Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Development of a Bead Immunoassay To Measure Vi Polysaccharide-Specific Serum IgG after Vaccination with the Salmonella enterica Serovar Typhi Vi Polysaccharide

Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 μg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.Typhoid fever is caused by Salmonella enterica serotype Typhi (32). Humans are the only natural host and reservoir of S. enterica serotype Typhi (32, 41). Typhoid fever represents a spectrum of diseases ranging from an acute uncomplicated disease—including fever, headache, malaise, and disturbances of bowel function (constipation in adults and diarrhea in children)—to a more severe, complicated form of disease in 10 to 20% of infected patients that includes bleeding in the gastrointestinal tract, intestinal perforation (in 1 to 3% of hospital typhoid fever cases) and an altered mental state (32, 41). The case fatality rate is highly variable, depending on the medical treatment available and geographic location. For example, the average fatality rate is less than 1% overall but may range between 2% fatality in hospitalized patients in Pakistan and Vietnam and 50% fatality in hospitalized patients in some parts of Indonesia and Papua New Guinea (32, 41). Worldwide, typhoid fever remains a significant public health problem, with an estimated 17,000,000 cases of typhoid fever each year and up to 600,000 deaths (2, 10, 32, 41).Typhoid vaccines currently available are composed of purified Vi polysaccharide or live attenuated S. enterica serotype Typhi (Ty21a) organisms (10, 39). The Vi polysaccharide vaccine induces protective serum antibody responses that reach a maximum at 28 days after a single intramuscular vaccination with 25 μg purified Vi polysaccharide (39), a capsular polysaccharide (Vi for virulence) that increases the virulence of S. enterica serotype Typhi (32). Protective antibody levels have been estimated to be 1 μg/ml anti-Vi IgG antibody in the serum (20). Protective efficacy of the Vi polysaccharide vaccine as determined by protection against disease is modest, with only 55 to 72% of subjects protected against disease through 3 years postvaccination (1, 20, 39). The live attenuated Ty21a vaccine is administered orally as three or four doses of enteric capsules (39). Due to its use as an oral, mucosally administered vaccine, the Ty21a vaccine induces protection against typhoid fever by induction of mucosal IgA and serum IgG antibodies specific for lipopolysaccharide antigens (39). The protective efficacy of the Ty21a vaccine at 3 years postvaccination was reported to range from 42 to 67% when using three doses of Ty21a enteric capsules (11, 39). Next-generation vaccines that utilize Vi conjugated to protein carriers that provide superior induction of anti-Vi antibodies are currently in development (14, 21, 25, 36).Despite its ability to induce protective immune responses when used alone or conjugated to protein carriers, the use of Vi polysaccharide as a coating antigen in enzyme-linked immunosorbent assay (ELISA) to measure vaccine-induced anti-Vi antibody responses has been reported to be problematic. The use of polysaccharides (lipopolysaccharide [LPS], Haemophilus influenzae type b capsular polysaccharide, Vi polysaccharide) as coating antigens for immunoassays is plagued by problems such as a poor binding of polysaccharides to ELISA plates and inconsistent results (3, 15, 16, 26, 33). To increase binding of Vi antigen to ELISA plates and produce more-robust assays, others have biotinylated Vi and then added it to streptavidin-coated plates (12) or conjugated Vi to tyramine (22, 26). However, some reports indicate that Vi was used without any additional treatment as an ELISA coating antigen (7, 19, 21) although a Vi ELISA performed on plates was less sensitive than a radioimmunoassay procedure (19).Immunoassays based on the use of fluorescent beads as the solid surface have recently been developed and compared to ELISA for the measurement of antigen-specific antibodies for polysaccharides from Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae type b (HiB) (5, 8, 23, 27, 34, 35). The fluorescent bead assays were comparable to ELISA and sometimes were noted as having enhanced dynamic ranges or increased sensitivity (5, 8, 27, 35). An additional benefit of fluorescent bead immunoassays is their ability to be multiplexed to permit the simultaneous measurement of antibodies specific for different antigens (8, 23, 27, 34, 35). This study was performed to evaluate a fluorescent bead immunoassay for its ability to measure vaccine-induced antibodies specific for Salmonella serotype Typhi Vi polysaccharide. The performance of the fluorescent bead assay was compared to that of ELISA.     

Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health problem in Central Asia, Southeast Asia, Africa, and Latin America (11, 52, 53). It was estimated that more than 21 million cases of typhoid fever and >200,000 deaths occurred in 2000 (10). The treatment of patients and management of asymptomatic carriers are becoming more difficult due to the worldwide emergence of multidrug-resistant (MDR) strains (2, 15, 29, 42, 43). Vaccination is considered the most promising strategy for the control of typhoid fever in developing countries (11, 19, 52, 53).Typhoid fever in children younger than 5 years old has often been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of less than optimal culture media (35, 46). Several studies have shown that the incidence of typhoid fever among children less than 5 years old is similar to that in school age children and young adults (14, 27, 34, 50, 51).The 3 licensed typhoid vaccines have limited efficacy, and none are suitable for young children under 5 years old. The use of heat-inactivated whole-cell vaccine was suspended in many countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were introduced in the late 1980s; both vaccines are well accepted and confer moderate protection (50 to 70%) in older children and adults. However, neither vaccine is licensed for routine immunization of infants (52).The Vi capsular polysaccharide is both an essential virulence factor and a protective antigen for S. Typhi (36, 38, 39). The concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is not suitable for routine immunization of infants and young children because of its age-related immunogenicity and T-cell independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae (12, 20), Vi covalently bound with protein conferred T-cell dependence and increased immunogenicity (48-50). To date, diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins (CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi polysaccharide in laboratory studies (16, 17, 32, 48-50; personal communications). An improved method was developed (24), utilizing adipic acid dihydrazide (ADH) as the linker and Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier. Clinical trials of Vi-rEPA conjugates conferred 89% protection in Vietnamese children 2 to 5 years old for 46 months (23, 26, 28). The level of serum IgG anti-Vi induced by Vi-rEPA conjugates was correlated with prevention of typhoid fever in these studies (7, 21-23, 26, 28).One limitation of using rEPA as the carrier protein is the lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by pharmaceutical companies in China and India were prepared (24, 48, 49). Modifications of conjugation procedures were made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility of stockpiling in disaster relief.Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule using alternating injections of polysaccharide and conjugate. Priming or boosting effects of polysaccharide on its conjugate vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no consistent conclusion about various types of polysaccharides studied (6, 9, 31, 40, 41). Here, we compared the immune response of Vi polysaccharide injected before or after the administration of Vi-DT with the responses of those receiving 2 injections of Vi-DT. We also investigated the dosage effect for the purpose of better formulation.     

