Showing papers in "Clinical and Vaccine Immunology in 2010"

Correlates of Protection Induced by Vaccination

Abstract: This paper attempts to summarize current knowledge about immune responses to vaccines that correlate with protection. Although the immune system is redundant, almost all current vaccines work through antibodies in serum or on mucosa that block infection or bacteremia/viremia and thus provide a correlate of protection. The functional characteristics of antibodies, as well as quantity, are important. Antibody may be highly correlated with protection or synergistic with other functions. Immune memory is a critical correlate: effector memory for short-incubation diseases and central memory for long-incubation diseases. Cellular immunity acts to kill or suppress intracellular pathogens and may also synergize with antibody. For some vaccines, we have no true correlates, but only useful surrogates, for an unknown protective response.

Real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever.

Abstract: The world's largest Q fever outbreak is ongoing in The Netherlands with around 3,000 confirmed cases since the first half of 2007. Increased awareness has resulted in early referral of patients for diagnostics. An important drawback to serological diagnosis of acute Q fever is the lag phase in antibody response. Therefore, we evaluated the performance of a real-time PCR for detection of Coxiella burnetii DNA using serum samples from patients with acute Q fever. PCR, targeting IS1111, was retrospectively performed on acute-phase and follow-up convalescent-phase serum samples from 65 patients with acute Q fever as diagnosed by immunofluorescence assay. The results obtained by PCR were related to disease stage as defined by subsequent appearance of phase II IgM, phase II IgG, phase I IgM, and phase I IgG (IgM-II, IgG-II, IgM-I, and IgG-I, respectively) antibodies and time since onset of disease. In addition, we analyzed seronegative acute-phase serum samples from patients with inconclusive Q fever serology, because no convalescent-phase serum samples were available. PCR was scored positive in 49/50 (98%) seronegative sera, 9/10 (90%) sera with isolated IgM-II antibodies, 3/13 (23%) sera with IgM-II/IgG-II antibodies, 2/41 (5%) sera with IgM-II/IgG-II/IgM-I antibodies, 0/15 (0%) sera with IgM-II/IgG-II/IgM-I/IgG-I antibodies, and 0/1 (0%) serum sample with IgM-II/IgG-II/IgG-I antibodies. The latest time point after onset of disease in which C. burnetii DNA could be detected was at day 17. In patients with inconclusive Q fever serology, PCR was positive in 5/50 (10%) cases. We conclude that real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever. C. burnetii DNA becomes undetectable in serum as the serological response develops.

Updated Postlicensure Surveillance of the Meningococcal C Conjugate Vaccine in England and Wales: Effectiveness, Validation of Serological Correlates of Protection, and Modeling Predictions of the Duration of Herd Immunity

Abstract: Meningococcal serogroup C conjugate (MCC) vaccines were licensed in the United Kingdom more than 10 years ago based on correlates of protection that had previously been established for serogroup C-containing polysaccharide vaccines by using the serum bactericidal antibody (SBA) assay. These correlates of protection were subsequently validated against postlicensure estimates of observed vaccine effectiveness up to 7 to 9 months after the administration of the MCC vaccine. Vaccine effectiveness was, however, shown to fall significantly more than 1 year after the administration of a 3-dose course in infancy. Despite this finding, the marked impact on serogroup C disease has been sustained, with the lowest recorded incidence (0.02 case per 100,000 population) in the 2008-2009 epidemiological year, mainly due to the indirect herd immunity effect of the vaccine in reducing carriage. Updated estimates of vaccine effectiveness through 30 June 2009 confirmed high short-term protection after vaccination in infancy, at 97% (95% confidence interval [CI], 91% to 99%), falling to 68% (95% CI, −63% to 90%) more than a year after vaccination. The observed vaccine effectiveness more than 12 months postvaccination was consistent with measured declining SBA levels, but confidence intervals were imprecise; vaccine effectiveness estimates were consistent with SBA titers of 1:4 or 1:8 as correlates of long-term protection after a primary course in infants. Modeling suggested that protection against carriage persists for at least 3 years and predicted the stabilization of serogroup C disease at low levels (fewer than 50 cases per year) up to 2015-2016.

Effects of Lactobacilli on Cytokine Expression by Chicken Spleen and Cecal Tonsil Cells

Abstract: Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius are all normal residents of the chicken gastrointestinal tract. Given the interest in using probiotic bacteria in chicken production and the important role of the microbiota in the development and regulation of the host immune system, the objective of the current study was to examine the differential effects of these bacteria on cytokine gene expression profiles of lymphoid tissue cells. Mononuclear cells isolated from cecal tonsils and spleens of chickens were cocultured with one of the three live bacteria, and gene expression was analyzed via real-time quantitative PCR. All three lactobacilli induced significantly more interleukin 1β (IL-1β) expression in spleen cells than in cecal tonsil cells, indicating a more inflammatory response in the spleen than in cecal tonsils. In cecal tonsil cells, substantial differences were found among strains in the capacity to induce IL-12p40, IL-10, IL-18, transforming growth factor β4 (TGF-β4), and gamma interferon (IFN-γ). In conclusion, we demonstrated that L. acidophilus is more effective at inducing T-helper-1 cytokines while L. salivarius induces a more anti-inflammatory response.

