Chloroform-methanol Residue Research Articles

Enhanced protection against Q fever in BALB/c mice elicited by immunization of chloroform-methanol residue of Coxiella burnetii via intratracheal inoculation

Human Q fever is recognized as a worldwide public health problem. It often occurs by inhalation of airborne aerosols contaminated with Coxiella burnetii, a gram-negative intracellular bacterium, mainly from domestic livestock. In this study, we analyzed the possibility to establish mucosal and systemic immunity against C. burnetii infection using a pulmonary delivery of chloroform-methanol residue of C. burnetii (CMR) vaccine. Mice were immunized by the intratracheal inoculation of CMR (IT-CMR) or the subcutaneous injection of CMR (SC-CMR), and the immunized mice were challenged with C. burnetii by the intratracheal route. The levels of IFN-γ, IL-12p70, IL-5, and IL-4 in the IT-CMR group in splenic T cells stimulated ex vivo were significantly higher than in the SC-CMR group. Significantly elevated sIgA to C. burnetii was detected in the bronchoalveolar lavage fluid of mice immunized by IT-CMR but not by SC-CMR, which might have contributed to the significant reduction in C. burnetii load and pathological lesions in the lungs of the mice after the challenge of C. burnetii. These results suggest that compared with SC-CMR in mice, IT-CMR was more efficient to elicit cellular and lung mucosal immune responses against aerosol infection of C. burnetii.

Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice.

The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.

Comparative efficacy and immunogenicity of Q fever chloroform:methanol residue (CMR) and phase I cellular (Q-Vax) vaccines in cynomolgus monkeys challenged by aerosol

Preliminary evidence gathered in rodents and livestock suggested that a phase I chloroform:methanol residue (CMR) extracted vaccine was safe and efficacious in protecting these animals from challenge with the obligate phagolysosomal pathogen ( Coxiella burnetii). Prior to the initiation of phase II studies in human volunteers, we compared, in non-human primates ( Macaca fascicularis), the efficacy of CMR vaccine with Q-Vax, a licensed cellular Australian Q fever vaccine that has been demonstrated to provide complete protection in human volunteers. Vaccine efficacy was assessed by evaluating thoracic radiographs and the presence of fever and bacteremia in monkeys challenged by aerosol with Coxiella burnetii. Changes in blood chemistries, hematology, behavior and pulmonary function were also examined. CMR, whether administered in single 30 or 100 μg doses or two 30 μg subcutaneous doses, gave equivalent protection in vaccine recipients as a single 30 μg dose of Q-Vax. In addition, vaccination resulted in significant, although temporary, increases in specific antibody titers against C. burnetii phases I and II antigens. The C. burnetii CMR vaccine may be an efficacious alternative to cellular Q fever vaccines in humans.

Antiviral properties of a crude extract from a green alga Caulerpa taxifolia (Vahl) C. Agardh.

A crude extract of Caulerpa taxifolia was tested for its antiviral activity. The chloroform-methanol residue showed an interesting inhibitor effect in vitro toward the feline immunodeficiency virus (FIV), a valid model for studying the acquired immunodeficiency syndrome. This extract reduced the virus-induced syncytia in the cultured cells, the viral reverse transcriptase activity and the viral capsid protein P24 expression.

Comparative efficacy of a Coxiella burnetii chloroform:methanol residue (CMR) vaccine and a licensed cellular vaccine (Q-Vax) in rodents challenged by aerosol

Q fever is an acute and self-limited febrile illness caused by the obligate intracellular bacterium Coxiella burnetii. While phase I cellular Q fever vaccines are efficacious in humans, vaccination of immune individuals may result in sterile abscesses and granulomas. The chloroform:methanol residue vaccine (CMR) was developed as a safer alternative. The efficacy of a licensed phase I cellular vaccine (Q-Vax) was compared with that of CMR vaccine in A/J mice and Hartley guinea pigs challenged with virulent phase I C. burnetii by aerosol. Both vaccines were efficacious. The CMR vaccine dose required to protect 50% of mice (PD 50) against lethal aerosol challenge (11 LD 50) was one-third of the Q-Vax dose. However, the PD 50 for CMR was four times the Q-Vax dose in guinea pigs challenged by aerosol (60 LD 50). It was concluded that CMR is an efficacious alternative to cellular Q fever vaccines for the prevention of Q fever. Published by Elsevier Science Ltd.