Serum Bactericidal Antibody Response 1 Year after Meningococcal Polysaccharide Vaccination of Patients with Common Variable Immunodeficiency

Some patients with common variable immunodeficiency (CVID) can generate an antibody response following vaccination with Neisseria meningitidis polysaccharide, but the duration of this protection is unknown. In this study, serum bactericidal antibody (SBA) responses to serogroup C N. meningitidis of 23 patients with CVID and 23 sex- and age-matched controls were measured 1 year after vaccination with the plain A/C meningococcal polysaccharide vaccine. The fold rise in serum bactericidal antibody geometric mean titers of the control group from prevaccination to 1 year postvaccination was significantly higher than that of the patient group (5.41- versus 2.96-fold, P = 0.009). Of 23 CVID patients, 8 had a poor response to vaccine (<4-fold rise) 3 weeks after vaccination, and low titers remained when measured 1 year later. Of the 15 CVID patients who had a normal response to vaccine (≥4-fold rise) 3 weeks after vaccination, 6 cases failed to maintain protective SBA titers, whereas the remaining 9 had protective titers 1 year after vaccination. Only one of the 23 controls, who developed protective SBA titers after 3 weeks, lost the protective titers after 1 year. Among the patients, the presence of bronchiectasis and/or splenomegaly at enrollment was associated with poor SBA response to vaccine at 3 weeks and/or failure to maintain protective levels at 1 year. The results of this study demonstrate that a number of CVID patients can produce protective antibody titers that can persist for 1 year after vaccination, which lends strong support to the inclusion of polysaccharide vaccine in the immunization program for CVID patients.Common variable immunodeficiency (CVID) is the commonest symptomatic primary immunodeficiency disease and is a heterogeneous group of disorders, characterized by severe reduction of serum levels of IgG and IgA, with normal or low numbers of B cells in the absence of any recognized genetic abnormality (2, 11, 16, 30). Patients with CVID usually experience recurrent bacterial infections (1, 14) and carry an increased risk of autoimmunity (12, 28) and malignancies (4, 24). Various defects of B cells, T cells, and dendritic cells have been reported for CVID (26, 29, 34-36); however, the exact pathophysiology of the disease is still unclear (3, 15).Deployment of polysaccharide and protein vaccines in CVID is a subject of active debate. Although it is intuitive that CVID patients should have poor antibody responses to vaccine, it is apparent that some patients can produce normal antibody titers (5, 18, 21, 32, 33). We have reported that a protective antibody response was achieved 3 weeks following vaccination with polysaccharide meningococcal vaccine of a group of CVID patients (32, 33). In the current study, we measured serum bactericidal antibody (SBA) titers (7) of the same cohort of patients 1 year after the initial vaccination.     

A DNA Vaccine-Encoded Nucleoprotein of Influenza Virus Fails To Induce Cellular Immune Responses in a Diabetic Mouse Model

Influenza virus infections cause yearly epidemics and are a major cause of lower respiratory tract illnesses in humans worldwide. Influenza virus has long been recognized to be associated with higher morbidity and mortality in diabetic patients. Vaccination is an effective tool to prevent influenza virus infection in this group of patients. Vaccines employing recombinant-DNA technologies are an alternative to inactivated virus and live attenuated virus vaccines. Internal highly conserved viral nucleoprotein (NP) can be delivered as a DNA vaccine to provide heterosubtypic immunity, offering resistance against various influenza virus strains. In this study, we investigated the efficacy of an NP DNA vaccine for induction of cell-mediated immune responses and protection against influenza virus infection in a mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized on days 0, 14, and 28 by injection of NP DNA vaccine. Two weeks after the last immunization, the cellular immune response was evaluated by gamma interferon (IFN-γ), lymphocyte proliferation, and cytotoxicity assays. The mice were challenged with influenza virus, and the viral titers in the lungs were measured on day 4. Diabetic mice showed significantly smaller amounts of IFN-γ production, lymphocyte proliferation, and cytotoxicity responses than nondiabetic mice. Furthermore, higher titers of the influenza virus were detected after challenge in the lungs of the diabetic mice. The present data suggest that the NP DNA vaccine with the protocol of immunization described here is not able to induce efficient cellular immune responses against influenza virus infection in diabetic mice.Influenza viruses cause highly contagious respiratory diseases with potentially fatal outcomes (29). The current licensed vaccine is only partially efficacious in healthy adults, and due to the impact of antigenic drift on vaccine effectiveness, components have to be reevaluated each year (12). Because of the significant impact that antigenic drift has on vaccine effectiveness, it is evident that new vaccines are needed to induce cross-reactive immunity against epidemic or pandemic influenza (2, 13). Vaccines employing recombinant-DNA technologies are an alternative to inactivated virus and live attenuated virus vaccines (4, 10). Many influenza DNA vaccine experiments have successfully demonstrated that DNA vaccines are effective for the induction of immune responses against the influenza virus (5, 7, 8, 27).Influenza virus nucleoprotein (NP), a core antigen of influenza virus, is a more conserved protein than its membrane glycoproteins (7, 19). DNA vaccination with influenza virus NP has been studied in animal models, and heterosubtypic immunity offering resistance against different influenza virus strains has been demonstrated (7, 22, 23, 26). On the other hand, Influenza usually causes high morbidity and mortality in patients with diabetes and is associated with a loss of metabolic control, development of ketoacidosis, and compromise of collectin-mediated host defense of the lung by glucose (1, 3, 21). Hence, vaccination against influenza infection is an important public health intervention for reducing morbidity and mortality among persons with diabetes (28). Although some experimental and clinical studies have investigated the capacity of inactivated-influenza vaccines in diabetic patients or animal models (3, 31), the potency of DNA immunization in induction of cellular immune responses of diabetic animal models has not been studied. In the present study, the DNA construct encoding influenza virus NP as a candidate for vaccination against influenza virus infection was used to immunize diabetic BALB/c mice, and its effectiveness in induction of cellular immunity was evaluated.     

Circulation of Mumps Virus Genotypes in Spain from 1996 to 2007

Although the WHO recommends the use of genotyping as a tool for epidemiological surveillance for mumps, limited data on mumps virus (MV) genotype circulation that may be used to trace the patterns of virus spread are available. We describe the first complete series of data from Spain. The small hydrophobic region was sequenced from 237 MV-positive samples from several regions of Spain collected between 1996 and 2007. Six different genotypes were identified: A, C, D (D1), G (G1, G2), H (H1, H2), and J. Genotype H1 was predominant during the epidemic that occurred from 1999 to 2003 but was replaced by genotype G1 as the dominant genotype in the epidemic that occurred from 2005 to 2007. The same genotype G1 strain caused concomitant outbreaks in different parts of the world (the United States, Canada, and the United Kingdom). The remaining genotypes (genotypes A, C, D, and J) appeared in sporadic cases or small limited outbreaks. This pattern of circulation seems to reflect continuous viral circulation at the national level, despite the high rates of vaccine coverage.Mumps is a highly transmissible but usually benign disease consisting of bilateral swelling of the salivary glands. In some instances, though, clinical complications can arise. Bilateral orchitis and clinical self-limited meningitis or more serious complications, such as encephalitis, deafness, male sexual sterility, and pancreatitis, may occur in rare cases (9).Mumps virus (MV), a virus belonging to the genus Rubulavirus of the subfamily Paramyxovirinae, is considered monotypic regarding its antigenicity. Thus, mumps vaccination is part of the regular immunization schedule of many countries, usually along with the measles and rubella vaccination (i.e., the MMR vaccine) in a single formulation. However, in contrast to rubella and measles, secondary vaccine failure frequently allows MV circulation within highly immunized populations (1, 5, 8, 24, 36). The use of a poorly immunogenic genotype A strain called Rubini has been proposed as a cause of these major failures, although the occurrence of mumps in patients immunized with other vaccine strains has also been described (26).Genetic variation in the small hydrophobic (SH) gene has led to the characterization of 12 genotypes, which are recognized by the WHO (13, 14, 23). Differential efficiency on cross neutralization among different genotypes has been suggested (20, 21), as have the differential capacities of certain strains to invade the neural system (25, 32). Finally, previous experience with the elimination programs for other preventable viral diseases, such as measles, rubella, and polio, suggest that genotyping would facilitate mumps surveillance (38), since the pattern of viral circulation can be traced. Consequently, molecular epidemiology studies have been performed around the world (2, 7, 10, 11, 12, 15, 17, 18, 23, 31, 32, 33, 34, 35, 41).Mumps vaccination was introduced in the Spanish national vaccination repertoire in 1981 as part of the triple viral vaccine (the MMR vaccine). As a result, the number of mumps cases fell from 286,887 in 1984 to 1,527 in 2004. However, the general descendant trend was interrupted by some peaks: in 1989 (from 48,393 in 1987 to 83,527 in 1989), 1996 (from 7,002 in 1995 to 14,411 in 1996), 2000 (from 2,857 in 1998 to 9,391 in 2000), and 2007 (from 1,527 in 2004 to 10,219 in 2007). Surprisingly, these peaks occurred despite a national vaccine coverage rate of over 95% of the population by 1999. As has been previously reported (29, 30), the occurrence of vaccinated individuals with MV RNA present in their saliva and/or urine with viral IgG but not IgM in acute-phase serum was frequent, suggesting secondary vaccine failure. Although most secondary vaccine failures were associated with the use of at least one dose of the Rubini strain, cases of mumps in patients vaccinated with two doses of the Jerryl Lynn strain were also observed (29, 30).In this report, we describe the MV genotypes circulating in Spain over the past 8 years. This represents the first series of data obtained for the national level. Interestingly, this series comprises three different epidemic peaks with two interepidemic periods with scarce viral circulation, allowing comparison of the pattern of genotype circulation under different epidemiological conditions.     