Prevalence of Antibodies to Four Human Coronaviruses Is Lower in Nasal Secretions than in Serum

Abstract: Little is known about the prevalence of mucosal antibodies induced by infection with human coronaviruses (HCoV), including HCoV-229E and -OC43 and recently described strains (HCoV-NL63 and -HKU1). By enzyme-linked immunosorbent assay, we measured anti-HCoV IgG antibodies in serum and IgA antibodies in nasal wash specimens collected at seven U.S. sites from 105 adults aged 50 years and older (mean age, 67 ± 9 years) with chronic obstructive pulmonary disease. Most patients (95 [90%]) had at least one more chronic disease. More patients had serum antibody to each HCoV strain (104 [99%] had antibody to HCoV-229E, 105 [100%] had antibody to HCoV-OC43, 103 [98%] had antibody to HCoV-NL63, and 96 [91%] had antibody to HCoV-HKU1) than had antibody to each HCoV strain in nasal wash specimens (12 [11%] had antibody to HCoV-229E, 22 [22%] had antibody to HCoV-OC43, 8 [8%] had antibody to HCoV-NL63, and 31 [31%] had antibody to HCoV-HKU1), respectively (P < 0.0001). The proportions of subjects with IgA antibodies in nasal wash specimens and the geometric mean IgA antibody titers were statistically higher for HCoV-OC43 and -HKU1 than for HCoV-229E and -NL63. A higher proportion of patients with heart disease than not had IgA antibodies to HCoV-NL63 (6 [16%] versus 2 [3%]; P = 0.014). Correlations were highest for serum antibody titers between group I strains (HCoV-229E and -NL63 [r = 0.443; P < 0.0001]) and between group II strains (HCoV-OC43 and -HKU1 [r = 0.603; P < 0.0001]) and not statistically significant between HCoV-NL63 and -OC43 and between HCoV-NL63 and -HKU1. Patients likely had experienced infections with more than one HCoV strain, and IgG antibodies to these HCoV strains in serum were more likely to be detected than IgA antibodies to these HCoV strains in nasal wash specimens.

Safety and Immunogenicity of a 13-Valent Pneumococcal Conjugate Vaccine Compared to Those of a 7-Valent Pneumococcal Conjugate Vaccine Given as a Three-Dose Series with Routine Vaccines in Healthy Infants and Toddlers

Abstract: A 13-valent pneumococcal conjugate vaccine (PCV13) has been developed to improve protection against pneumococcal disease beyond that possible with the licensed 7-valent vaccine (PCV7). This study compared the safety and immunogenicity of PCV13 with those of PCV7 when given as part of the pediatric vaccination schedule recommended in Italy. A total of 606 subjects were randomly assigned to receive either PCV13 or PCV7 at 3, 5, and 11 months of age; all subjects concomitantly received diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated polio-Haemophilus influenzae type B (DTaP-HBV-IPV/Hib) vaccine. Vaccine reactions were monitored. Antibody responses to DTaP-HBV-IPV/Hib antigens, serotype-specific anticapsular polysaccharide IgG responses, and antipneumococcal opsonophagocytic assay (OPA) activity were measured 1 month after the two-dose primary series and 1 month after the toddler dose. Overall, the safety profile of PCV13 was similar to that of PCV7. The response to DTaP-HBV-IPV/Hib antigens was substantially the same with both PCV13 and PCV7. PCV13 elicited antipneumococcal capsular IgG antibodies to all 13 vaccine serotypes, with notable increases in concentrations seen after the toddler dose. Despite a lower immunogenicity for serotypes 6B and 23F after the primary series of PCV13, responses to the seven common serotypes were comparable between the PCV13 and PCV7 groups when measured after the toddler dose. PCV13 also elicited substantial levels of OPA activity against all 13 serotypes following both the infant series and the toddler dose. In conclusion, PCV13 appeared comparable to PCV7 in safety profile and immunogenicity for common serotypes, demonstrated functional OPA responses for all 13 serotypes, and did not interfere with immune responses to concomitantly administered DTaP-HBV-IPV/Hib vaccine.