Efficacy of Coxiella burnetii and its chloroform-methanol residue (CMR) fraction against Rift Valley fever virus infection in mice

Strains of Coxiella burnetii phase I and II whole cells (WC-I and WC-II) or whole cell fractions were assessed for their potential to induce long-lasting protection in endotoxin-non-responder C3H HeJ or CD-1 mice against Rift Valley fever (RVF) virus challenge. Among the whole cell fractions, only the chloroform-methanol residue (CMR), administered as a single dose (100 μg per mouse) 24 h before viral challenge, effectively protected 100% of the mice from RVF virus; the CMR of the Ohio strain of C. burnetii was not protective. Most of the RVF virus-infected mice treated with other C. burnetii cell fractions died, although their times to death varied. Lipopolysaccharide (LPS) associated with CMR preparations used in these studies, did not protect against RVF virus challenge. A single dose of 100 μg of CMR given 24 h before viral challenge completely eradicated 4-t logs of RVF virus in the serum, liver, spleen, and central nervous system. Compared to several other immunomodulators, CMR was an equally effective antiviral agent. Efficacy of CMR of both Henzerling and Ohio strains disappeared or was marginal when treatment was initiated 2–3 days before RVF viral challenge, even when a second or a third dose of CMR was administered after challenge. A single dose of liposome-encapsulated CMR to RVF virus-infected mice extended the range of therapeutic efficacy of this biologically active component of C. burnetii to 4 days before infection.

Crude extracts and two linear diterpenes from Cystoseira balearica and their activity.

Crude extracts of Cystoseira balearica were submitted to pharmacological investigations. The chloroform-methanol residue caused relaxation of the rat aortic musculature and an inhibitory action on the guinea pig atria. The fractionation of this crude extract gave two active diterpenoids, eleganolone (1) and elegandiol (2), the latter has not previously been isolated from this alga. These pure components, never before tested, showed a higher activity when compared with the crude residue.

Safety and immunogenicity in human volunteers of a chloroform-methanol residue vaccine for Q fever

Current Q fever vaccines, consisting of Formalin-inactivated phase I whole Coxiella burnetii, are highly efficacious in preventing disease in high-risk settings but are associated with a risk of unacceptable local reactions in previously immune individuals and require cumbersome preliminary immunologic evaluation of potential vaccinees. A vaccine prepared from the residue of chloroform-methanol extraction of phase I Henzerling strain C. burnetii (CMR) has been shown to be less reactogenic but still immunogenic and protective in small animals and sheep. In a placebo-controlled trial, we immunized 35 healthy adults unscreened for markers of prior C. burnetii immunity with a single subcutaneous CMR dose of 30, 60, 120, or 240 micrograms. None of those receiving the 30- or 60-micrograms CMR dose and none of the placebo recipients experienced any adverse effects. Five of 15 120-micrograms dose CMR recipients complained of transient discomfort in the inoculated arm; erythema or induration of > or = 100 mm2 was noted in three and four, respectively, and two had malaise and low-grade fever (< 101 degrees F, orally). No 240-micrograms dose vaccinee reported limb discomfort, but 7 of 10 had erythema and/or induration of > or = 100 mm2 (P < 0.001 versus placebo). Two reported malaise, and one had low-grade fever. All adverse effects were self-limited. Serum immunoglobulin M responses were optimally detected with CMR antigen and occurred in 50, 60, 73, and 90% of recipients of the 30-, 60-, 120-, and 240-micrograms doses, respectively; results with phase I whole-cell antigen were similar. Serum immunoglobulin G responses were best detected with phase II antigen and were seen in 20, 20, and 40% of those receiving the 60-, 120-, and 240-micrograms doses, respectively. Peripheral blood T-cell proliferative responses to C. burnetii recall antigens were transient and of low magnitude but were seen with CMR antigen in 33% of 120-micrograms dose recipients and 40% of 240-micrograms dose recipients. Data from this study and those from comparative-efficacy trials in primates should provide the basis for field trials of the CMR vaccine.

