'Rescue' of mini-genomic constructs and viruses by combinations of morbillivirus N, P and L proteins.

Chloramphenicol acetyltransferase (CAT)-expressing negative-sense mini-genomic constructs of measles virus (MV) and rinderpest virus (RPV) were rescued by standard technology with helper plasmids expressing the nucleocapsid (N), phospho- (P) and large (L) proteins of MV, canine distemper virus (CDV) or RPV in order to determine whether the proteins of different viruses can function together. Homogeneous sets consisting of N, P and L plasmids derived from one virus were able to generate reporter gene expression from either mini-genomic construct. Heterogeneous sets of proteins from different viruses were not functional, with the exception that a low level of activity was obtained when MV N and P protein were combined with RPV L protein in the rescue of the MV mini-genomic construct, or CDV N was combined with RPV P and L in the rescue of the RPV mini-genome. However, only homogeneous sets of plasmids were able to rescue infectious virus from full-length anti-genome-expressing plasmids.

Susceptibility of mice deficient in the MHC class II transactivator to infection with Mycobacterium tuberculosis.

Major histocompatibility complex (MHC) class II antigen presentation and subsequent CD4+ T-cell activation are critical for acquired immunity to Mycobacterium tuberculosis infection. MHC class II gene expression is primarily controlled by the master transactivator CIITA protein. Without functional CIITA protein, MHC class II expression is lost, impairing immune responses and increasing susceptibility to infection. In this study, we compared protective immune responses of CIITA-deficient mice and wild-type C57BL/6 controls with low dose aerosol M. tuberculosis infection. After aerogenic challenge, CIITA-/- mice failed to limit mycobacterial growth (2.5 and 2.0 log10 > WT lung and spleen CFUs, respectively, at day 58). Lung histopathology involved extensive necrosis, severe pneumonitis and overwhelming inflammation in the gene knockout mice. Mean survival time for CIITA-/- mice was significantly reduced (57 versus >300 days for WT). This extreme sensitivity to tuberculous infection was largely attributed to the absence of CD4+ cells. Flow cytometric studies detected virtually no CD4+ cells in CIITA-/- mouse spleens after infection versus elevated numbers in WT spleens. Failed CD4+ T-cell expansion markedly reduced interferon-gamma (IFN-gamma production in CIITA-/- mice versus WT controls. These results suggest the necessity of a functional CIITA pathway for controlling tuberculous infections and that interventions targeting CIITA expression may be useful antimycobacterial therapeutics.

Progressive pulmonary tuberculosis is not due to increasing numbers of viable bacilli in rabbits, mice and guinea pigs, but is due to a continuous host response to mycobacterial products.

Tuberculosis (TB) kills more people in the world today than any other infectious disease. A better vaccine to prevent clinical tuberculosis is greatly needed. Candidate vaccines are often evaluated by infecting rabbits, mice and guinea pigs by an aerosol of virulent tubercle bacilli and culturing their lungs for viable bacilli at various times thereafter. In all three species, however, the number of viable bacilli usually does not continuously increase until the host succumbs. The number of viable bacilli increases logarithmically for only about 3 weeks. Then, the host develops delayed-type hypersensitivity (DTH) and cell-mediated immunity (CMI), which keep the number of viable bacilli rather constant during the subsequent weeks. In the immunized host, DTH and CMI stop the logarithmic increase sooner than in the unimmunized controls, so that the stationary bacillary levels that follow are lower. This review analyzes host-parasite interactions in the lungs of rabbits, mice and guinea pigs. All three species cannot prevent inhaled fully virulent tubercle bacilli from establishing an infection, but they differ markedly in the type of the disease produced once it is established.

Establishment of a rescue system for canine distemper virus.

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.

DNA vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity.

Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-gamma)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-gamma, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice with Mycobacterium tuberculosis indicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-gamma-secreting cells, and significant resistance to a tuberculous challenge.

An aerosol challenge mouse model for Moraxella catarrhalis.

A simple, reproducible, and non-invasive mouse pulmonary clearance model for Moraxella catarrhalis via aerosol challenge was established. All of eight tested strains could be inoculated into mice at more than 10(5) colony-forming units (CFU)/lung with a challenge concentration of 1x10(9)-6x10(9) CFU/ml in a nebulizer. The number of bacteria retained at 6 h postchallenge was more than 10(4) CFU/lung while at 24 h postchallenge, approximate 10(3) CFU/ml or less remained in the lungs. A maximum of 100 mice could be challenged per aerosol exposure. The number of bacteria inoculated in the lungs could be adjusted by the bacterial challenge concentration, the exposure time, and the negative pressure. Lung tissue sections revealed that bacteria were evenly distributed in the lungs. Passive immunization significantly enhanced pulmonary clearance of the homologous strain in this model. These data indicate that this model will be useful for evaluating M. catarrhalis vaccine candidates and studying roles of immunity against M. catarrhalis.

Propelling novel vaccines directed against tuberculosis through the regulatory process.