How Innate Immune Mechanisms Contribute to Antibody-Enhanced Viral Infections

Abstract: Preexisting antibodies may enhance viral infections. In dengue, nonneutralizing antibodies raised by natural infection with one of four dengue viruses (DENVs) may enhance infection with a different virus by a process we term “intrinsic antibody-dependent enhancement” (iADE). In addition, nonprotective antibodies raised by formalin-inactivated respiratory syncytial virus (RSV) and measles virus vaccines have led to enhanced disease during breakthrough infections. Infections under iADE conditions not only facilitate the process of viral entry into monocytes and macrophages but also modify innate and adaptive intracellular antiviral mechanisms, suppressing type 1 interferon (IFN) production and resulting in enhanced DENV replication. The suppression observed in vitro has been documented in patients with severe (dengue hemorrhagic fever [DHF]) but not in patient with mild (dengue fever [DF]) secondary dengue virus infections. Important veterinary viral infections also may exhibit iADE. It is thought that use of formalin deconforms viral epitopes of RSV, resulting in poor Toll-like receptor (TLR) stimulation; suboptimal maturation of dendritic cells with reduced production of activation factors CD40, CD80, and CD86; decreased germinal center formation in lymph nodes; and the production of nonprotective antibodies. These antibodies fail to neutralize RSV, allowing replication with secondary stimulation of RSV-primed Th2 cells producing more low-avidity antibody, resulting in immune complexes deposited into affected tissue. However, when formalin-inactivated RSV was administered with a TLR agonist to mice, they were protected against wild-type virus challenge. Safe and effective vaccines against RSV/measles virus and dengue virus may benefit from a better understanding of how innate immune responses can promote production of protective antibodies.

Improved diagnosis of Strongyloides stercoralis using recombinant antigen-based serologies in a community-wide study in northern Argentina.

Abstract: The serodiagnosis of Strongyloides stercoralis infection by enzyme-linked immunosorbent assays based on crude antigen (CrAg-ELISA), while useful, has been limited by the reliance on crude parasite extracts. Newer techniques such as the luciferase immunoprecipitation system assay (LIPS), based on a 31-kDa recombinant antigen (termed NIE) from S. stercoralis and/or the recombinant antigen S. stercoralis immunoreactive antigen (SsIR), or the NIE-ELISA have shown promise in controlled settings. We compared each of these serologic assays in individuals from both regions of the world in which S. stercoralis is endemic and those in which it is not. A comprehensive stool evaluation (sedimentation concentration, Baermann concentration with charcoal cultures, agar plate, and Harada-Mori) and four different serologic techniques using CrAg-ELISA or recombinant NIE-ELISA as well as LIPS using NIE alone or in combination with a second recombinant antigen (NIE/SsIR-LIPS) were compared among individuals with parasitologically proven infection (n = 251) and healthy controls from regions of the world in which the infection is nonendemic (n = 11). Accuracy was calculated for each assay. The prevalence of S. stercoralis infection was 29.4% among Argentinean stool samples (n = 228). Sedimentation concentration and Baermann were the most sensitive stool-based methods. NIE-LIPS showed the highest sensitivity (97.8%) and specificity (100%) of the serologic assays. The calculated negative predictive value was highest for both the NIE-LIPS and CrAg-ELISA (>97%) irrespective of disease prevalence. No cross-reactivity with soil-transmitted helminths was noted. NIE-LIPS compares favorably against the current CrAg-ELISA and stool evaluation, providing additional accuracy and ease of performance in the serodiagnosis of S. stercoralis infections irrespective of disease prevalence.

Kinetics of Antibody Persistence following Administration of a Combination Meningococcal Serogroup C and Haemophilus influenzae Type b Conjugate Vaccine in Healthy Infants in the United Kingdom Primed with a Monovalent Meningococcal Serogroup C Vaccine

Abstract: The kinetics of antibody persistence following the administration of a combination meningococcal serogroup C and Haemophilus influenzae type b (Hib) conjugate vaccine (Menitorix) in the second year of life in children primed with two doses of one of three monovalent meningococcal serogroup C (MCC) vaccines was investigated. The study subjects were administered either Menitorix at 12 to 15 months of age, followed by the seven-valent pneumococcal conjugate vaccine (PCV7) and the measles, mumps, and rubella vaccine 4 to 6 weeks later, or all three vaccines concomitantly at 12 to 15 months of age. Blood samples were collected before and 1, 2, 12, and 24 months after the boosting. Sera were analyzed for meningococcal serogroup C serum bactericidal antibody (SBA) and IgG as well as Hib-polyribosylribitol phosphate (PRP)-specific IgG. The antibody persistence data from this study were compared to those of a prior study of Southern et al. (Clin. Vaccine Immunol. 14:1328-1333, 2007) in which children were given three primary doses of a vaccine containing both the MCC and the Hib vaccines but were boosted only with a Hib conjugate vaccine. The magnitude of the meningococcal SBA geometric mean titer was higher for those subjects primed with the MCC vaccine conjugated to tetanus toxoid (NeisVac-C) than for those primed with one of two MCC vaccines conjugated to CRM 197 (Menjugate or Meningitec) up to 1 year following boosting. Two years after boosting, the percentages of subjects with putatively protective SBA titers of ≥8 for children primed with NeisVac-C, Menjugate, and Meningitec were 43%, 22%, and 23%, respectively. Additional booster doses of the MCC vaccine may be required in the future to maintain good antibody levels; however, there is no immediate need for a booster during adolescence, as mathematical modeling has shown that persisting herd immunity is likely to control disease for a number of years.

