Christian Brandt

Influence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime‐boost strategies in mice

The transfer of maternal antibodies to the offspring and their inhibitory effects on active infant immunization is an important factor hampering the use of certain vaccines, such as measles or respiratory syncytial virus vaccine, in early infancy. The resulting delay in protection by conventional or novel vaccines may have significant public health consequences. To define immunization approaches which may circumvent this phenomenon, experiments were set up to further elucidate its immunological bases. The influence of maternal antibodies on antibody and T cell responses to measles hemagglutinin (MV‐HA) were analyzed following MV‐HA immunization of pups born to immune or control BALB/c mothers using four different antigen delivery systems: live or inactivated conventional measles vaccine, a live recombinant canarypox vector and a DNA vaccine. High levels (>u20095 log10) of maternal anti‐HA antibodies totally inhibited antibody responses to each of the vaccine constructs, whereas normal antibody responses were elicited in presence of lower titers of maternal antibodies. However, even high titers of maternal antibodies affected neither the induction of vaccine‐specific Th1/Th2 responses, as assessed by proliferation and levels of IFN‐γ and IL‐5 production, nor CTL responses in infant mice. On the basis of these unaltered T cell responses, very early priming and boosting (at 1 and 3 weeks of age, respectively) with live measles vaccine allowed to circumvent maternal antibody inhibition of antibody responses in pups of immune mothers. This was confirmed in another immunization model (tetanus toxoid). It suggests that effective vaccine responses may be obtained earlier in presence of maternal antibodies through the use of appropriate immunization strategies using conventional or novel vaccines for early priming.

Determinants of infant responses to vaccines in presence of maternal antibodies.

Presence of maternally-derived antibodies at time of immunization is known to often interfere with active infant immunization, although with variable degrees of clinical significance. In order to progressively decipher the rules that form the basis for these inhibitory effects on infant vaccine responses, two antigens (measles, tetanus) and various antigen presentation systems were evaluated in murine early life immunization models either in absence or presence of maternal antibodies. Both conventional (proteins, conjugate vaccines) and new (live viral vectors, DNA plasmids) antigen presentation systems were found to be similarly susceptible to the inhibitory influence of maternal antibodies. Factors emerging as crucial determinants of maternal antibody-mediated effects on responses to both live and non-live vaccines include (i) the level of maternal antibodies present at immunization, (ii) the use of distinct vaccines in mothers and pups and (iii) their distinct influence on B cell and T cell vaccine responses.

Protective immunity against respiratory syncytial virus in early life after murine maternal or neonatal vaccination with the recombinant G fusion protein BBG2Na.

Maternal and neonatal immunization were evaluated for their capacity to induce protective immunity against respiratory syncytial virus (RSV) lower respiratory tract infections in early life. Murine models were studied by use of a novel recombinant vaccine candidate, designated BBG2Na, which was derived in part from the RSV (Long) G protein. Maternal immunization resulted in the passive transfer of high levels of RSV-A antibodies to the offspring, which protected them from RSV challenge for up to 14 weeks. Indeed, protection correlated with the detection of RSV antibodies in the serum. Neonatal immunization with BBG2Na induced significant antibody responses even in the first week of life. Most importantly, these neonatal responses were not inhibited by the presence of RSV maternal antibodies. Consequently, the combination of maternal and neonatal immunization with BBG2Na resulted in the continual presence of protective levels of antibodies in the offspring.

Differences in survival, brain damage, and cerebrospinal fluid cytokine kinetics due to meningitis caused by 3 different Streptococcus pneumoniae serotypes: Evaluation in humans and in 2 experimental models