Immunisation de chiens avec des vaccins contre la fièvre Q: comparaison entre des vaccins de &lt;em&gt;Coxiella burnetii&lt;/em&gt; de phase I, phase II et du RCM de phase I

Q fever vaccines were tested in mixed breed dogs by vaccinating them with formalin-killed Coxiella burnetii whole cells (WC) in either phase I (WCI) or phase II (WCII), or the chloroform: methanol residue (CMR) subunit of phase I cells. Phase I vaccines mixed (1:1) with Freund's incomplete adjuvant (FIA) induced humoral immune responses to phases I and II antigens as measured by microagglutination assay. The CMR vaccine mixed (1:1) with FIA induced greater antigen-specific antibody levels to both phases I and II antigens than the corresponding WCI vaccine. The WCII vaccine induced antibody responses to only phase II antigens. The time course of erythema and induration after skin testing with C. burnetii antigens were suggestive of cell-mediated immunity (CMI). Although granulomas were observed with only WCI and WCII, none of the skin test antigens induced abscesses at the injection site. In contrast, axillary nodes draining the vaccine injection site developed sterile draining abscesses in all dogs by days 19 to 24 for the WCI and CMR, and day 104 for the WCII vaccines. The abscesses had resolved within 30 days after first appearance. Responses to Con A and PHA and recall antigens of lymphocytes from the blood, axillary and mesenteric nodes, and spleen at 222 days after vaccination were variable among dogs. Lymphocytes from various organs responded to one or more of the recall antigens and to both mitogens in the absence or presence of indomethacin. Although these Q fever vaccines induced humoral and CMI, either the antigens or FIA caused sterile draining abscesses.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting

The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure-resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle.

Immune modulation by Coxiella burnetii: characterization of a phase I immunosuppressive complex differentially expressed among strains.

Coxiella burnetii, the etiological agent of Q fever, possesses immunomodulatory activity which positively and negatively regulates host immune responses. We wish to determine the Coxiella strain differences and the chemical nature of cellular components suppressing lymphocyte responsiveness. The bacterial components responsible for the immunomodulatory activity are associated with phase I cells. In its natural state, the phase I cell-associated, immunosuppressive complex (ISC) was resistant to chemical and enzymatic treatment. The ISC was inactivated and rendered accessible by chloroform-methanol (CM) (4:1) extraction of phase I cells which produced a CM residue (CMRI) and CM extract (CME). The suppressive components in either CMRI or CME did not induce ISC activity in the host when injected separately. Reconstitution of the CMRI with CME prior to injection produced the same pathological reactions characteristic of phase I cells. The CMRI suppressive component was sensitive to alkali, acid, periodate, lysozyme, and neuraminidase, but resistant to lipase and protease. An active component of CMRI was attached to the cell matrix by disulphide bonds. The amphipathic, lipophilic, CME suppressive component was ubiquitously distributed in procaryotes and eukaryotes because ISC activity of CMRI was regained after association with reagent-grade lipids and different CMEs. The ISC was expressed by phase I strains with smooth lipopolysaccharide (LPS) but not by phase II strains with rough LPS. Phase I heart valve strains carrying significant amounts of rough LPS did not express all of the biological properties of the ISC. The LPS molecule induced immune enhancement without immunosuppression. Thus, expression of the ISC showed strain variation and may be under genetic control. The complete details of the chemical composition and active components of the ISC should prove useful for biological-response-modification studies.