Interacting Roles of Immune Mechanisms and Viral Load in the Pathogenesis of Crimean-Congo Hemorrhagic Fever

Abstract: Until now, the pathogenesis of Crimean-Congo hemorrhagic fever (CCHF) has not been well described. However, it has been hypothesized that it could be a result of the direct injury of virus-infected tissues in combination with the indirect effects of host immune responses, including cytokines. To shed more light on the role of viral load and cytokines, differential influences of CCHF virus (CCHFV) RNA load, antibody response, and cytokine production on severity and outcome of the disease were studied in sera of 46 patients with confirmed acute CCHF from Kosovo. In this study, viral load proved to be strongly related to the severity and outcome of the disease, with higher viral loads detected in patients with fatal outcomes than in surviving patients. Also, patients with fatal outcome had on average a weaker antibody response, if one was present at all. High levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) were associated with poor outcome, since detected concentrations were highest in patients with fatal outcome and lowest in patients with moderate disease course. Additionally, a positive linear dependence between viral load and these cytokines was observed. Interestingly, reduced levels of IL-12 were detected in all CCHF patients. Our study favors the hypothesis that CCHF could be a result of a delayed and downregulated immune response caused by IL-10, which leads to an increased replication and spread of CCHFV throughout the body. This consequently triggers increased production of IFN-γ and TNF-α, cytokines mediating vascular dysfunction, disseminated intravascular coagulation, organ failure, and shock.

Establishment of an Aerosol Challenge Model of Tuberculosis in Rhesus Macaques and an Evaluation of Endpoints for Vaccine Testing

Abstract: The establishment of an aerosol challenge model in nonhuman primates (NHPs) for the testing of vaccines against Mycobacterium tuberculosis would assist the global effort to optimize novel vaccination strategies. The endpoints used in preclinical challenge studies to identify measures of disease burden need to be accurate and sensitive enough to distinguish subtle differences and benefits afforded by different tuberculosis (TB) vaccine regimens when group sizes are inevitably small. This study sought to assess clinical and nonclinical endpoints as potentially sensitive measures of disease burden in a challenge study with rhesus macaques by using a new protocol of aerosol administration of M. tuberculosis. Immunological and clinical readouts were assessed for utility in vaccine evaluation studies. This is the first example of TB vaccine evaluation with rhesus macaques where long-term survival was one of the primary endpoints. However, we found that in NHP vaccine efficacy studies with maximum group sizes of six animals, survival did not provide a valuable endpoint. Two approaches used in human clinical trials for the evaluation of the gamma interferon (IFN-gamma) response to vaccination (enzyme-linked immunospot [ELISpot] assay and enzyme-linked immunosorbent assay [ELISA]) were included in this study. The IFN-gamma profiles induced following vaccination were found not to correlate with protection, nor did the level of purified protein derivative (PPD)-specific proliferation. The only readout to reliably distinguish vaccinated and unvaccinated NHPs was the determination of lung lesion burden using magnetic resonance (MR) imaging combined with stereology at the end of the study. Therefore, the currently proposed key markers were not shown to correlate with protection, and only imaging offered a potentially reliable correlate.

Characterization of fHbp, nhba (gna2132), nadA, porA, and Sequence Type in Group B Meningococcal Case Isolates Collected in England and Wales during January 2008 and Potential Coverage of an Investigational Group B Meningococcal Vaccine

Abstract: Invasive disease caused by meningococcal capsular groups A, C, W-135, and Y is now preventable by means of glycoconjugate vaccines that target their respective polysaccharide capsules. The capsule of group B meningococci (MenB) is poorly immunogenic and may induce autoimmunity. Vaccines based on the major immunodominant surface porin, PorA, are effective against clonal epidemics but, thus far, have a limited scope of coverage against the wider MenB population at large. In an alternative approach, the first-generation, investigational, recombinant MenB (rMenB) plus outer membrane vesicle (OMV) (rMenB-OMV) vaccine contains a number of relatively conserved surface proteins, fHBP, NHBA (previously GNA2132), and NadA, alongside PorA P1.4-containing OMVs from the New Zealand MeNZB vaccine. MenB currently accounts for approximately 90% of cases of meningococcal disease in England and Wales. To assess potential rMenB-OMV vaccine coverage of pathogenic MenB isolates within this region, all English and Welsh MenB case isolates from January 2008 (n = 87) were genetically characterized with respect to fHBP, NHBA, NadA, and PorA. Alleles for fHbp, nhba, and porA were identified in all of the isolates, of which 22% were also found to harbor nadA alleles. On the basis of genotypic data and predicted immunological cross-reactivity, the potential level of rMenB-OMV vaccine coverage in England and Wales ranges from 66% to 100%.