Evaluating the Orthopoxvirus Type I Interferon-Binding Molecule as a Vaccine Target in the Vaccinia Virus Intranasal Murine Challenge Model

The biological threat imposed by orthopoxviruses warrants the development of safe and effective vaccines. We developed a candidate orthopoxvirus DNA-based vaccine, termed 4pox, which targets four viral structural components, A33, B5, A27, and L1. While this vaccine protects mice and nonhuman primates from lethal infections, we are interested in further enhancing its potency. One approach to enhance potency is to include additional orthopoxvirus immunogens. Here, we investigated whether vaccination with the vaccinia virus (VACV) interferon (IFN)-binding molecule (IBM) could protect BALB/c mice against lethal VACV challenge. We found that vaccination with this molecule failed to significantly protect mice from VACV when delivered alone. IBM modestly augmented protection when delivered together with the 4pox vaccine. All animals receiving the 4pox vaccine plus IBM lived, whereas only 70% of those receiving a single dose of 4pox vaccine survived. Mapping studies using truncated mutants revealed that vaccine-generated antibodies spanned the immunoglobulin superfamily domains 1 and 2 and, to a lesser extent, 3 of the IBM. These antibodies inhibited IBM cell binding and IFN neutralization activity, indicating that they were functionally active. This study shows that DNA vaccination with the VACV IBM results in a robust immune response but that this response does not significantly enhance protection in a high-dose challenge model.The potential for variola virus (VARV, causing smallpox), or a genetically modified orthopoxvirus pathogenic to humans, to be accidentally or maliciously released into the environment has prompted a renewed interest in the development of orthopoxvirus countermeasures. A live-virus vaccine against orthopoxviruses is available and indeed was used to eradicate smallpox in the 20th century. However, this vaccine is associated with moderate to severe side effects, including myocarditis, eczema vaccinatum, and death (4, 21). As such, this vaccine is contraindicated for large portions of the population. Because the vaccine is also capable of spreading virus to nonvaccinated persons, those living with persons who are contraindicated for the vaccine are advised not to get vaccinated. Accordingly, safer alternative vaccines are being sought. These include highly attenuated live-virus vaccines, such as MVA and Lc16m8 (13, 23), and molecular vaccines. Molecular approaches include protein- and DNA-based subunit vaccines targeting various protective immunogens (9, 10, 14, 16, 17, 29, 34). Ideally, these vaccines will provide cross-protective immunity against all members of the orthopoxvirus family, including genetically modified strains. Subunit vaccines targeting structural molecules (A33, B5, L1, A27, H3, and D8) located on the two infectious forms of orthopoxvirus particles, the mature virion (MV) and the enveloped virion (EV), have shown protective efficacy in independent laboratories (6, 8-10, 14, 16, 17, 29, 34). Combinations of the MV and EV immunogens have been shown to elicit more complete protection than that elicited by vaccination with EV or MV targets alone (9, 15, 16). We have focused on a gene-based molecular vaccine, termed 4pox, targeting the EV immunogens A33 and B5 plus the MV targets L1 and A27 (11, 12, 15-17). This vaccine protects mice and nonhuman primates from lethal vaccinia virus (VACV) or monkeypox virus (MPXV) challenges, respectively (16, 17, 19). Recent studies have revealed that complete protection from lethality can be established after a single boost (12, 17).Orthopoxviruses express a multitude of immune evasion strategies, including soluble decoy receptors, complement-inactivating molecules, and intracellular inhibitors of interferon (IFN) (for reviews, see references 26 and 31). The VACV interferon-binding molecule (IBM) (B19R/B18R) is a type I interferon-binding decoy receptor expressed by VACV (5, 32). The molecule is secreted from infected cells, whereupon it aids in virus replication within the infected host by inhibiting the antiviral activity of type I IFNs by direct binding (2, 32). There are three immunoglobulin superfamily (IgSF) domains within the protein; however, the function of these domains and their role in either cell binding or type I IFN neutralization are unclear. Deletion of IBM results in ∼100-fold attenuation of VACV in vivo (32). Xu et al. recently reported that the ectromelia virus (ECTV) molecule EVM166, the IBM ortholog, is critical for virus replication in vivo (35). Deletion of EVM166 results in a 10⁷-fold decrease in infectivity in vivo. Furthermore, protein vaccination with the EVM166 molecule plus adjuvant afforded complete protection against ECTV (35). This finding demonstrates that a viral nonstructural molecule can function as an efficacious vaccine target. Thus, targeting viral immune response modifiers (IRMs) that alter the immune response in favor of viral replication may represent a hitherto-untapped group of vaccine targets (35). Whereas EVM166 is capable of protecting mice from ECTV, the ability of IBM, the VACV EVM166 ortholog, to function as a vaccine target has not been established.Vaccination with live virus confers significant protection after a single dose. However, vaccination with subunit vaccines targeting orthopoxvirus structural molecules consistently requires two or more vaccinations. Live-virus vaccines contain many vaccine targets, both structural targets and IRMs. Accordingly, it is possible that targeting IRMs, in addition to the structural proteins, confers superior protection compared to solely targeting structural molecules. The study by Xu et al. clearly demonstrates that viral IRMs are potent targets in at least some orthopoxvirus models (35). Therefore, we hypothesized that it might be possible to increase the efficacy of our structural-gene-based vaccine by including a nonstructural target previously shown to play a role in pathogenesis. Here, we investigated the protective efficacy of a DNA vaccine targeting the VACV IBM molecule alone and combined with the 4pox structural immunogens.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Salmonella enterica Serovar Typhimurium Vaccine Strains Expressing a Nontoxic Shiga-Like Toxin 2 Derivative Induce Partial Protective Immunity to the Toxin Expressed by Enterohemorrhagic Escherichia coli