BACKGROUNDnExperimental meningitis with Streptococcus pneumoniae serotypes 1, 3, and 9 has resulted in pronounced differences in disease severity, but clinical meningitis studies addressing serotype-related differences in case-fatality rates are lacking.nnnMETHODSnStudy subjects were Danish patients with pneumococcal meningitis due to serotype 1 (n=38), 3 (n=69), or 9V (n=59) during 1990-2002 for whom clinical information was available. The 3 serotypes were tested for brain damage and cerebrospinal fluid (CSF) inflammatory kinetics in 2 experimental models of meningitis.nnnRESULTSnPatients with serotype 1 had a significantly lower case-fatality rate (3%), compared with patients with serotypes 3 (23%) and 9V (32%) (P=.0047, log-rank test). Age and serotype were independent prognostic factors for fatal outcome. In experimental meningitis, the median number of areas per brain slide with brain damage was significantly lower in rats infected with serotype 1 than in rats infected with serotypes 3 and 9V. Three distinct patterns of brain damage were observed: serotype 1, cortical hemorrhage; serotype 3, cortical necrosis and abscess formation; and serotype 9V, subcortical (callosal) abscess formation. Serotype 1 caused the poorest bacterial growth and lowest CSF levels of white blood cells, tumor necrosis factor- alpha, and interleukin-8 (P<.05).nnnCONCLUSIONnCase-fatality rates of patients with pneumococcal meningitis, the degree and pattern of brain damage, and CSF cytochemical alterations in experimental pneumococcal meningitis differ according to serotype.

CD4+ T-Cell-Mediated Antiviral Protection of the Upper Respiratory Tract in BALB/c Mice following Parenteral Immunization with a Recombinant Respiratory Syncytial Virus G Protein Fragment

ABSTRACT We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4+, but not CD8+, T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967–1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4+ T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.

Attenuation of the Bacterial Load in Blood by Pretreatment with Granulocyte-Colony-Stimulating Factor Protects Rats from Fatal Outcome and Brain Damage during Streptococcus pneumoniae Meningitis

ABSTRACT A model of pneumococcal meningitis in young adult rats receiving antibiotics once the infection was established was developed. The intent was to mimic clinical and histopathological features of pneumococcal meningitis in humans. The primary aim of the present study was to evaluate whether medical boosting of the peripheral neutrophil count affected the outcome of the meningitis. The risk of terminal illness over the first 7 days after infection was significantly reduced for rats who had elevated peripheral white blood cell counts after receiving granulocyte-colony-stimulating factor (G-CSF) prior to the infection compared to that for untreated rats (P = 0.039 by the log rank test). The improved outcome was associated with reduced signs of cerebral cortical damage (P = 0.008). Furthermore, the beneficial effects of G-CSF were associated with reduced bacterial loads in the cerebrospinal fluid (median, 1.1 × 105 versus 2.9 × 105 CFU/ml; P = 0.023) and in blood (median, 2.9 × 102 versus 6.3 × 102 CFU/ml; P = 0.024), as well as attenuated pleocytosis (median, 800 × 106 versus 1,231 × 106 cells/liter; P = 0.025), 24 h after the infection. Conversely, initiation of G-CSF therapy 28 h postinfection did not alter the clinical or histological outcome relative to that for non-G-CSF-treated rats. The magnitude of bacteremia and pretreatment with G-CSF were found to be prognostic factors for both outcome and brain damage. In summary, elevated neutrophil levels prior to the development of meningitis result in reduced risks of death and brain damage. This beneficial effect is most likely achieved through improved control of the systemic disease.

In silico serine β-lactamases analysis reveals a huge potential resistome in environmental and pathogenic species.

The secretion of antimicrobial compounds is an ancient mechanism with clear survival benefits for microbes competing with other microorganisms. Consequently, mechanisms that confer resistance are also ancient and may represent an underestimated reservoir in environmental bacteria. In this context, β-lactamases (BLs) are of great interest due to their long-term presence and diversification in the hospital environment, leading to the emergence of Gram-negative pathogens that are resistant to cephalosporins (extended spectrum BLsu2009=u2009ESBLs) and carbapenems (carbapenemases). In the current study, protein sequence databases were used to analyze BLs, and the results revealed a substantial number of unknown and functionally uncharacterized BLs in a multitude of environmental and pathogenic species. Together, these BLs represent an uncharacterized reservoir of potentially transferable resistance genes. Considering all available data, in silico approaches appear to more adequately reflect a given resistome than analyses of limited datasets. This approach leads to a more precise definition of BL clades and conserved motifs. Moreover, it may support the prediction of new resistance determinants and improve the tailored development of robust molecular diagnostics.

Mutagenesis of the CTX-M-type ESBL-is MIC-guided treatment according to the new EUCAST recommendations a safe approach?