Impact of a Pneumococcal Conjugate Vaccination Program on Carriage among Children in Norway

Abstract: In July 2006, the seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in Norway with a reduced (2 doses + 1 boost) dose schedule. Post-PCV7 shifts in pneumococcal reservoirs were assessed by two point prevalence studies of nasopharyngeal colonization among children in day care centers, before (2006) and after (2008) widespread use of PCV7. Nasopharyngeal swabs were obtained from 1,213 children, 611 in 2006 and 602 in 2008. A total of 1,102 pneumococcal isolates were recovered. Serotyping, multilocus sequence typing, and antimicrobial drug susceptibility testing were performed on all isolates. Although carriage of PCV7 serotypes decreased among both vaccinated and unvaccinated children, the overall prevalence of pneumococcal carriage remained high (80.4%) after vaccine introduction. The pneumococcal populations were diverse, and in the shift toward non-PCV7 serotypes, expansion of a limited number of established clonal complexes was observed. While non-antimicrobial-susceptible clones persisted among PCV7 serotypes, antimicrobial resistance did not increase among non-PCV7 serotypes. Direct and indirect protection of PCV7 against nasopharyngeal colonization was inferred from an overall decrease in carriage of PCV7 serotypes. No preference was found for nonsusceptible clones among the replacing non-PCV7 serotypes.

Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation

Abstract: The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN-γ], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8+ T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation.

Immunologic indicators of clinical progression during canine Leishmania infantum infection.

Abstract: In both dogs and humans Leishmania infantum infection is more prevalent than disease, as infection often does not equate with clinical disease. Previous studies additively indicate that advanced clinical visceral leishmaniasis is characterized by increased production of anti-Leishmania antibodies, Leishmania-specific lymphoproliferative unresponsiveness, and decreased production of gamma interferon (IFN-γ) with a concomitant increase of interleukin-10 (IL-10). In order to differentiate infection versus progressive disease for better disease prognostication, we temporally evaluated humoral and cellular immunologic parameters of naturally infected dogs. The work presented here describes for the first time the temporal immune response to natural autochthonous L. infantum infection in foxhounds within the United States. Several key changes in immunological parameters should be considered when differentiating infection versus clinical disease, including a dramatic rise in IgG production, progressive increases in antigen-specific peripheral blood mononuclear cell proliferation, and IFN-γ production. Polysymptomatic disease is precluded by increased IL-10 production and consistent detection of parasite kinetoplast DNA in whole blood. This clinical presentation and the immuno-dysregulation mirror those observed in human patients, indicating that this animal model will be very useful for testing immunomodulatory anti-IL-10 and other therapies.

Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector

Abstract: Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.

Phenotypic Definition of Effector and Memory T-Lymphocyte Subsets in Mice Chronically Infected with Mycobacterium tuberculosis

Abstract: The bacterium Mycobacterium tuberculosis remains one of the world9s most successful pathogens, a situation that is aggravated by the fact that the existing vaccine, Mycobacterium bovis BCG, is not effective in adults. As with any vaccine, the purpose of giving BCG vaccination is to establish a long-lived state of memory immunity, but whether this is successfully completely established is still unclear. It is generally accepted that memory T cells can be divided into central and effector memory populations by function and by phenotype; however, the majority of data supporting this division have been generated using transgenic mouse models or mice that have recovered from acute viral infections. Tuberculosis, on the other hand, represents a persistent, chronic state of immunity in which the presence of memory T cells is far less well defined. We show here that mice vaccinated with BCG or chronically infected with M. tuberculosis establish antigen-specific populations of cells within the lungs that predominantly express a cellular phenotype consistent with their being effector or effector memory cells. In contrast, cells with a central memory phenotype exist in much lower numbers in the lungs but can be found in significantly larger numbers in the spleen, where they may represent a potential reservoir. These data suggest that the effector-to-central-memory T-cell transition may well be minimal in these persisting mycobacterial infections, and they support a novel hypothesis that this may explain the fundamental basis of the failure of the BCG vaccine in humans.

Enzyme-Linked Immunosorbent Assay for Detection of Filovirus Species-Specific Antibodies

Abstract: Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.

Immunization of mice with single PspA fragments induces antibodies capable of mediating complement deposition on different pneumococcal strains and cross-protection.

Abstract: PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal α-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

Disseminated penicilliosis, recurrent bacteremic nontyphoidal salmonellosis, and burkholderiosis associated with acquired immunodeficiency due to autoantibody against gamma interferon.