Shiga-like toxin 2 (Stx2)-producing enterohemorrhagic Escherichia coli (referred to as EHEC or STEC) strains are the primary etiologic agents of hemolytic-uremic syndrome (HUS), which leads to renal failure and high mortality rates. Expression of Stx2 is the most relevant virulence-associated factor of EHEC strains, and toxin neutralization by antigen-specific serum antibodies represents the main target for both preventive and therapeutic anti-HUS approaches. In the present report, we describe two Salmonella enterica serovar Typhimurium aroA vaccine strains expressing a nontoxic plasmid-encoded derivative of Stx2 (Stx2ΔAB) containing the complete nontoxic A2 subunit and the receptor binding B subunit. The two S. Typhimurium strains differ in the expression of flagellin, the structural subunit of the flagellar shaft, which exerts strong adjuvant effects. The vaccine strains expressed Stx2ΔAB, either cell bound or secreted into the extracellular environment, and showed enhanced mouse gut colonization and high plasmid stability under both in vitro and in vivo conditions. Oral immunization of mice with three doses of the S. Typhimurium vaccine strains elicited serum anti-Stx2B (IgG) antibodies that neutralized the toxic effects of the native toxin under in vitro conditions (Vero cells) and conferred partial protection under in vivo conditions. No significant differences with respect to gut colonization or the induction of antigen-specific antibody responses were detected in mice vaccinated with flagellated versus nonflagellated bacterial strains. The present results indicate that expression of Stx2ΔAB by attenuated S. Typhimurium strains is an alternative vaccine approach for HUS control, but additional improvements in the immunogenicity of Stx2 toxoids are still required.Shiga-like toxins (Stx) play a crucial role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, which may lead to hemorrhagic colitis, central nervous system disturbances, and hemolytic-uremic syndrome (HUS) (27, 33). HUS involves acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, with mortality rates ranging from 1% to 4% (45, 50). EHEC strains may express different serotypes, including the widely distributed O157:H7 serotype, and infection correlates with the ingestion of contaminated ground beef and cow manure-contaminated water, vegetables, juices, and other products (13, 18). The incidence of EHEC-associated HUS cases is particularly high in developed countries, and high incidence rates have been recorded in Argentina, where cultural and diverse epidemiological factors contribute to the widespread dissemination of the disease among children and teenagers (38).EHEC strains may express two different Stx types. Stx1 is virtually identical to Stx produced by Shigella dysenteriae, while Stx2 shows only 56% homology to Stx1 at the amino acid sequence level (14, 33, 51). Both toxin types are formed by one A subunit and five B subunits, which bind to glycosphingolipid receptors, such as globotriaosyl ceramide (Gb3), on host cell membranes and promote retrograde toxin transport through the Golgi complex and endoplasmic reticulum. In the cell cytoplasm, Stx2 subunit A is processed into two fragments; one of them (A1) is endowed with N-glycosidase activity, which depurinates a specific adenine residue of the eukaryotic 28S rRNA, inhibits protein synthesis, and induces apoptosis of the target cell (18, 51).After ingestion and gut colonization, Stx molecules are released by the bacterial cells and translocate across the gut epithelium to reach, via the bloodstream, capillary endothelial cells at renal glomeruli, where the most relevant tissue damage occurs (33, 45, 50). Epidemiological data indicate that individuals infected with Stx2-producing bacterial strains, and some closely related variants, have a high probability of developing HUS (45, 50). In addition, Stx2 expression has been shown to increase gut colonization by bacterial cells due to induction of increased receptor expression by enterocytes (39).So far, there is no effective prophylactic or therapeutic approach for the prevention of HUS development among EHEC-infected individuals. The treatments available involve platelet transfusion in cases of severe anemia, hemodialysis, and supportive care (7, 50). A more direct anti-Stx treatment under clinical or preclinical evaluation involves the use of synthetic Stx glycolipid receptor analogs and humanized anti-Stx monoclonal antibodies (44, 52).Attempts to develop prophylactic anti-HUS vaccines are focused on the generation of Stx-neutralizing antibodies or the blockade of gut colonization. The vaccine strategies based on Stx2 that have been tested under experimental conditions have included DNA vaccines (5, 12), protein-conjugated polysaccharides (28), purified recombinant B subunits (24, 25, 29, 30, 47, 53, 55, 58), and B-subunit-derived synthetic peptides (19, 20). Anti-EHEC vaccine approaches based on the blockade of gut colonization have employed intimin and type III secreted proteins, such as EspA and EspB (3, 37, 54).Live bivalent anti-Stx vaccines based on genetically modified, attenuated Vibrio cholerae or Salmonella enterica serovar Typhimurium strains have been reported to induce anti-StxB antibody responses following oral administration to mice or rabbits (1, 10, 49). Attenuated Salmonella strains, used as orally administered vaccine vectors for the expression of heterologous antigens, show several advantages over conventional parenterally delivered cellular or acellular vaccine formulations (15, 16). Attenuated Salmonella strains are safe, are easily administered by untrained personnel, and, more relevantly, may induce systemic and secreted antigen-specific antibody and cell-based immune responses against self and heterologous antigens. In addition, whole bacterial cells carry on their surfaces several molecular structures known to activate both innate and adaptive immune responses. These molecules, such as lipopolysaccharide and flagellin, act as strong adjuvants, both systemically and at mucosal surfaces.Flagellins, the structural subunit of flagellar filaments, contribute both to the virulence of bacterial pathogens and to the activation of inflammatory responses in mammalian hosts. Bacterial flagellins have been shown to bind both extracellular and intracellular receptors of antigen-presenting cells, leading to inflammation and increased adaptive immune responses, including the generation of antigen-specific antibodies and T cells (2, 26). The strong adjuvant effects of Salmonella flagellins, either when admixed with purified antigens or when used as hybrid proteins genetically fused to the target antigens, have been demonstrated recently (4, 8, 22, 23, 36). However, there is no clear evidence that the expression of flagellin affects the immunogenicity of heterologous antigens expressed by attenuated Salmonella vaccine strains.In the present study, we generated new experimental anti-HUS vaccine formulations based on two recombinant attenuated S. Typhimurium aroA vaccine strains differing in the expression of flagellin. The two strains were genetically modified in order to express a nontoxic Stx2 derivative consisting of the whole Stx2 B subunit and a partially deleted A subunit encompassing the first amino acid of the A1 subunit genetically fused to the whole A2 subunit (Stx2ΔAB). The Stx2ΔAB protein was previously tested in mice immunized with a DNA vaccine (5). The results of the present study show that the S. Typhimurium vaccine strains express and secrete the recombinant toxin and induce both systemic and mucosal anti-StxB antibodies with anti-Stx2 neutralization activity, conferring partial protection against intravenous (i.v.) challenge with Stx2.     

