Lethal Respiratory Infections Research Articles

Using the C57BL/6 and SKH1 Strains to Evaluate the Efficacy of CMX001 Following Lethal Respiratory Infections with Ectromelia Virus

Using the C57BL/6 and SKH1 Strains to Evaluate the Efficacy of CMX001 Following Lethal Respiratory Infections with Ectromelia Virus

Lethal pulmonary infection with Francisella novicida is associated with severe sepsis.

The bacterial or host determinants of lethality associated with respiratory Francisella infections are currently unknown. No exo- or endotoxins that contribute to the severity of this disease have been identified. However, a deregulated host immune response upon infection is characterized by an initial 36- to 48-h delay followed by a rapid and excessive inflammatory response prior to death at 72-120 h. Here, we extend these findings by comparing host immune responses between sublethal and lethal respiratory infections of mice with an attenuated transposon mutant (Mut) of F. novicida (F.n.) strain U112 (sublethal) versus the wild-type (WT) strain (lethal). Infection with WT bacteria, but not the Mut, was characterized by sustained bacteremia and systemic dissemination of the pathogen with temporal increases in bacterial burdens in liver and spleen. Severe pathology with large foci of infiltrates associated with extensive tissue damage was evident in WT-infected lungs, and Mut-infected mice displayed much reduced pathology with intact lung architecture. Similar to other experimental models of severe sepsis, WT- but not the Mut-infected mice exhibited a robust increase in numbers of Gr1+ and CD11b+ cells, while displaying a significant depletion of alphabeta T cells. Further, a dramatic up-regulation of multiple cytokines and chemokines was observed only in lethal WT infection. In addition, an earlier and larger increased expression of S100A9, a known mediator of sepsis, was observed in WT-infected mice. Taken together, these results show that a hyperinflammatory host immune response, culminating in severe sepsis, is responsible for the lethal outcome of respiratory tularemia.

IL‐12 p80‐dependent macrophage recruitment primes the host for increased survival following a lethal respiratory viral infection

A protective immune response to a respiratory viral infection requires a series of coordinated cellular and molecular responses. We have previously demonstrated that increased expression of airway epithelial cell interleukin (IL)-12 p80, a macrophage chemoattractant, is associated with human respiratory viral infection and mediates post-viral mortality in the mouse. To better understand the role of IL-12 p80-dependent macrophage chemotaxis in mediating viral immunity, we generated a transgenic mouse strain utilizing a promoter to drive IL-12 p40 gene expression in the airway epithelium. This transgenic strain secreted biologically active IL-12 p80 in a lung-specific manner, and demonstrated a selective increase in the number of resident, unactivated airway macrophages at baseline. Following infection with a sublethal dose of mouse parainfluenza virus type 1 (Sendai virus), the transgenic mice demonstrated an earlier peak and decline in the number of airway inflammatory cells. The transgenic mice were resistant to a lethal dose of virus and this viral resistance was dependent on the increased number of airway macrophages at baseline as partial depletion prior to infection abrogated this phenotype. The survival advantage in the transgenic mice was independent of viral load but was associated with a more rapid decline in the number of airway inflammatory cells and concentrations of multiple chemokines including the CC chemokine ligand 2 (CCL2)/JE, CCL3/macrophage inflammatory protein (MIP)-1alpha, CCL4/MIP-1beta, and CCL5/RANTES. Collectively, these results suggest that IL-12 p80-driven increases in the number of resident airway macrophages prime the host for a protective immune response that can confer increased survival following a lethal respiratory viral infection.

Mucosal Vaccine Using CTL Epitope-Pulsed Dendritic Cell Confers Protection for Intracellular Pathogen