Abstract: Acquired immunodeficiency due to autoantibody against gamma interferon has recently been associated with opportunistic nontuberculous mycobacteriosis, especially among Southeast Asians. We report another 8 cases, all except one apparently immunocompetent hosts who suffered from concomitant or sequential infections by other intracellular pathogens causing penicilliosis, extraintestinal nontyphoidal salmonellosis, and burkholderiosis. The only case with an underlying immunodeficiency syndrome had systemic lupus erythematosus that was quiescent throughout the multiple infective episodes. Eight out of 10 (80.0%) patients with serological evidence of penicilliosis, 5 out of 7 (71.4%) with culture-positive extraintestinal nontyphoidal salmonellosis, 5 out of 28 (17.9%) with serological evidence of melioidosis, and 7 out of 13 (53.8%) with culture-positive nontuberculous mycobacteriosis possessed autoantibody against gamma interferon, whereas only 1 out of 100 patients with systemic lupus erythematosus did. Our study represents the first and largest case series linking this emerging immunodeficiency syndrome with these atypical infections in apparently immunocompetent hosts. Thus, we advocate that any patient with unexplained recurrent or polymicrobial infections due to these intracellular pathogens should be screened for acquired immunodeficiency due to autoantibody against gamma interferon.

Dynamic changes in inflammatory cytokines in pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus.

Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces both humoral and cellular immune responses. In this study, we investigated the changes in cytokine levels in peripheral blood between the highly pathogenic PRRSV HuN4 strain and its derivative strain HuN4-F112 obtained by serial propagation in MARC145 cells to 112 passages. The results demonstrated that pigs infected with HuN4 showed a loss of appetite, decrease in body weight, raised body temperature, and respiratory symptoms, along with interstitial pneumonia lesions. The PRRSV amounts in the pigs infected with HuN4 were 10(5) to 10(9) copies/ml in the blood and 10(10) to 10(11) copies/g in the lung tissues, whereas the virus amounts with HuN4-F112 were 10(2.15) to 10(3.13) copies/ml in the blood and 10(3.0) to 10(3.6) copies/g in the lungs. Moreover, the levels of interleukin 1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-alpha), and alpha interferon (IFN-alpha) in peripheral blood were upregulated 7 days postinoculation with HuN4, which was earlier than in the HuN4-F112 group. Furthermore, cytokine levels in the pigs infected with HuN4 returned to normal on the 21st day postinoculation, while the levels in those infected with HuN4-F112 continued to increase. These results demonstrated that the pigs infected with the highly pathogenic PRRSV HuN4 strain generated earlier and higher levels of inflammatory cytokines, and the results also indicated that HuN4 may aggravate inflammation and damage tissues and organs. The low-pathogenic PRRSV HuN4-F112 strain induced lower levels of inflammatory cytokines, which may enhance the immune responses against the infection.

Increased Allergic Immune Response to Sarcoptes scabiei Antigens in Crusted versus Ordinary Scabies

Abstract: Scabies, a parasitic skin infestation by the burrowing "itch" mite Sarcoptes scabiei, causes significant health problems for children and adults worldwide. Crusted scabies is a particularly severe form of scabies in which mites multiply into the millions, causing extensive skin crusting. The symptoms and signs of scabies suggest host immunity to the scabies mite, but the specific resistant response in humans remains largely uncharacterized. We used 4 scabies mite recombinant proteins with sequence homology to extensively studied house dust mite allergens to investigate a differential immune response between ordinary scabies and the debilitating crusted form of the disease. Subjects with either disease form showed serum IgE against recombinant S. scabiei cysteine and serine proteases and apolipoprotein, whereas naive subjects showed minimal IgE reactivity. Significantly (P < 0.05) greater serum IgE and IgG4 binding to mite apolipoprotein occurred in subjects with crusted scabies than in those with ordinary scabies. Both subject groups showed strong proliferative responses (peripheral blood mononuclear cells) to the scabies antigens, but the crusted scabies group showed increased secretion of the Th2 cytokines interleukin 5 (IL-5) and IL-13 and decreased Th1 cytokine gamma interferon (IFN-gamma) in response to the active cysteine protease. These data confirm that a nonprotective allergic response occurs in the crusted disease form and demonstrate that clinical severity is associated with differences in the type and magnitude of the antibody and cellular responses to scabies proteins. A quantitative IgE inhibition assay identified IgE immunoreactivity of scabies mite antigens distinct from that of house dust mite antigens, which is potentially important for specific scabies diagnosis and therapy.

Novel Double-Antigen Sandwich Immunoassay for Human Hepatitis B Core Antibody

Abstract: A novel diagnostic immunoassay testing procedure for hepatitis B virus core antibody (anti-HBc) using homogeneous purified full-length hepatitis B virus core antigen (HBcAg) capsids obtained from Escherichia coli was compared with Abbott Architect anti-HBc chemiluminescent microparticle immunoassay (CMIA; indirect method) against a library of specimens. A monoclonal anti-HBc neutralization confirmatory assay was then used to determine the degree of discordance between specimens. The new assay was found to be superior in both sensitivity and specificity.