A Fusion Protein Vaccine Containing OprF Epitope 8, OprI,and Type A and B Flagellins Promotes Enhanced Clearance of Nonmucoid Pseudomonas aeruginosa

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this study was to establish whether a multivalent vaccine containing P. aeruginosa type A and B flagellins as well as the outer membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscular immunization with flagellins and OprI (separate) or OprI-flagellin fusion proteins generated significant antiflagellin immunoglobulin G (IgG) responses. However, only the fusions of OprI with type A and type B flagellins generated OprI-specific IgG. Immunization with a combination of OprF epitope 8 (OprF_311-341), OprI, and flagellins elicited high-affinity IgG antibodies specific to flagellins, OprI, and OprF that individually promoted extensive deposition of C3 on P. aeruginosa. Although these antibodies exhibited potent antibody-dependent complement-mediated killing of nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice immunized with the OprF_311-341-OprI-flagellin fusion had a significantly lower bacterial burden three days postchallenge and cleared the infection significantly faster than control mice. In addition, mice immunized with the OprF_311-341-OprI-flagellin fusion had significantly less inflammation and lung damage throughout the infection than OprF-OprI-immunized mice. Based on our results, OprF_311-341-OprI-flagellin fusion proteins have substantial potential as components of a vaccine against nonmucoid P. aeruginosa, which appears to be the phenotype of the bacterium that initially colonizes CF patients.Cystic fibrosis (CF) is a hereditary disease that is linked to a defective CF transmembrane receptor (CFTR) (48). In CF patients, the presence of a defective CFTR protein leads to dehydrated mucosal surfaces and disruption of ion transport. In the initial stages of disease, CF patients are infected with Staphylococcus aureus and Haemophilus influenzae but eventually become infected with nonmucoid Pseudomonas aeruginosa, a gram-negative opportunistic pathogen that is the major cause of morbidity and mortality in these patients (5, 27, 28, 61). Following colonization, P. aeruginosa undergoes a mucoid conversion to an alginate-overexpressing phenotype that is associated with biofilm development and enhanced resistance to antibiotic therapy (28). CF is characterized by lung inflammation mediated, in part, by chronic P. aeruginosa infection. P. aeruginosa possesses numerous virulence factors that facilitate evasion of the immune system (15, 37, 43, 49). For example, P. aeruginosa secretes enzymes such as alkaline protease and elastase, which degrade complement components and thus limit the role of complement in the clearance of early pulmonary P. aeruginosa infections (16). The critical role of complement in the clearance of P. aeruginosa is evidenced by the observation that C3 and C5 knockout mice were unable to clear P. aeruginosa after challenge (40, 69). In addition, P. aeruginosa expresses lipopolysaccharide variants that interfere with C3b deposition (52).Initial efforts to develop a P. aeruginosa vaccine focused primarily on lipopolysaccharide. Although vaccination with P. aeruginosa lipopolysaccharide was effective in several animal models and led to the production of highly opsonic antibodies, the efficacy in human trials was limited by antigenic diversity of O antigens among P. aeruginosa isolates (11). Since flagellin, OprI, and OprF exhibit conserved amino acid sequences, more recent studies have focused on these proteins as potential vaccine antigens (14, 26, 31, 67, 68).P. aeruginosa possesses two types of flagellins, type A and type B, that differ in amino acid composition and length of the hypervariable region. P. aeruginosa flagellins have the unique property of being potent adjuvants as well as protective antigens (8, 32, 42, 50). Previous work has established flagellin as a potent adjuvant in mice (1, 3, 9, 10, 23, 33-35, 45, 53, 56) as well as cynomolgus and African green monkeys (24, 36). A phase III clinical trial of P. aeruginosa flagellins in CF patients demonstrated that the vaccine was well tolerated and caused a 30% reduction in the incidence of infection (12). In related studies, immunization with the OprI antigen of P. aeruginosa and an appropriate adjuvant elicited a protective response in mice that correlated with the titer of OprI-specific immunoglobulin G (IgG) (14). In addition, an adenovirus expressing epitope 8 (amino acids 311 to 341) of OprF (i.e., the OprF_311-341 protein) provided protection against acute P. aeruginosa infection (67, 68). Several investigators have focused on a fusion peptide containing OprF and OprI as a potential vaccine candidate. Although large amounts of this protein were required for an optimal response, immunization with an OprF-OprI fusion protein resulted in a 95-fold increase in the 50% lethal dose for mice. A subsequent study in burn patients revealed that an OprF-OprI fusion protein was immunogenic and well tolerated (26, 31).Although these experimental P. aeruginosa vaccines have shown promise in initial clinical trials, none have achieved the level of response required for protection against P. aeruginosa in CF patients. After a critical review of the literature, we have identified several features that are critical for an effective P. aeruginosa vaccine: the presence of a potent adjuvant, the ability to induce high-titer antigen-specific IgG that exhibits a high degree of functional activity (for example, complement activation), multivalency, and the ability to induce a robust memory response. To that end, we generated a multivalent vaccine containing type A and B flagellins, OprF, and OprI and have evaluated its immunogenicity and protective potential. A key feature of the vaccine is the presence of flagellin, a potent adjuvant that signals via Toll-like receptor 5 (TLR5).     

emm and C-Repeat Region Molecular Typing of Beta-Hemolytic Streptococci in a Tropical Country: Implications for Vaccine Development