The development of novel vaccines for use in the prevention and immunotherapy of tuberculosis is an area of intense interest for scientific researchers, public health agencies and pharmaceutical manufacturers. Development of effective anti-tuberculosis vaccines for use in specific target populations will require close cooperation among several different international organizations including agencies responsible for evaluating the safety and effectiveness of new biologics for human use. In this review, the major issues that are addressed by regulatory agencies to ensure that vaccines are pure, potent, safe, and effective are discussed. It is hoped that the comments provided here will help accelerate the development of new effective vaccines for the prevention and treatment of tuberculosis.

Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis.

Mycobacterium smegmatis is typically used as a bacterial host for cloning and expressing single genes or genomic libraries of the human pathogen Mycobacterium tuberculosis. To study virulence of M. tuberculosis, we set out to ask the question, whether a genomic library derived from M. tuberculosis H37Rv confers virulence to the non-virulent M. smegmatis. A representative library from the M. tuberculosis H37Rv genome was generated and transformed into wild-type M. smegmatis. Mice were challenged with recombinant clones by intravenous, aerogenic and intranasal infection. We were unable to detect either growth or persistence of recombinant clones in tissues of infected mice; instead, the infection was cleared. Since the concern that virulent traits might be transferred, bio-safety regulations often require the handling of these experiments at bio-safety Level 3. However, we failed to find any evidence that the M. tuberculosis library confers virulence when expressed in M. smegmatis. We suggest that the results, presented here, should fundamentally alter the containment requirements for similar experiments in the future.

Growth of a highly virulent strain of Mycobacterium tuberculosis in mice of differing susceptibility to tuberculous challenge.

Mycobacterium tuberculosis strain CDC 1551 exhibited unusually high levels of infectivity and virulence during a recent outbreak of tuberculosis in a rural community. Mice infected intravenously or aerogenically with M. tuberculosis CDC 1551 developed infections in the lungs and spleen which were almost identical with those for M. tuberculosis strains Erdman, H37Rv and Indian. There was also no significant difference in the survival rates of mice infected intravenously with CDC 1551, Erdman or H37Rv over a 3-month period. Studies designed to detect differences in the growth rates of CDC 1551 and Erdman in 3 inbred strains of mice failed to explain the high level of infectiousness and virulence expressed by CDC 1551 in human populations exposed to this organism.

Mycobacterial pathogenesis: a historical perspective.

Tuberculosis is an age-old human affliction which continues to flourish worldwide despite the development of effective drugs for its treatment and a vaccine (BCG) for its prevention. At least 8 million people die from this disease each year, a figure which is likely to increase as the AIDS epidemic continues its relentless spread into Africa and Southeast Asia. Consumption was shown to be caused by Mycobacterium tuberculosis more than a century ago, yet we still know very little about the mechanisms used by this organism to elude the normally effective cellular host defenses as it establishes a progressive infection within the lung. The majority of individuals exposed to tuberculous infection are able to limit the primary infection to the lungs and its lymph nodes, resulting in a latent form of the disease which can provide the host with a lifelong immunity to reinfection. While a great deal is known about the cellular mediators of this immune response (together with the cytokines which modulate them) we lack a clear understanding of the role that they play during the establishment of the dormant form of the disease. Live BCG vaccine has been widely used in many Third World countries as a major component of their tuberculosis control programs. However, several carefully controlled human trials have shown little protection achieved in vaccinated individuals. Development of improved vaccines, both for the prevention and therapy of this disease is an urgent research priority and a number of potential immunogens are under active investigation. However, our limited understanding of the pathogenesis of this chronic disease, together with a lack of data on the role played by different bacterial components in the modulation of the immune response, continues to severely limit our ability to develop a rational approach to this project. To achieve this goal, it will be necessary to establish innovative approaches to the presentation of protective antigens by taking advantage of recent advances in the molecular biology of this complex and enigmatic group of organisms.

Cerebrovascular consequences of repeated exposure to NG-nitro-L-arginine methyl ester.