OBJECTIVESnSpurred by the latest EUCAST and CLSI recommendation to adjust antibiotic therapy on the basis of MICs instead of resistance mechanisms, we aimed to investigate the ability of CTX-M-1 and CTX-M-14 to achieve ceftazidime resistance under selective conditions.nnnMETHODSnWe exposed Escherichia coli transconjugants bearing natural plasmids that express CTX-M-1, CTX-M-14 or CTX-M-15 to various selective culture conditions and tracked their growth and mutational frequencies. For selected mutants we analysed the sequences of the bla CTX-M genes, determined the altered MICs of cefotaxime, cefepime, ceftazidime and meropenem, and measured the efflux properties and the changes in transcriptional levels of bla CTX-M genes.nnnRESULTSnThe CTX-M-1- and CTX-M-14-bearing clones switched from ceftazidime-susceptible to ceftazidime-resistant phenotypes under selective conditions within 24 h. However, no mutations within the bla CTX-M genes were found, and the efflux was unlikely to be involved in the increased ceftazidime MICs. In CTX-M-1-bearing clones bla CTX-M-1 expression was 19-fold increased under ceftazidime-selective conditions but there was a high variance within the clones. Reasons for increased ceftazidime MICs of CTX-M-bearing clones remain unclear but might be the increased enzymatic activity or other intrachromosomal mutations.nnnCONCLUSIONSnIt can be speculated that different strategies to survive under selective conditions can be adopted by E. coli, thereby establishing an optimal mechanism with the lowest energy demand for each transconjugant. Based on our in vitro findings, we cannot fully recommend the use of ceftazidime, particularly in critically ill patients with infections due to ESBL producers, regardless of susceptibility to ceftazidime.

A Nosocomial Foodborne Outbreak of a VIM Carbapenemase-Expressing Citrobacter freundii

BackgroundnA foodborne outbreak of VIM carbapenemase-expressing Citrobacter freundii (CPC) occurred between February 2016 and June 2016 at a major university hospital in Germany.nnnMethodsnAn explosive increase in CPC isolated from rectal swabs of patients during weekly routine screening led to the declaration of an outbreak. A hospital-wide prevalence screening was initiated as well as screening of all patients on admission and before transfer to another ward, canteen staff, patient rooms, medical and kitchen inventory, and food. Swabs were streaked out on selective plates. All CPC isolates were analyzed using mass spectrometry, and selected isolates were analyzed using whole-genome sequencing.nnnResultsnA total of 76 were identified; most were unrelated cases in different wards. The CPC was isolated from retained samples of prepared vegetable salads and puddings and from a mixing machine used to prepare these foods only after an overnight culture. The immediate ban on serving potential source food resulted in a sharp decline and finally disappearance of novel cases. Repeated testing of presliced vegetables showed a high degree of contamination with C. freundii without a carbapenemase, indicating a possible source.nnnConclusionsnAn explosive increase in carbapenemase-expressing Enterobacteriaceae contamination may have been caused by a foodborne source, and presliced vegetables should be taken into account as a putative pathogen repository. These findings underline the importance of appropriate cooling, transport, reheating, and distribution of meals and indicate that probing of nonorganic surfaces is limited by low sensitivity, which may be increased by additional overnight cultivation in appropriate media.

Development of a rapid diagnostic assay based on magnetic bead purification of OXA-β-lactamase mRNA.

AIMnTo countermeasure the global spread of β-lactamases, we developed a rapid molecular test for the highly variable OXA-β-lactamases that allows minimizing the time to effective treatment.nnnMETHODSnOXA-mRNA was specifically enriched from total RNA using group-specific biotinylated DNA probes and streptavidin-coated magnetic beads. Phylogenetic OXA groups were distinguished by PCR product size.nnnRESULTSnThis mRNA fishing method is highly sensitive, yielding specific results from as little as 1 ng total RNA. It enables discrimination of OXA-extended substrate spectrum β-lactamases and carbapenemases and the semi-quantitative detection of highly expressed ISAba1-controlled variants.nnnCONCLUSIONnTargeting mRNA with specific probes on magnetic beads will allow for adaptation to automated systems, such as point-of-care diagnostics.