We designed a study to investigate the molecular epidemiology of group A streptococcal (GAS) and group C and G streptococcal (GCS and GGS) disease in Fiji, a country which is known to have a high burden of streptococcal disease. Molecular typing of the N-terminal portion (emm typing) of the M protein was performed with 817 isolates (535 GAS and 282 GCS/GGS). We also performed genotyping of the C-repeat region in 769 of these isolates to identify J14 sequence types. The profile of emm types for Fiji was very different from that found for the United States and Europe. There were no dominant emm types and a large number of overlapping types among clinical disease states. Commonly found GAS emm types in industrialized countries, including emm1, emm12, and emm28, were not found among GAS isolates from Fiji. Over 93% of GAS isolates and over 99% of GCS/GGS isolates that underwent J14 sequence typing contained either J14.0 or J14.1. Our data have implications for GAS vaccine development in developing countries and suggest that a vaccine based upon the conserved region of the M protein may be a feasible option for Fiji and potentially for other tropical developing countries.The group A streptococcus (GAS) is an important cause of morbidity and mortality globally, with variation in disease burden between populations (9). A greater burden of GAS disease occurs in developing countries, particularly those located in the tropics, than in industrialized nations (9). The spectrums of GAS disease also differ between developed and developing countries. In many developing countries, GAS impetigo is often endemic, with resultant high rates of acute poststreptococcal glomerulonephritis, acute rheumatic fever, and rheumatic heart disease leading to at least 200,000 deaths annually, and the burden of invasive disease has probably been underestimated (9, 43). In industrialized countries, a massive number of cases of GAS pharyngitis leads to significant economic impact (27) and invasive disease leads to a significant number of deaths (25, 26).The molecular epidemiologies of GAS disease appear to differ between industrialized and developing nations, although there is a paucity of data from developing nations (5, 9). There are a number of different methods used to characterize GAS, with sequence typing of the 5′ N-terminal end of the M protein gene (emm) the most widely used (3, 17, 18). There have been recent large epidemiologic studies that have used emm sequence typing to investigate the molecular epidemiology of GAS pharyngitis and invasive GAS disease in industrialized nations, most notably in the United States, Canada, and Europe (16, 26, 37) (R. R. Tanz, S. T. Shulman, W. Kabat, E. Kabat, E. Cederlund, D. Patel, Z. Li, V. Sakota, J. B. Dale, and B. Beall, presented at the XVIth Lancefield International Symposium on Streptococci and Streptococcal Diseases, Palm Cove, Queensland, Australia, 2005). Far fewer studies have been conducted in developing nations. The limited available data suggest that numerous new emm types and subtypes have been discovered that have not previously been observed in industrialized countries (40, 45), that the diversity of emm types in developing countries is greater than that in industrialized countries (23, 32-34, 40, 45), and that the majority of isolates are of emm types traditionally associated with impetigo, irrespective of the clinical site of recovery of the isolates (5, 6, 23, 33, 40).Molecular epidemiologic data have implications for vaccine design. Although a number of antigens have shown promise as potential vaccine candidates, only one vaccine, a 26-valent M-protein-based vaccine, has reached clinical trials in recent times (15, 24). Serotypes for this vaccine were chosen if they were known to be common causes of invasive GAS disease or uncomplicated pharyngitis in the United States or if they were associated with rheumatic fever in classical studies from the United States in the mid-20th century (14). While this vaccine is likely to be efficacious in the United States, concerns have been raised about the transferability of this vaccine to developing-country settings (8).Alternative approaches to a multivalent vaccine strategy include the development of a conserved-epitope vaccine. A number of conserved epitopes have been identified and are under investigation, including some within the portion of the M protein closest to the cell wall (the C-repeat region) which appear to be relatively conserved (2, 11, 31, 36). An example is the J8 peptide, a B-cell epitope, contained within the larger sequence J14 (named J14.0 in this article for clarity) (28, 29). Following the discovery of J14.0, a number of J14 sequence types have been identified (47). To date, across all C-repeat regions, there have been 55 different J14 sequence types described, which have been named in the order that they have been discovered (J14.0 to J14.54) (47). Typing of the C-repeat region by the J14 sequence type of GAS has been employed previously (47). J14 sequence type is relevant to J8 because antibodies raised against J8 in mice provide cross-protective immunity against GAS isolates containing J14.0 and J14.1 (Michael Batzloff, Queensland Institute of Medical Research, unpublished data). Antibodies raised against the J14.0 peptide in mice have been shown to opsonize GAS strains belonging to a variety of emm subtypes that contain J14 sequences other than the J14.0 sequence type, including J14.2 (47).Group C streptococci (GCS) and group G streptococci (GGS) are emerging infectious agents, particularly as a cause of invasive disease and of epidemic pharyngitis (19, 35, 48). We have observed higher than expected rates of invasive GCS/GGS in Fiji, as well as high pharyngeal carriage among school children (43). There is also some evidence to suggest that these organisms may play a role in the pathogenesis of acute rheumatic fever and poststreptococcal glomerulonephritis (13, 30). There are very few data regarding emm sequence typing of GCS/GGS and no available data regarding sequence typing of the C-repeat region of these organisms (22).We designed a study to investigate the molecular epidemiology of GAS and GCS/GGS disease in a tropical setting known to have a high burden of invasive, pharyngeal, and impetiginous streptococcal disease (42-44). Because of the implications for vaccine development, we included molecular typing of both the 5′ end of the M protein (emm sequence typing) and the C-repeat region of the M protein (J14 sequence typing).     

Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.Chronic obstructive pulmonary disease (COPD) is currently the fourth leading cause of death in the United States and Europe. The morbidity and mortality associated with exacerbations of COPD together with the associated health care-related costs of $32 billion reported in the United States in 2002 demonstrate that there is a need for a better understanding of the etiology and pathogenesis of these events (6, 36, 43). Bacteria have been isolated in large numbers from the lower respiratory tract during exacerbations, and up to 50% of COPD exacerbations are due to a bacterial agent, primarily nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (31). M. catarrhalis accounts for up to 10% of these infections in adults, and this strictly human pathogen is currently among the three most prominent causes of otitis media in children (13, 15, 28). Some of the primary reasons why M. catarrhalis continues to cause disease can be attributed to the fact that greater than 90% of the clinical isolates express beta-lactamase, there is a high frequency of recurrent disease observed for children that have recovered from infection, and there is a lack of a vaccine (13, 27, 42, 47). Thus, the identification of potential drug targets and vaccine antigens is clearly a priority.One of the major problems hindering the identification of putative vaccine antigens involves the fact that M. catarrhalis is a strictly human pathogen, and the human immune response to this bacterium remains poorly understood. Previous studies investigated the production of new antibodies against different bacterial pathogens in patients suffering from COPD and lower respiratory tract infections. These patients exhibited increased antibody responses to bacterial outer membrane proteins (OMP) and surface-exposed lipooligosaccharides (LOS) after clearing the bacterial strain following an exacerbation (4, 29, 38, 51). Human serum immunoglobulin G (IgG), sputum IgA, or salivary IgA antibodies against M. catarrhalis surface proteins such as UspA1, UspA2, Hag, TbpB, CopB, OMP CD, OMP E, and OMP G1b have been developed (1, 3, 25, 28, 29). In addition, new antibodies to LOS have also been detected in some COPD patients (3, 28, 29).The LOS structure of M. catarrhalis has been well studied. There are three major serotypes, serotypes A, B, and C, previously defined by polyclonal antisera and structural analyses (8-10, 23, 46). The LOS glycosyltransferase (lgt) genes that encode the enzymes required to transfer carbohydrate residues to the M. catarrhalis LOS molecule were previously identified and characterized (11, 33, 41, 49). In addition, the lgt present in a given strain of M. catarrhalis can be used to identify the specific LOS serotype of that isolate using our previously described multiplex PCR method (12). Serotypes A and B are the predominant glycoforms expressed by most clinical isolates analyzed to date (12, 46). In recently reported animal studies, other researchers suggested that detoxified M. catarrhalis LOS has potential as a vaccine antigen in a mouse pulmonary clearance model (16, 19, 52, 53). While these data are both valuable and interesting, it is sometimes difficult to link observations of animals to those of humans (34).Currently, we have constructed a panel of defined LOS mutants that are defective in the expression of each specific glycosyltransferase gene identified in all three major M. catarrhalis LOS serotypes. These truncations are a comprehensive set of mutations with various oligosaccharide (OS) chain lengths representing most, if not all, possible LOS epitopes (11, 33, 41, 49). Purified LOS samples from these mutants were used in enzyme-linked immunosorbent assays (ELISAs) to assess the development of new human antibodies to all LOS epitopes developed following an M. catarrhalis infection. ELISAs were previously employed to determine levels of antibody to Neisseria meningitidis lipopolysaccharide (LPS), including inner core mutations, in patients following disease (35). Thus, this LOS-based ELISA system with the full set of mutations has the potential to determine the regions of the LOS molecule that elicit new antibodies in both children and adults following infection, providing a unique opportunity to analyze the human immune response to these major surface glycolipids in the native host.     