Effective protective immunity against respiratory infections with intracellular pathogens requires pathogen-specific cytotoxic T cells (CTL) in the lung. However, vaccines that induce pathogen-specific CTL in the lung are poorly explored. Dendritic cells (DC) have increasingly been exploited as vaccines against infections. However, few studies have investigated the ability of mucosal DC vaccines to elicit protective CTL responses in the lung. Our objective was to develop an efficacious mucosal DC vaccine to generate protective CTL against respiratory infections with intracellular pathogens. Bone marrow-derived DC (BM-DC) pulsed with a single immunodominant CTL epitope, listeriolysin O (LLO) 91-99, of Listeria monocytogenes (LM) were intratracheally administered into mice. The frequency and function of epitope-specific CTL in mediastinal lymph nodes (MLN) and spleen were assessed for their ability to protect against LM infection. After intratracheal administration, lipopolysaccharide (LPS)-treated LLO 91-99-loaded BM-DC (LPS-LLO DC) more frequently migrated to MLN than LPS-untreated LLO 91-99-loaded BM-DC (LLO DC). Using tetrameric H2-K(d)/LLO 91-99 peptide complex, specific CD8(+) T cells were found in MLN as well as the spleen in LPS-LLO DC-immunized mice, but not in LLO-DC-immunized mice. Both MLN and spleen cells obtained from LPS-LLO DC-immunized mice produced large amounts of IFN-gamma in response to LLO 91-99 with high epitope-specific CTL activities. Vaccination with LPS-LLO DC, but not LLO DC, protected mice against lethal respiratory infection with LM. These data suggest that mucosal vaccination with LPS-treated immunodominant CTL epitope-loaded DC is a promising strategy for generating protective CTL against respiratory infections with intracellular pathogens.

Mousepox in the C57BL/6 strain provides an improved model for evaluating anti-poxvirus therapies

The intranasal lethal mousepox model employing the A/Ncr mouse strain is used to evaluate anti-orthopoxvirus therapies. These infections mimic large droplet transmission and result in 100% mortality within 7-10 days with as little as 1 PFU of ectromelia virus. Unlike the A/Ncr model, humans are less susceptible to lethal respiratory infections with variola virus and monkeypox virus as demonstrated by their lower mortality rates. In this study we show that a low dose intranasal infection of C57BL/6 mice results in 60-80% mortality and better models smallpox. Comparing CMX001 (HDP-cidofovir) efficacy in the A/Ncr strain and the C57BL/6 strain revealed that delayed treatment with CMX001 is more efficacious at preventing severe disease in the C57BL/6 strain. The increased efficacy of CMX001 in C57BL/6 over A/Ncr following an intranasal infection with ectromelia appears to be mediated by a stronger Th1 cell mediated response. Following footpad infection we show that the C57BL/6 strain has earlier and more robust transcriptional activity, Th1 cytokine secretions, antigen presenting activity and IFNγ splenic CD8+ T cell responses as compared to the A/Ncr strain. As a result of the enhanced immune response in the C57BL/6 strain, non-lethal intradermal ectromelia infections can therapeutically protect up to 3 days following a homologous, lethal intranasal infection – much like how smallpox vaccination can protect humans for up to 4 days following intranasal variola infection.

Combinatorial variation of branching length and multivalency in a large (390 625 member) glycopeptide dendrimer library: ligands for fucose-specific lectins

A new approach to dendrimer property tuning is demonstrated by combinatorial variation of dendrimer branch length and multivalency level, using a large (390625-member) self-encoded glycopeptide dendrimer library. Ligands for the fucose lectins of Ulex europaeus were identified by screening a combinatorial library of dendrimers with variable arm length (0–4 amino acids) and multivalency (2–5 end groups). The ligands identified by this approach also bind to the fucose lectin LecB (PA-IIL) of Pseudomonas aeruginosa, and might provide a possible starting point to develop anti-infective agents against this antibiotic resistant pathogen which causes lethal respiratory tract infections in cystic fibrosis patients.

Psittacid Herpesvirus 1 and Infectious Laryngotracheitis Virus: Comparative Genome Sequence Analysis of Two Avian Alphaherpesviruses

Psittacid herpesvirus 1 (PsHV-1) is the causative agent of Pacheco's disease, an acute, highly contagious, and potentially lethal respiratory herpesvirus infection in psittacine birds, while infectious laryngotracheitis virus (ILTV) is a highly contagious and economically significant avian herpesvirus which is responsible for an acute respiratory disease limited to galliform birds. The complete genome sequence of PsHV-1 has been determined and compared to the ILTV sequence, assembled from published data. The PsHV-1 and ILTV genomes exhibit similar structural characteristics and are 163,025 bp and 148,665 bp in length, respectively. The PsHV-1 genome contains 73 predicted open reading frames (ORFs), while the ILTV genome contains 77 predicted ORFs. Both genomes contain an inversion in the unique long region similar to that observed in pseudorabies virus. PsHV-1 is closely related to ILTV, and it is proposed that it be assigned to the Iltovirus genus. These two avian herpesviruses represent a phylogenetically unique clade of alphaherpesviruses that are distinct from the Marek's disease-like viruses (Mardivirus). The determination of the complete genomic nucleotide sequences of PsHV-1 and ILTV provides a tool for further comparative and functional analysis of this unique class of avian alphaherpesviruses.