1. Acute treatment with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) produces cerebral oligaemia. The effects of repeated exposure to L-NAME upon cerebral blood flow were examined to determine whether the enhanced NOS inhibition reported following chronic treatment might reduce cerebral perfusion to ischaemic levels. 2. Rats were treated with L-NAME (75 mg kg-1, i.p.) once daily for 10 days. Local cerebral blood flow and glucose utilization were measured by [14C]-iodoantipyrine and [14C]-2-deoxyglucose quantitative autoradiography respectively, either 1 h or 15 h after the last injection. A second group of rats was injected (i.p.) only once with L-NAME, either 1 h or 15 h prior to the measurement procedures. 3. Mean arterial blood pressure (MABP) was significantly increased (+35%) 1 h after a single injection of L-NAME. Although the hypertension was reduced 15 h after the injection (+13%), MABP remained significantly higher than control. 4. Local cerebral blood flow was significantly decreased 1 h after a single injection of L-NAME (ranging from -45% to -54%), and remained so even after 15 h (-39% to -48%). At neither time-point was there any change in glucose utilization. 5. At 15 h after the final injection of the chronic L-NAME treatment protocol, MABP was significantly elevated from control (+58%) and was also significantly higher than at 1 h following a single injection (+20%). There was no effect upon the established hypertension when rats treated chronically with L-NAME were challenged with a further injection of the drug and MABP was measured 1 h later, suggesting saturation of NOS inhibition. 6. Although reduced, cerebral blood flow was not significantly different from control when measured 15 h after the last injection of the chronic L-NAME treatment. When rats treated chronically with L-NAME were subjected to a further challenge with the drug, cerebral blood flow was reduced when measured 1 h after the acute injection (ranging from -34% to -41%). There was however evidence of some attenuation in the response when compared to that measured 1 h after a single injection of L-NAME (ranging from -45% to -54%). Thus, the cerebral circulation shows no evidence of either sustained L-NAME-induced vasoconstriction or saturated NOS inhibition following 10 daily injections of L-NAME. Chronic L-NAME treatment had no effect upon cerebral glucose use. 7. The trend towards re-establishment of cerebrovascular dilator tone and the normalization of cerebral flow-metabolism relationships could explain the lack of any ischaemic damage found in chronically treated rats, but the loss of an extended autoregulatory range afforded by acute L-NAME treatment may be responsible for an increased incidence of stroke.

The immune response to mycobacterial infection: development of new vaccines.

Pulmonary tuberculosis continues to flourish worldwide despite our most vigorous attempts to control it. After nearly a century of study we still know very little about the virulence factors of M. tuberculosis of M. bovis or how they trigger the protective immune response within the infected host. This anti-tuberculous response is mediated by a population of specifically sensitised T lymphocytes which activate the monocytes entering the developing lesion from the bloodstream. The immunologically activated macrophage induces a persistent bacteriostasis which is usually sufficient to protect the host although it will not eliminate the infection altogether so that reactivation can occur whenever the cellular defences are depleted as a result of aging or immunosuppressive chemotherapy. Protective immunogens released by actively growing tubercle bacilli give rise to a protective cell-mediated, rather than a humoral (non-protective) immunity. The genes responsible for the production of these "protective" antigens are being cloned and transferred to suitable mycobacterial vectors by means of the newly developed "shuttle phasmid". Development of such recombinants constitute the first step in preparing more effective anti-tuberculous vaccines for future use against these important human and animal pathogens.

Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence.

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

Differential release of tumor necrosis factor-alpha from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria.

The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-alpha compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-alpha; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-alpha secretion. The TNF-alpha response was dose-dependent, related directly to the percentage of infected cells, and peaked 6-12 h post-infection. An early and vigorous TNF-alpha response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.

Identification of a genomic fragment of Mycobacterium tuberculosis responsible for in vivo growth advantage.

The purpose of this work was to develop a system to identify virulence determinants of M. tuberculosis by genetic complementation. The ability to grow in mouse spleen and/or lung was defined as a potential phenotype for virulence. Enrichment for growing recombinant clones from a pool of H37Ra transformants containing the integrating pYUB178::H37Rv cosmid library was accomplished by in vivo selection. A molecular strategy was devised to isolate and clone the 25-kb H37Rv genomic fragment ivg that conferred in vivo growth advantage to H37Ra. This study is a first step toward understanding the genetics of virulence in M. tuberculosis. A detailed description of these experiments has been submitted for publication in Infection and Immunity.

Tuberculosis: the return of an old enemy.

Tuberculosis is an ancient human scourge that continues to be an important public health problem worldwide. The increasing number of multidrug-resistant (MDR) M. tuberculosis isolates from both AIDS and non-AIDS patients is an ominous trend that threatens tuberculosis eradication programs both in the U.S. and overseas. New antituberculosis vaccines with therapeutic properties are urgently needed for human immunodeficiency virus-infected individuals, as well as health care professionals likely to be exposed to MDR tubercle bacilli. Recombinant DNA vaccines bearing protective genes from virulent M. tuberculosis are being developed using shuttle phasmids to transfer genetic material from one mycobacterial species to another. Improved assay procedures are needed to measure the protection afforded by these new vaccines under experimental and field test conditions. Tuberculosis vaccine development should be given a high priority in current medical research goals.

Immunization of mice with the antigen A60 of Mycobacterium bovis BCG.

Antigen 60 (A60) is a thermostable component of the cytoplasm of Mycobacterium tuberculosis and BCG which can be fractionated into at least 15 protein bands when analysed by Western blot. Normal B6D2 mice were immunized subcutaneously with 20 micrograms of the A60 protein suspended in Freund's incomplete adjuvant (FIA) or in saline. Three weeks later the mice received a second dose of vaccine followed 2 weeks later by an aerogenic challenge with approximately 10(3) CFU of M. tuberculosis Erdman. The mice receiving the adjuvanted A60 showed a significant reduction (P less than 0.05) in the number of viable organisms recovered from the lungs and the spleen 3 weeks after challenge. However, this response was less than that seen in BCG vaccinated controls.