Epithelial-fibroblast interactions in IPF: Lessons from in vitro co-culture studies.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial disease that is characterized by increased cellular proliferation and differentiation together with excessive extracellular matrix (ECM) deposition leading to buildup of scar tissue (fibrosis) and remodeling in the lungs. The activated and differentiated (myo)fibroblasts are one of the main sources of tissue remodeling in IPF and a crucial mechanism known to contribute to this feature is an aberrant crosstalk between pulmonary fibroblasts and the abnormal or injured pulmonary epithelium. This epithelial-fibroblast interaction mimics the temporal, spatial and cell-type specific crosstalk between the endoderm and mesoderm in the so-called epithelial-mesenchymal trophic unit (EMTU) during lung development that is proposed to be activated in healthy lung repair and dysregulated in various lung diseases including IPF. To study the dysregulated lung EMTU in IPF, various complex in vitro models have been established. Hence, in this review, we will provide a summary of studies that have used complex (3-dimensional) in vitro co-culture, and organoid models to assess how abnormal epithelial-fibroblast interactions in lung EMTU contribute to crucial features of the IPF including defective cellular differentiation, proliferation and migration as well as increased ECM deposition.

Understanding fibroblast-immune cell interactions via co-culture models and their role in asthma pathogenesis.

Asthma is a chronic lung disease involving airway inflammation and fibrosis. Fibroblasts are the main effector cells important for lung tissue production which becomes abnormal in asthmatics and is one of the main contributors to airway fibrosis. Although fibroblasts were traditionally viewed solely as structural cells, they have been discovered to be highly active, and involved in lung inflammatory and fibrotic processes in asthma. In line with this, using 2D and 3D in vitro co-culture models, a complex interaction between lung fibroblasts and various immune cells important for the pathogenesis of asthma have been recently uncovered. Hence, in this review, we provide the first-ever summary of various studies that used 2D and 3D in vitro co-culture models to assess the nature of aberrant immune cell-fibroblast interactions and their contributions to chronic inflammation and fibrotic mechanisms in asthma pathogenesis.

Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles.

Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remain an elusive goal. The highly conserved 30 amino-acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:Boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to present HIV-1 envelope glycoprotein neutralizing determinants to the immune system.

Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets.

Vaccine-induced antibodies that interfere with viral entry are the protective correlate of most existing prophylactic vaccines. However, for highly variable viruses such as HIV-1, the ability to elicit broadly neutralizing antibody responses through vaccination has proven to be extremely difficult. The major targets for HIV-1 neutralizing antibodies are the viral envelope glycoprotein trimers on the surface of the virus that mediate receptor binding and entry. HIV-1 has evolved many mechanisms on the surface of envelope glycoproteins to evade antibody-mediated neutralization, including the masking of conserved regions by glycan, quaternary protein interactions and the presence of immunodominant variable elements. The primary challenge in the development of an HIV-1 vaccine that elicits broadly neutralizing antibodies therefore lies in the design of suitable envelope glycoprotein immunogens that circumvent these barriers. Here, we describe neutralizing determinants on the viral envelope glycoproteins that are defined by their function in receptor binding or by rare neutralizing antibodies isolated from HIV-infected individuals. We also describe the nonvariable cellular receptors involved in the HIV-1 entry process, or other cellular proteins, and ongoing studies to determine if antibodies against these proteins have efficacy as therapeutic reagents or, in some cases, as vaccine targets to interfere with HIV-1 entry.

Rational modifications of HIV-1 envelope glycoproteins for immunogen design.

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will likely require the elicitation of broadly neutralizing antibodies as well as cellular responses. The HIV exterior envelope glycoprotein trimers, gp120, and the transmembrane glycoprotein, gp41, mediate entry and are the sole viral targets for neutralizing antibodies. However, as subunit immunogens the envelope glycoproteins do not efficiently elicit antibodies capable of neutralizing the extremely diverse array of viruses circulating in the human population. The preponderance of data suggest that inefficient generation of broadly neutralizing antibodies is due to naturally evolved mechanisms of immune evasion inherent in the unmodified HIV envelope glycoproteins. Because the established modes of anti-viral vaccine development, live-attenuation and virus inactivation have not yet been successful for HIV, we and others have focused on subunit vaccine design. In this review, we describe current approaches of rational modification of the envelope glycoproteins based upon structure, antigenicity, biochemistry and biophysics to alter the properties of the envelope glycoproteins such that, as subunit immunogens, they now better elicit broadly neutralizing antibodies. The application of structure-assisted, rational subunit vaccine design may be a general paradigm for future efforts to develop vaccines against emerging human pathogens.

A four-element based transposon system for allele specific tagging in plants--theoretical considerations.

The two-element transposon constructs, utilizing either Ac/Ds or Spm/dSpm, allow random tagging of genes in heterologous model species, but are inadequate for directed tagging of specific alleles of agronomic importance. We propose the use of Ac/Ds in conjunction with Spm/dSpm to develop a four-element system for directed tagging of crop-specific alleles. The four-element based construct would include both Ds and dSpm along with relevant marker genes and would function in two steps. In the first step dSpm(Ds) stocks (a minimum of two) would be crossed to a line containing transposases of Spm and unlinked integrations would be selected from segregating population by the use of a negative selection marker to develop stocks representing integration of dSpm(Ds) at a large number of locations in the genome. Selections would be made for a line in which dSpm(Ds) shows partial or complete linkage to the allele of interest. In the second step selected line would be crossed to a line containing Ac transposase to induce transpositions of Ds element to linked sites thereby exploiting the natural tendency of Ds element to jump to linked sites. Unlinked jumps of dSpm(Ds) and linked jumps of Ds could be monitored by appropriate marker genes. The proposed model would allow tagging of allele of interest in chromosome addition lines and also help in the efficient use of genic male sterility systems for hybrid seed production by tightly marking the fertility restorer gene with a negative selection marker.