Characterization of Unique Regions of Borrelia burgdorferi Surface-Located Membrane Protein 1

The pathogen of Lyme disease, Borrelia burgdorferi, produces a putative surface protein termed “surface-located membrane protein 1” (Lmp1). Lmp1 has been shown previously to assist the microbe in evasion of host-acquired immune defenses and in the establishment of persistent infection of mammals. Here, we show that Lmp1 is an integral membrane protein with surface-exposed N-terminal, middle, and C-terminal regions. During murine infection, antibodies recognizing these three protein regions were produced. Separate immunization of mice with each of the discrete regions exerted differential effects on spirochete survival during infection. Notably, antibodies against the C-terminal region primarily interfered with B. burgdorferi persistence in the joints, while antibodies specific to the N-terminal region predominantly affected pathogen levels in the heart, including the development of carditis. Genetic reconstitution of lmp1 deletion mutants with the lmp1 N-terminal region significantly enhanced its ability to resist the bactericidal effects of immune sera and also was observed to increase pathogen survival in vivo. Taken together, the combined data suggest that the N-terminal region of Lmp1 plays a distinct role in spirochete survival and other parts of the protein are related to specific functions corresponding to pathogen persistence and tropism during infection that is displayed in an organ-specific manner. The findings reported here underscore the fact that surface-exposed regions of Lmp1 could potentially serve as vaccine targets or antigenic regions that could alter the course of natural Lyme disease.Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted by ticks belonging to the Ixodes scapularis complex (2, 31). Upon introduction to the host dermis, spirochetes disseminate from the site of infection to distal cutaneous areas and navigate to a variety of internal host organs (6). The colonization of the pathogen in a select set of infected host organs eventually leads to robust inflammatory responses resulting in multiple clinical complications, such as arthritis, carditis, and neurological disorders (31, 48). In mammalian hosts, B. burgdorferi infection may persist in diverse tissues despite the development of a vigorous immune response (5, 8, 13, 16, 24, 44). Understanding the mechanism by which spirochetes selectively persist in certain tissues and induce a severe inflammatory response is important for the development of preventive and therapeutic strategies against Lyme borreliosis.B. burgdorferi survives in a complex enzootic life cycle. The varied metabolic and immune host environments have been shown to dramatically influence spirochete gene expression (11, 14, 26, 29, 32-34, 41, 43, 49, 51). Microbial antigens that are produced in a time- or tissue-specific manner might assist B. burgdorferi to overcome host defenses and to persist in local environments. Differentially expressed gene products, particularly surface antigens, could directly participate in host-pathogen interaction or host immune evasion, contributing to microbial survival and organ-specific pathogenesis (36, 51). In recent years, a few spirochete gene products have been identified that are either indispensable or contribute significantly to host or vector infectivity and transmission through the tick-mouse infection cycle (9, 23, 25, 26, 28, 37, 39, 40, 45, 47, 52, 53). However, in most cases, the genes identified encoded proteins that lack orthologs in other bacteria; therefore, their molecular functions in spirochete biology or infectivity remain unclear.Recently, a protein termed Lmp1, a chromosomally encoded antigen with an approximate molecular mass of 128 kDa, was shown to be induced in infected murine tissues, especially at early phases of infection in the heart (51). Lmp1 has been suggested to be integral to pathogen persistence and to be involved in evading the host adaptive immune response during infection (51). The antigen is localized to the microbial surface, is immunogenic during animal or human infection (3, 51), and is conserved across orthologs in other B. burgdorferi sensu lato isolates. Computer algorithms suggest that Lmp1 contains a typical type I leader peptide, although whether the signal sequence is cleaved remains unknown. Lmp1 contains three possible separate functional regions located at the N-terminal, middle, and C-terminal portions of the protein. Although the overall structure of Lmp1 is unrelated to known proteins, the middle region of the protein contains several peptide repeats which may be related to adhesins (3). The C-terminal region contains several tetratricopeptide repeats (TPRs), which are motifs that are well documented to play important roles in protein-protein interactions (17, 22, 42). Despite earlier studies, the molecular function of Lmp1 and the possible unique role(s) of its individual protein regions with regard to B. burgdorferi virulence and Lyme disease pathogenesis remain unclear. Characterization of functional protein regions of novel spirochete virulence determinants, such as Lmp1, will likely shed further light into how Lmp1 could potentially serve as a vaccine target or how antibodies against antigenic regions of Lmp1 could alter the course of a natural Lyme disease infection.     

Vaccination of Rhesus Macaques with the Anthrax Vaccine Adsorbed Vaccine Produces a Serum Antibody Response That Effectively Neutralizes Receptor-Bound Protective Antigen In Vitro

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.Anthrax is caused by infection with Bacillus anthracis, and its pathogenesis is associated with an antiphagocytic poly-d-glutamic acid capsule and a binary anthrax toxin (ATx). The ATx comprises two protein exotoxins: lethal toxin (LTx) and edema toxin (ETx). The two toxins both have a binding protein called protective antigen (PA) but have separate effector proteins, edema factor (EF) and lethal factor (LF) (3, 20, 56). LTx is composed of PA and LF, and ETx is composed of PA and EF. In the initial stages of infection by B. anthracis, full-length 83-kDa PA (PA83) secreted by the bacterium binds to either one or both of at least two host cell ATx receptors (ATRs): tumor endothelial marker 8 (TEM8) (7, 27, 57) or capillary morphogenesis protein 2 (CMG2) (51).Vaccines containing PA as the major component confer protective efficacy in various animal models of multiple routes of infection (5, 14-16, 29, 42-44, 59). PA has four domains: an amino-terminal domain (domain 1, which is composed of subregions 1a and 1b) that contains two calcium ions and the S₁₆₃RKKRS₁₆₈ cleavage site for activating proteases, a heptamerization domain (domain 2) that contains a large flexible loop implicated in membrane insertion, a small domain (domain 3) hypothesized to aid in oligomerization, and a carboxy-terminal receptor-binding domain (domain 4) (26, 41). Upon binding to an ATR, PA is proteolytically cleaved by the cell surface protease furin to a 63-kDa polypeptide (PA63), releasing a 20-kDa amino-terminal fragment (domain 1a). Cleavage and release of domain 1a facilitate assembly of the PA prepore; a heptameric ring-shaped structure with a negatively charged lumen. Assembly of the prepore exposes a large hydrophobic surface for binding of LF and/or EF molecules to form ATx (9, 17, 19, 26, 33, 35, 37, 41). One PA63 heptamer is able to bind up to three LF and/or EF molecules. The ATx is then endocytosed by a lipid raft-mediated clathrin-dependent process (1, 34). The low-pH conditions (pH 5.5) in the endosome induce the prepore to undergo a conformational switch that translocates ATx to the target cell cytosol (6, 17, 18, 24, 32, 37, 41, 55).The current licensed vaccine for use in humans is anthrax vaccine adsorbed (AVA [BioThrax]; Emergent BioSolutions, Lansing, MI). AVA is a cell-free filtrate from a toxigenic, nonencapsulated B. anthracis strain, V770-NP1-R (2, 10, 45). The primary immunogen is PA (59) adsorbed to aluminum hydroxide adjuvant (10, 29). The current AVA vaccination schedule consists of five 0.5-ml intramuscular (i.m.) injections at 0 and 4 weeks and 6, 12, and 18 months, with annual boosters (10, 30).There are various potential molecular targets in which the host humoral antibody response to vaccination with AVA or PA can interfere with ATx-mediated cytotoxicity. These targets include, but are not limited to, (i) blocking of free PA83 binding to the host cell ATx receptor (TEM8 or CMG2); (ii) inhibition of PA83 proteolytic cleavage by the host cell surface furin-like enzyme or serum proteases, leaving the PA unprocessed and thus unable to form toxin complexes; (iii) interruption of PA63 heptamerization to form the prepore on the host cell surface; (iv) blocking the binding of LF and EF monomers to the PA heptamer prepore; and (v) disruption of internalization and translocation of the ATx. Consequently, PA has become a focal point in developing immunotherapies and next-generation vaccines for the prevention and treatment of anthrax (4, 13, 21, 22, 31, 36, 39, 40, 53, 58, 60).Most of the anti-PA therapies under development specifically target PA domains 2 and 4, with domain 4 being the most frequent target (21, 53, 60). The therapeutic effects of antibodies targeted against domain 4 are considered to be based primarily on blocking the interaction of PA with its host cell receptor (26, 49). However, in active immunization, there will be multiple epitopes presented to the host immune system that are critical to mounting a protective immune response and, likewise, others that may make little or no contribution. Although PA20 is cleaved from PA83 and has no described role in the intoxication process, recent reports have proposed that in AVA-vaccinated humans, the PA20 fragment (domain 1a) contains immunodominant epitopes (48, 61). Therefore, it was postulated that vaccines containing full-length PA (PA83) may be suboptimal due to the dominance of PA20 and that perhaps PA63-based vaccines may be more advantageous (47, 48).To address the question of suboptimal immune responses in PA83-based vaccine and therapeutic design, we developed two low-temperature anthrax lethal toxin (LTx) neutralization activity (TNA) assays, the noncomplexed TNA (NC-TNA) and receptor-bound TNA (RB-TNA) assays. These assays allow comparison of antibody-mediated neutralization of LTx both before and after receptor binding by PA. The goal of this work was to evaluate the ability of anti-PA antibody responses in AVA-vaccinated and inhalation anthrax-challenged rhesus macaques (Macaca mulatta) to neutralize anthrax LTx in vitro both before and after PA has bound to, and been processed at, the cell surface receptor.     