Improved protection of mice against lethal respiratory infection with Coccidioides posadasii using two recombinant antigens expressed as a single protein

Two recombinant antigens which individually protect mice from lethal intranasal infection were studied in combination, either as a mixture of two separately expressed proteins or as a single chimeric expression product. Mice vaccinated with either combination survived longer than mice given single antigens. Immunized mice also exhibited specific IgG immunoglobulins and yielded splenocytes which produced interferon-γ in response to either antigen. The chimeric antigen has the practical advantage of offering enhanced protection from multiple components without increasing production costs.

Inactivation of the viral interleukin 1 receptor improves CD8+ T-cell memory responses elicited upon immunization with modified vaccinia virus Ankara

Interleukin 1 (IL1) is an important regulator of inflammatory responses and contributes to host immune defence against infection. Vaccinia virus encodes a viral soluble IL1beta receptor (IL1betaR), which modulates the acute-phase host response to infection and might influence the induction of immune responses against virus-associated antigens. Here, modified vaccinia virus Ankara (MVA) mutants defective in IL1betaR production were produced by insertion of selectable marker gene sequences that precisely deleted the IL1betaR coding sequences from the MVA genome (MVA-DeltaIL1betaR). Analysis of MVA mutants indicated that deletion of the IL1betaR gene did not abrogate the formation of MVA progeny upon tissue culture propagation. After high-dose intranasal infection with MVA-DeltaIL1betaR, mice showed no signs of fever or other illness, suggesting that the avirulent phenotype remained preserved for MVA-DeltaIL1betaR. Following vaccination of mice, MVA-DeltaIL1betaR or non-mutated MVA induced similar acute-phase immune responses. Importantly, when monitored at the memory phase, significantly higher vaccinia virus-specific total CD8(+) and HLA-A*0201-binding peptide epitope-specific T-cell responses were found after vaccination of HLA-A*0201-transgenic and non-transgenic mice with MVA-DeltaIL1betaR. Moreover, 4-6 months after vaccination, MVA-DeltaIL1betaR provided higher levels of protection against lethal respiratory challenge infection with virulent vaccinia virus strain Western Reserve compared with wild-type MVA. These data suggest that deletion of the viral IL1betaR gene may be considered a relevant approach to amplify the virus-specific CD8+ memory T-cell response and duration of protective immunity obtained after MVA vaccination.

Intranasal Interleukin-12 Treatment for Protection against Respiratory Infection with theFrancisella tularensisLive Vaccine Strain

Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F. tularensis was investigated. It was found that gamma interferon (IFN-gamma) and IL-12 were strictly required for protection, since mice deficient in IFN-gamma, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-gamma-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8-/- mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-gamma and CD8 T cells.

Anti‐cowpox Virus Activities of Certain Adenosine Analogs, Arabinofuranosyl Nucleosides, and 2′‐Fluoro‐arabinofuranosyl Nucleosides †

Nucleoside analogs were investigated for their potential to inhibit cowpox virus (a surrogate for variola and monkeypox viruses) in cell culture and in lethal respiratory infections in mice. Cell culture antiviral activity was determined by plaque reduction assays, with cytotoxicity determined by cell proliferation assays. Selectivity indices (SI's, 50% cytotoxic concentration divided by 50% virus‐inhibitory concentration) were determined for 15 compounds. Three arabinofuranosyl (Ara) nucleosides showed activity in mouse mammary tumor (C127I) cells: guanine (Ara‐G), thymine (Ara‐T), and adenine (Ara‐A) with SI's of 113, 61, and 95, respectively. The 2′‐fluoro‐Ara nucleosides of 5‐F‐cytosine (FIAC), 5‐methyluracil (FMAU), and 5‐iodouracil (FIAU) exhibited SI's of 148, 77, and 29, respectively. Other potent compounds included cidofovir (a positive control) and 3′‐O‐methyladenosine, with SI values of 164 and 56, respectively. In general, assays performed in African green monkey kidney (Vero) cells produced lower SI's than in C127I cells, except for 5‐iodo‐2′‐deoxyuridine (IDU) which had an SI of > 71 in Vero cells and 3.1 in C127I cells. Intranasal infection of mice with cowpox virus was followed a day later by twice daily intraperitoneal treatment with compounds for 5 days. Ara‐A was active at 300 mg/kg/day (40% survival), FMAU at 100 mg/kg/day (70% survival), and cidofovir (given for 1 day only) at 100 mg/kg (80–100% survival). None of the other compounds, including IDU, prevented death nor delayed the time to death. Cidofovir had the best potential for treating orthopoxvirus infections of those tested. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Prevention of lethal respiratory vaccinia infections in mice with interferon-α and interferon-γ