Options for inactivation, adjuvant, and route of topical administration of a killed, unencapsulated pneumococcal whole-cell vaccine

Abstract: We previously reported that ethanol-killed cells of a noncapsulated strain of Streptococcus pneumoniae, given intranasally with cholera toxin as an adjuvant, protect rats against pneumonia and mice against colonization of the nasopharynx and middle ear by capsulated pneumococci of various serotypes. The acceleration of pneumococcal clearance from the nasopharynx in mice is CD4+ T cell-dependent and interleukin 17A (IL-17A) mediated and can be antibody independent. Here, anticipating human studies, we have demonstrated protection with a new vaccine strain expressing a nonhemolytic derivative of pneumolysin and grown in bovine-free culture medium. Killing the cells with chloroform, trichloroethylene, or beta-propiolactone--all used without postinactivation washing--produced more-potent immunogens than ethanol, and retention of soluble components released from the cells contributed to protection. Two sequential intranasal administrations of as little as 1 microg of protein (total of cellular and soluble combined) protected mice against nasopharyngeal challenge with pneumococci. Nontoxic single and double mutants of Escherichia coli heat-labile toxin were effective as mucosal adjuvants. Protection was induced by the sublingual and buccal routes, albeit requiring larger doses than when given intranasally. Protection was likewise induced transdermally with sonicates of the killed-cell preparation. Thus, this whole-cell antigen can be made and administered in a variety of ways to suit the manufacturer and the vaccination program and is potentially a solution to the need for a low-cost vaccine to reduce the burden of childhood pneumococcal disease in low-income countries.

Field evaluation of the efficacy of Mycobacterium bovis bacillus Calmette-Guerin against bovine tuberculosis in neonatal calves in Ethiopia.

Abstract: In developing countries, the conventional test and slaughter strategy for the control of bovine tuberculosis is prohibitively expensive, and alternative control methods such as vaccination are urgently required. In this study, the efficacy of Mycobacterium bovis bacillus Calmette-Guerin (BCG) for protection against bovine tuberculosis (bTB) was evaluated in Holstein calves under field conditions in Ethiopia. Thirteen neonatally vaccinated and 14 control calves were exposed for 10 to 23 months to skin test reactor cows. Gamma interferon (IFN-γ) testing, comparative intradermal tuberculin testing, postmortem examination, and bacteriological culture were used for the evaluation of BCG efficacy. The overall mean pathology score was significantly (P < 0.05) higher in control calves than in vaccinated calves. Culture positivity for Mycobacterium bovis was higher in the control calves than in the vaccinated calves, and significantly more BCG-vaccinated animals would have passed a standard meat inspection (P = 0.021). Overall, the protective efficacy of BCG was between 56% and 68%, depending on the parameters selected. Moreover, by measuring gamma interferon responses to the antigens ESAT-6 and CFP-10, which are present in M. bovis but absent from BCG, throughout the experiment, we were able to distinguish between vaccinated animals that were protected against bTB and those animals that were not protected. In conclusion, the present trial demonstrated an encouraging protective effect of BCG against bTB in a natural transmission setting in Ethiopia.

Construction and Immunological Evaluation of Multivalent Hepatitis B Virus (HBV) Core Virus-Like Particles Carrying HBV and HCV Epitopes

Abstract: A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.

Expression of Homing Receptors on IgA1 and IgA2 Plasmablasts in Blood Reflects Differential Distribution of IgA1 and IgA2 in Various Body Fluids

Abstract: Although secretory IgA is the most abundantly produced Ig isotype, the mechanisms underlying the differential distribution of IgA subclasses in various body fluids remain unclear. To explore these mechanisms, we examined the distribution of IgA subclasses, the influence of the nature and sites of encounters with antigens, and the correlation between IgA subclass distribution and homing potentials of circulating IgA plasmablasts. IgA1 predominated in serum, tears, nasal wash fluid, and saliva; the levels of IgA1 and IgA2 were comparable in vaginal wash fluid; and IgA2 predominated in intestinal lavage fluids. Seventy-one percent of circulating IgA plasmablasts secreted IgA1. The intestinal homing receptor (HR), α4β7, was expressed more frequently on IgA2 than on IgA1 plasmablasts, with no differences in the expression of other HRs. IgA subclass distribution among circulating antigen-specific antibody-secreting cells (ASC) was dependent on the nature of the antigen: following vaccination with Salmonella enterica serovar Typhi, unconjugated pneumococcal polysaccharide, or Haemophilus influenzae polysaccharide-diphtheria toxoid conjugate, the proportions of specific IgA1 ASC were 74%, 47%, 56%, and 80%, respectively. HR expression depended on the route of administration: expression of HRs was different after oral than after parenteral vaccination, while no difference was seen between HR expression of antigen-specific IgA1 and IgA2 ASC induced via the same route. The key factors determining IgA subclass distribution in a given secretion are the nature of the antigens encountered at a particular site and the site-specific homing instructions given to lymphocytes at that site. These two factors are reflected as differences in the homing profiles of the total populations of circulating IgA1 and IgA2 plasmablasts.

