Function, Fc receptor, antibody-dependent phagocytosis, monocytes
Measuring the Induced Membrane Voltage with Di-8-ANEPPS
Institutions: University of Ljubljana.
Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ.
Cellular Biology, Issue 33, induced transmembrane voltage, induced membrane voltage, induced transmembrane potential, potentiometric dyes, di-8-ANEPPS, electroporation, electropermeabilization
Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients
Institutions: San Paolo Hospital University of Milan, Italy.
The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development
of the humoral and cellular mucosal immune system1,2
Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal
gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and
bacterial DNA fragments, which contribute to systemic immune activation4-7
. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene
followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in
these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric
16S ribosomial gene9
. Following PCR product purification, cloning and sequencing analyses are performed.
Medicine, Issue 52, Plasma DNA extraction, 16S rRNA gene PCR, sequencing analysis, HIV
Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production
Institutions: Keck School of Medicine, University of Southern California.
In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine γHV68 is a natural pathogen of murine rodents. γHV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo
virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that γHV68 hijacks MAVS and IKKβ, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NFΚB activation and antiviral cytokine production. Specifically, γHV68 infection activates IKKβ and that activated IKKβ phosphorylates RelA to accelerate RelA degradation. As such, γHV68 efficiently uncouples NFΚB activation from its upstream activated IKKβ, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.
Immunology, Issue 85, Herpesviridae, Cytokines, Antiviral Agents, Innate, gamma-HV68, mice infection, MEF, antiviral cytokine
Murine Superficial Lymph Node Surgery
Institutions: Maisonneuve-Rosemont Hospital Research Center, University of Montreal, University of Montreal.
In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4
. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106
cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11
. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.
Physiology, Issue 63, Immunology, mouse, lymph node, surgery, immune response, lymphocytes
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays
Institutions: NCI-Frederick, University of Pittsburgh, Tuffts University.
Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART).
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19
and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15
,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19
. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.
Immunology, Issue 55, single genome sequencing, SGS, real-time PCR, single-copy assay, SCA, HIV-1, ultra-sensitive, RNA extraction
Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty
Institutions: University of Arizona, University of Arizona.
A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2
. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.
Medicine, Issue 77, Infection, Virology, Infectious Diseases, Anatomy, Physiology, Molecular Biology, Biomedical Engineering, Retroviridae Infections, Body Weight Changes, Diagnostic Techniques and Procedures, Physical Examination, Muscle Strength, Behavior, Virus Diseases, Pathological Conditions, Signs and Symptoms, Diagnosis, Musculoskeletal and Neural Physiological Phenomena, HIV, HIV-1, AIDS, Frailty, Depression, Weight Loss, Weakness, Slowness, Exhaustion, Aging, clinical techniques
An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells
Institutions: CHUL (CHUQ), Quebec City, Quebec, Canada.
, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1
. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3
. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro
studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum
schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5
. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7
, but that it decreased HIV-1 infection of macrophages8
. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum
-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env
gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.
Immunology, Issue 66, Infection, Medicine, Malaria, HIV-1, Monocyte-Derived Macrophages, PBMC, Red blood cells, Dendritic Cells, Co-infections, Parasites, Plasmodium falciparum, AIDS
Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors
Institutions: National Cancer Institute.
Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.
Immunology, Issue 86, HIV, cytotoxicity, infectivity, luciferase, drug resistance, integrase, reverse transcriptase
Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery
Institutions: Beckman Research Institute of City of Hope, Beckman Research Institute of City of Hope, Beckman Research Institute of City of Hope.
The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery.
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).
Immunology, Issue 52, SELEX (Systematic Evolution of Ligands by EXponential enrichment), RNA aptamer, HIV-1 gp120, RNAi (RNA interference), siRNA (small interfering RNA), cell-type specific delivery
Isolation of Mouse Peritoneal Cavity Cells
Institutions: Blood Research Institute.
The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of naïve macrophages in the peritoneal cavity makes it a preferred site for the collection of naïve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+
B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.
JoVE Immunology, Issue 35, Immune cells, Peritoneal cavity, Macrophage, B cell, B1 cell, isolation procedure
Isolation of Mouse Lung Dendritic Cells
Institutions: Louisiana State University .
Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2
as well as in the control of tolerogenic responses 3
in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4
, plasmacytoid DC (pDC) 5
and the IFN-producing killer DC (IKDC) 6,7
. The cDC subset is the most prominent DC subset in the lung 8
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (β2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9
. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+
cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4/80, an splenic macrophage marker, the CD11chi
lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.
Immunology, Issue 57, Lung, dendritic cells, classical, conventional, isolation, mouse, innate immunity, pulmonary
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Peptide-based Identification of Functional Motifs and their Binding Partners
Institutions: Morehouse School of Medicine, Institute for Systems Biology, Universiti Sains Malaysia.
Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.
Virology, Issue 76, Biochemistry, Immunology, Infection, Infectious Diseases, Molecular Biology, Medicine, Genetics, Microbiology, Genomics, Proteins, Exosomes, HIV, Peptides, Exocytosis, protein trafficking, secretion, HIV-1, Nef, Secretion Modification Region, SMR, peptide, AIDS, assay
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice
Institutions: Virginia Polytechnic Institute and State University.
The host immune response to pathogens is a complex biological process. The majority of in vivo
studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.
Infection, Issue 86, LPS, Lipopolysaccharide, mouse, pneumonia, gram negative bacteria, inflammation, acute lung inflammation, innate immunity, host pathogen interaction, lung, respiratory disease
Identification of Post-translational Modifications of Plant Protein Complexes
Institutions: University of Warwick, Norwich Research Park, The Australian National University.
Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via
the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.
Plant Biology, Issue 84, plant-microbe interactions, protein complex purification, mass spectrometry, protein phosphorylation, Prf, Pto, AvrPto, AvrPtoB
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation
Institutions: Université de Montréal, Université de Montréal, Université de Montréal.
Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-γ production by T lymphocytes in response to in vitro
stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens.
Immunology, Issue 89, Varicella zoster virus, cell-mediated immunity, T cells, interferon gamma, ELISpot, umbilical cord blood transplantation
In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function
Institutions: The Ragon Institute of MGH, MIT and Harvard, Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM).
T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro
assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro
experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10Rα and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 °C, 5% CO2
. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.
Immunology, Issue 80, Virus Diseases, Immune System Diseases, HIV, CD4 T cell, CD8 T cell, antigen-presenting cell, Cytokines, immunoregulatory networks, PD-1: IL-10, exhaustion, monocytes
Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation
Institutions: La Jolla Institute for Allergy and Immunology.
Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens. Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells. The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells. Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy. Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.
Immunology, Issue 10, Natural Killer T cells, NKT cells, CD1 Tetramers, antigen presentation, glycolipid antigens, CD1d, Mucosal Immunity, Translational Research
Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase
Institutions: Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg.
HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.
Medicine, Issue 16, HIV, Cell Biology, Recombinase, provirus, HeLa Cells