The antiviral efficacy of interferons (IFNs) was evaluated using a vaccinia intranasal infection model in mice in this study. We provide evidence that intranasal administration of IFN-α and IFN-γ (days −1 to +3) resulted in 100 and 90% survival against a lethal respiratory vaccinia infection (8 LD 50) in mice, respectively; whereas no animals in the placebo group survived through the study period (21 days). The IFN treatment consisted of a single daily dose of 5×10 3 U per mouse for 5 consecutive days. The efficacy of IFN-γ was evident even when the IFN-γ treatments started 1–2 days after infection and when a lower dose (2×10 3 U per mouse) was used. The treatment of IFN-α and IFN-γ reduced the virus titers in the lungs of infected mice by 1000–10 000-fold, when the administration started 1 day after infection. Our data suggest that IFN-α and IFN-γ are effective in protecting vaccinia-infected mice from viral replication in lungs and mortality, and may be beneficial in other human orthopoxvirus infections.

A review of compounds exhibiting anti-orthopoxvirus activity in animal models

Several animal models using mice (most frequently), rabbits, or monkeys have been used to identify compounds active against orthopoxvirus infections. The treatment of vaccinia virus infections has been well studied in models involving infection of scarified skin or eyes, or resulting from intravenous, intraperitoneal, intracerebral, or intranasal virus inoculation. Cowpox virus has been used in intranasal or aerosol infection studies to evaluate the treatment of lethal respiratory infections. Rabbitpox, monkeypox, and variola viruses have been employed to a lesser extent than the other viruses in chemotherapy experiments. A review of the literature over the past 50 years has identified a number of compounds effective in treating one or more of these infections, which include thiosemicarbazones, nucleoside and nucleotide analogs, interferon, interferon inducers, and other unrelated compounds. Substances that appear to have the greatest potential as anti-orthopoxvirus agents are the acyclic nucleotides, ( S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (cidofovir, HPMPC) and 1-[(( S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine (cyclic HPMPC), and the acyclic nucleoside analog, 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242). Other classes of compounds that have not been sufficiently studied in lethal infection models and deserve further consideration are thiosemicarbazones related to methisazone, and analogs of adenosine- N 1-oxide and 1-(benzyloxy)adenosine.

Serious and lethal respiratory tract infections of viral etiology in children

Viruses may lead to serious and lethal pulmonary infections in immunocompetent and immunocompromised children. Series of children with acute respiratory distress syndrome and series of children requiring extracorporeal membrane oxygenation, as well as reported series of nosocomial viral illness, offer an insight into the extent of serious viral disease documented in the medical literature. Series of children with specific viral respiratory illness also will be reviewed, as will the means of diagnosis in these groups of patients. Copyright © 2000 by W.B. Saunders Company

Characterization of Putative Defective Interfering (DI) A/WSN RNAs Isolated from the Lungs of Mice Protected from an Otherwise Lethal Respiratory Infection with Influenza Virus A/WSN (H1N1): A Subset of the Inoculum DI RNAs

Defective interfering (DI) influenza virus A/WSN (H1N1) grown in embryonated eggs protected adult mice from a lethal respiratory infection with A/WSN virus. Eighteen bands of putative DI RNA, ranging in size from about 230 to 1020 nt, were identified in this preparation by reverse transcription and polymerase chain reaction using a segment-specific 3′ primer and a segment-universal 5′ primer. Every virion RNA segment was represented by one to four bands of putative DI RNA. However, only five bands of putative DI RNA could be isolated, using the same conditions, from lungs of WSN-infected mice protected from death by co-inoculation with egg-grown DI WSN These five bands originated from PB1, PB2, PA, and M virion RNAs end were all about 350-450 nt in length. Four putative DI RNAs originating from PB1, PB2, and PA virion RNAs were sequenced, and three were identical (including deletion junctions and base substitutions) to putative DI RNAs from the inoculum. These data suggest that the mouse lung was highly selective for a subset of inoculum DI RNAs and that one or more of these DI RNAs was responsible for protection in vivo. All putative DI RNAs had a single internal deletion.