Characterization of a phase I Coxiella burnetii chloroform-methanol residue vaccine that induces active immunity against Q fever in C57BL/10 ScN mice.

The effect of phase I Coxiella burnetii chloroform-methanol residue vaccine (CMRV) on the response of murine splenic lymphocytes to mitogenic and antigenic stimuli was evaluated in C57BL/10 ScN endotoxinnonresponder mice with an in vitro lymphocyte proliferation assay. Intraperitoneal injection of phase I CMRV resulted in antibody production against phases I and II antigens. Lymphocytes were responsive in vitro to concanavalin A, phytohemagglutinin, pokeweed mitogen, and specific recall antigens. Antibodies against phases I and II antigens were not detected after intraperitoneal injection of chloroform-methanol extract (CME). Lymphocytes also were only slightly hyporesponsive to mitogens. Reconstitution of the CMRV with the CME of phase I whole cells restored the immunopathological reactions that were associated with the phase I whole cell vaccine (WCV). The CMRV was more mitogenic than the WCV for lymphocytes from mice injected with saline. Lymphocytes from phase I WCV-injected mice were negatively modulated with nontoxic concentrations of homologous WCV or CMRV. Lymphocytes from phase I CMRV-injected mice were only slightly hyporesponsive to mitogens and were significantly stimulated by antigens of either WCV or CMRV as recall antigens. Vaccination of mice with 100 micrograms of CMRV, CME, or WCV provided 80, 0, or 50% protection, respectively, against a lethal intraperitoneal challenge with viable phase I C. burnetii. The epitopes which induce immunological hyporesponsiveness, negative modulation, and the death of lymphocytes were fractionated into the CMRV and CME. The CMRV provides at least one of the determinants which induce immunosuppression, whereas CME contains specific or nonspecific components or both. Collectively, these results show that the CMRV may be a potential candidate to replace the WCV currently used for human vaccination.

Biological and immunological properties of Coxiella burnetii vaccines in C57BL/10ScN endotoxin-nonresponder mice.

Vaccines prepared from Formalin-killed whole cells of Coxiella burnetii (Ohio strain) or from chloroform-methanol residue (CMR) and extract (CME) of such cells were examined for biological and immunological properties in male C57BL/10ScN endotoxin nonresponder mice. Vaccines containing killed whole cells induced a high incidence of gross pathology, as evidenced by liver necrosis, significant splenomegaly, and significant hepatomegaly in mice. The degree and onset of these pathological changes were directly and inversely proportional, respectively, to the dosage of killed organisms administered. Conversely, no splenomegaly, hepatomegaly, or liver necrosis were observed in mice inoculated with CMR or CME. Moreover, killed whole cells were lethal for mice at dosages of 150 to 1,200 micrograms, whereas no deaths were seen in animals given 1,200 micrograms of CMR. In addition, antibodies against phase I and phase II antigens of C. burnetii were detected in the sera of mice inoculated with either whole cells or CMR. Enhanced blastogenic response of splenic lymphocytes was observed when animals were vaccinated with killed whole cells and CMR but not with CME. Moreover, 80 to 90% of mice inoculated with 300 micrograms of the CMR were protected against a lethal challenge of viable rickettsiae, whereas only 50% of the animals given 300 micrograms of killed whole cells were protected. Treatments with CME were essentially without value, since no antibodies were detectable and no significant protection was elicited. Collectively, these results show that, although killed whole cells induced immunity in C57BL/10ScN mice, they induced deleterious tissue reactions, whereas CMR, which also induced immunity, was essentially nondeleterious, based on the parameters employed. These observations suggest that the chloroform-methanol-extractable component(s) is implicated in the deleterious tissue reactions and that the phase I and II antigens may not be involved in the induction of the pathology observed in C57BL/10ScN mice.