Interactive Changes between Macrophages and Adipocytes

Abstract: Obesity is associated with a proinflammatory state, with macrophage infiltration into adipose tissue. We tested the hypothesis that communication between macrophages and adipocytes affects insulin resistance by disrupting insulin-stimulated glucose transport, adipocyte differentiation, and macrophage function. To test this hypothesis, we cocultured 3T3-L1 adipocytes with C2D macrophages or primary peritoneal mouse macrophages and examined the impacts of macrophages and adipocytes on each other. Adipocytes and preadipocytes did not affect C2D macrophage TNF-α, IL-6, or IL-1β transcript concentrations relative to those obtained when C2D macrophages were incubated alone. However, preadipocytes and adipocytes increased PEC-C2D macrophage IL-6 transcript levels, while preadipocytes inhibited IL-1β transcript levels compared to those obtained when PEC-C2D macrophages were incubated in medium alone. We found that adipocyte coculture increased macrophage consumption of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and, in some cases, IL-6. C2D macrophages increasingly downregulated GLUT4 transcript levels in differentiated adipocytes. Recombinant TNF-α, IL-1β, and IL-6 also downregulated GLUT4 transcript levels relative to those for the control. However, only IL-6 was inhibitory at concentrations detected in macrophage-adipocyte cocultures. IL-6 and TNF-α, but not IL-1β, inhibited Akt phosphorylation within 15 min of insulin stimulation, but only IL-6 was inhibitory 30 min after stimulation. Lastly, we found that adipocyte differentiation was inhibited by macrophages or by recombinant TNF-α, IL-6, and IL-1β, with IL-6 having the most impact. These data suggest that the interaction between macrophages and adipocytes is a complex process, and they support the hypothesis that the macrophage-adipocyte interaction affects insulin resistance by disrupting insulin-stimulated glucose transport, adipocyte differentiation, and macrophage function.

Safety and Immunogenicity of a Recombinant Nonglycosylated Erythrocyte Binding Antigen 175 Region II Malaria Vaccine in Healthy Adults Living in an Area Where Malaria Is Not Endemic

Abstract: Erythrocyte binding antigen region II (EBA-175) is a conserved antigen of Plasmodium falciparum that is involved in binding of the parasite to the host's erythrocytes. We evaluated the safety and immunogenicity of a recombinant EBA-175 vaccine with aluminum phosphate adjuvant in healthy young adults living in the United States. Eighteen subjects/group received ascending doses (5, 20, 80, or 160 μg) of the vaccine at 0, 1, and 6 months; 8 subjects received placebo. Most of the injection site and systemic reactions were mild to moderate in intensity. After 2 or 3 doses of the vaccine at any concentration, antibody levels measured by enzyme-linked immunosorbent assay were significantly higher than those for the placebo group. Sera from subjects who received 3 doses of the vaccine at any concentration inhibited the growth of erythrocyte-stage P. falciparum at low levels compared to sera from placebo recipients or preimmune sera. In conclusion, the EBA-175 vaccine with adjuvant was safe and immunogenic in malaria-naive subjects.

Seroprevalences to Viral Pathogens in Free-Ranging and Captive Cheetahs (Acinonyx jubatus) on Namibian Farmland

Abstract: Cheetah populations are diminishing rapidly in their natural habitat. One reason for their decline is thought to be a high susceptibility to (infectious) diseases because cheetahs in zoos suffer from high disease-induced mortality. Data on the health status of free-ranging cheetahs are scarce, and little is known about their exposure and susceptibility to infectious diseases. We determined seroprevalences to nine key viruses (feline herpesvirus 1, feline calicivirus, feline parvovirus, feline coronavirus, canine distemper virus, feline immunodeficiency virus [FIV], puma lentivirus, feline leukemia virus, and rabies virus) in 68 free-ranging cheetahs on east-central Namibian farmland, 24 nonvaccinated Namibian captive cheetahs, and several other wild carnivore species and conducted necropsies of cheetahs and other wild carnivores. Eight of 11 other wild carnivores were seropositive for at least one of the viruses, including the first record of an FIV-like infection in a wild felid west of the Kalahari, the caracal (Felis caracal). Seroprevalences of the free-ranging cheetahs were below 5% for all nine viruses, which is significantly lower than seroprevalences in nonvaccinated captive cheetahs and those for five of seven viruses in previously studied free-ranging cheetahs from north-central Namibia (L. Munson, L. Marker, E. Dubovi, J. A. Spencer, J. F. Evermann, and S. J. O'Brien, J. Wildl. Dis. 40:23-31, 2004). There was no clinical or pathological evidence of infectious diseases in living or dead cheetahs. The results suggest that while free-ranging wild carnivores may be a source of pathogens, the distribution of seroprevalences across studies mirrored local human population density and factors associated with human habitation, probably reflecting contact opportunities with (nonvaccinated) domestic and feral cats and dogs. They also suggest that Namibian cheetahs respond effectively to viral challenges, encouraging consistent and sustainable conservation efforts.