Specific anti-cytomegaloviral activity of methotrexate associated with preferential accumulation of drug by infected cells: M. Wachsman, F.M. Hamzeh, H. Saito, and P. S. Lietman. 1 Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] has since 1996 been licensed for clinical use in the treatment of cytomegalovirus (CMV) retinitis in AIDS patients. Cidofovir has broad-spectrum activity against virtually all DNA viruses, including herpes-, adeno-, polyoma-, papilloma- and poxviruses. Among the poxviruses, vaccinia, variola (smallpox), cowpox, monkeypox, camelpox, molluscum contagiosum and orf have proven sensitive to the inhibitory effects of cidofovir. In vivo, cidofovir has shown high efficacy, even after administration of a single systemic (intraperitoneal) or intranasal (aerosolized) dose, in protecting mice from a lethal respiratory infection with either vaccinia or cowpox. Cidofovir has also demonstrated high effectiveness in the treatment of vaccinia virus infection in severe combined immune deficiency mice. In humans, cidofovir has been used successfully in the treatment, by both the topical and intravenous route, of recalcitrant molluscum contagiosum and orf in immunocompromised patients. Taken together, these data indicate that cidofovir should be effective in the therapy and short-term prophylaxis of smallpox and related poxvirus infections in humans, as well as the treatment of the complications of vaccinia that may arise in immunocompromised patients inadvertently inoculated with the smallpox vaccine (vaccinia).

Protective effects of anti-Bordetella pertussisadenylate cyclase antibodies against lethal respiratory infection of the mouse

Abstract Experimental respiratory infection of mice with a Bordetella pertussis clone producing and releasing high yields of pertussis toxin and adenylate cyclase was lethal, inducing complete edematous hemorrhagic alveolitis within 48–72 h. By preincubating the challenge inoculum with polyclonal antibodies against the filamentous haemagglutinin (FHA), the pertussis toxin (Ptx), the adenylate cyclase (AC) or a monoclonal anti-AC antibody, we found that only anti-FHA and anti-AC antibodies were protective against the lethal lesions. These results provide direct evidence that B. pertussis AC might be incriminated in the pathogenesis of whooping cough as a major toxin responsible for the local cytopathic syndrome in the respiratory tract.

Protective effects of anti-Bordetella pertussis adenylate cyclase antibodies against lethal respiratory infection of the mouse

Protective effects of anti-Bordetella pertussis adenylate cyclase antibodies against lethal respiratory infection of the mouse

Branhamella catarrhalis as a human pathogen and a possible hospital infectant

Branhamella catarrhalis, previously named Neisseria catarrhalis and supposed to be a saprophytic inhabitant of the human upper respiratory tract, is a facultative pathogen. It was already proved by other authors to be able to cause serious and even lethal respiratory infections and purulent meningitis. Our aim was only to call attention to its repeated and massive isolation of pure culture from nose and throat swabs of hospitalized children who mostly suffered from asthma bronchiale and spastic bronchitis. The isolation of this bacterium often occurred in clusters of 3 to 4 in the same ward. Some other observation indicate occasional spreading of this organisms from person to person at home and in the hospital. In most cases Branhamella catarrhalis behaved only as a mild infectant, but in immunodeficient or otherwise weakened patient it can cause serious hospital infection.

Immunization with an Induced Avirulent Auxotrophic Mutant of Pseudomonas Pseudomallei

Summary A virulent strain of Pseudomonas pseudomallei was subjected to ultraviolet irradiation and a mutant clone requiring preformed adenine or hypoxanthine for growth was isolated. The imposition of auxotrophism was associated with a striking decline in virulence for mice which could be restored by permitting the strain to revert to purine independence in vitro. The mutant persisted in mice without detected reversions or or untoward effects for approximately 20 days following inoculation of 107 cells. After repeated inoculations, a significant immune response was demonstrated by parenteral challenge with diverse strains of this species. However, animals so immunized remained susceptible to lethal respiratory infection.