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A₁ peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A₂ peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A₂ sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens representing the major etiological agents of hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease characterized by hemolytic anemia, thrombocytopenia, and renal failure (19). The infection correlates with ingestion of contaminated meat or vegetables but is also transmitted by water or even person-to-person contact (8, 14, 44). Sporadic or massive outbreaks have been reported in several developed countries but, in Argentina, HUS is endemic and represents a serious public health problem with high morbidity and mortality rates (29, 40). Production of verocytotoxin or Shiga-like toxin (Stx) is the basis of EHEC pathogenesis (18, 20). The toxin is formed by a single A subunit, which possesses N-glycosidase activity to the 28S rRNA and promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells (9, 28). Although two major types (Stx1 and Stx2) and several subtypes have been described, Stx2 and Stx2c are the most frequently found toxins in severe HUS cases among EHEC-infected subjects (12, 41). The degree of antigenic cross-reactivity between Stx2 and Stx1 is low, and several authors have reported that the two toxins do not provide heterologous protection, particularly concerning the B subunits (45, 47). On the other hand, Stx2c and Stx2d variants are readily neutralized by antibodies against Stx2 (27).Despite the magnitude of the social and economic impacts caused by EHEC infections, no licensed vaccine or effective therapy is presently available for human use. So far, attempts to develop vaccine formulations against EHEC-associated sequelae have relied mainly on induction of serum anti-Stx antibody responses. Several approaches have been pursed to generate immunogenic anti-Stx vaccine formulations and include the use of live attenuated bacterial strains (2, 32), protein-conjugated polysaccharides (21), purified B subunit (33, 48), B-subunit-derived synthetic peptides (15), and mutated Stx1 and Stx2 nontoxic derivatives (5, 6, 13, 16, 37, 39, 42, 45).In a previous report we described anti-Stx2 DNA vaccines encoding either the B subunit or a fusion protein between the B subunit and the first N-terminal amino acid of the A₁ subunit (8). The DNA vaccine encoding the hybrid protein elicited Stx-specific immune responses in mice and partial protection to Stx2 challenge (1, 33). Recent data have indicated that epitopes leading to generation of Stx-neutralizing antibodies are present on both the B as well as the A subunit (34, 45, 46). In addition, further evidence indicates that the A₂ subunit contains some of the most immunogenic epitopes of the Stx2 toxin (4). Thus, in line with such evidence, we attempted the construction of a new DNA vaccine encoding the last 32 amino acids from the A₂ subunit, in addition to the complete B subunit of Stx2, as separated polypeptides which could enhance the immunogenicity and protective effects of the vaccine formulation. In the present report, we describe the generation of a new DNA vaccine encoding both Stx2 A2 and B subunits as an approach to elicit protective antibody responses to Stx2. The results obtained demonstrate that immunization with this vaccine formulation results in systemic antibody responses to Stx2 A and B subunits and toxin neutralization activity both in vitro and in vivo.     

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-γ) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-γ-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-γ and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-γ and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.The attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) is the currently used vaccine against tuberculosis (TB). In spite of its wide use, the BCG vaccine only protects against severe forms of childhood TB and generally does not prevent adult pulmonary TB (11, 30, 47).Considering that one-third of the world population is thought to be infected with Mycobacterium tuberculosis and that only a small proportion of these individuals will develop active disease, new vaccine candidates to prevent the establishment of infection could also boost and improve the cellular immunity of already latently infected individuals. Vaccine candidates currently in clinical trials include improved recombinant BCG vaccines, virus-based recombinant vaccines, and subunit vaccines comprised of dominant secreted antigens (1, 32). Secreted proteins, regularly described as culture filtrate proteins (CFP), are the main targets of the T-cell response in mice, both at the height of infection and in a state of memory immunity, as well as in humans with active TB (1, 4, 5, 7, 23). Immunization with these antigens in the presence of different adjuvants provided protection in mice challenged with M. tuberculosis, and protection was mediated by gamma interferon (IFN-γ)-producing CD4⁺ cells (29, 38).We previously showed that a homologous immunization schedule based on three doses of CFP antigens plus CpG oligodeoxynucleotide adjuvant stimulated significant IFN-γ production by spleen cells and in the lungs of challenged mice. In spite of high IFN-γ concentrations, immunized and challenged mice were not protected and indeed had extensive lung damage (16).Since IFN-γ is the best indicator of protective immunity defined thus far, we changed the schedule of homologous immunization to heterologous immunization, also known as a prime-boost regimen, to induce protection.Several studies have demonstrated the efficacy of prime-boost vaccination strategies in generating cellular immunity to a variety of pathogens (3, 10, 14, 17, 34, 36, 44, 45, 49). Recently, our group also showed that a single dose of a DNA-HSP65 vaccine booster significantly enhanced the protection conferred against TB by a single subcutaneous dose of BCG (18). In addition, secreted antigens such as the 6-kDa early-secretion antigen target (ESAT-6), 85A or 85B antigens, and Mtb72F have proven to be promising candidates for BCG-boosting vaccines in mice, guinea pigs, and nonhuman primates (6, 9, 12, 19, 33, 37, 46, 48). Because a single dominant antigen may not confer the same level of protection to all vaccinated individuals, and based on high CFP antigen-mediated IFN-γ production in the presence of CpG adjuvant, in this study we used CFP plus CpG oligodeoxynucleotides to boost BCG vaccination in order to improve protection and lung preservation following M. tuberculosis challenge.