M.I. de Jonge

Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage.

The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal immunoglobulin G (IgG) to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model, samples were collected from PCV and control-vaccinated adults. In PCV-vaccinated subjects, IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared with pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS-specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity.

Patterns in Bacterial- and Viral-Induced Immunosuppression and Secondary Infections in the ICU

Abstract Immunosuppression renders the host increased susceptible for secondary infections. It is becoming increasingly clear that not only bacterial sepsis, but also respiratory viruses with both severe and mild disease courses such as influenza, respiratory syncytial virus, and the human rhinovirus may induce immunosuppression. In this review, the current knowledge on (mechanisms of) bacterial- and virus-induced immunosuppression and the accompanying susceptibility toward various secondary infections is described. In addition, the frequently encountered secondary pathogens and their preferred localizations are presented. Finally, future perspectives in the context of the development of diagnostic markers and possibilities for personalized therapy to improve the diagnosis and treatment of immunocompromised patients are discussed.

Characteristics of RSV-Specific Maternal Antibodies in Plasma of Hospitalized, Acute RSV Patients under Three Months of Age

Respiratory syncytial virus (RSV) is the leading cause for respiratory illness that requires hospitalization in infancy. High levels of maternal antibodies can protect against RSV infection. However, RSV-infected infants can suffer from severe disease symptoms even in the presence of high levels of RSV-specific antibodies. This study analyzes several serological characteristics to explore potential deficiencies or surpluses of antibodies that could relate to severe disease symptoms. We compare serum antibodies from hospitalized patients who suffered severe symptoms as well as uninfected infants. Disease severity markers were oxygen therapy, tachypnea, oxygen saturation, admission to the intensive care unit and duration of hospitalization. Antibodies against RSV G protein and a prefusion F epitope correlated with in vitro neutralization. Avidity of RSV-specific IgG antibodies was lower in RSV-infected infants compared to uninfected controls. Severe disease symptoms were unrelated to RSV-specific IgG antibody titers, avidity of RSV-IgG, virus neutralization capacity or titers against pre- and postfusion F or G protein ectodomains and the prefusion F antigenic site Ø. In conclusion, the detailed serological characterization did not indicate dysfunctional or epitope-skewed composition of serum antibodies in hospitalized RSV-infected infants suffering from severe disease symptoms. It remains unclear, whether specific antibody fractions could diminish disease symptoms.

Complement Factor H Serum Levels Determine Resistance to Pneumococcal Invasive Disease

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.

A novel flow cytometry-based assay for the quantification of antibody-dependent pneumococcal agglutination

The respiratory pathogen Streptococcus pneumoniae is a major cause of diseases such as otitis media, pneumonia, sepsis and meningitis. The first step towards infection is colonization of the nasopharynx. Recently, it was shown that agglutinating antibodies play an important role in the prevention of mucosal colonization with S. pneumoniae. Here, we present a novel method to quantify antibody-dependent pneumococcal agglutination in a high-throughput manner using flow cytometry. We found that the concentration of agglutinating antibodies against pneumococcal capsule are directly correlated with changes in the size and complexity of bacterial aggregates, as measured by flow cytometry and confirmed by light microscopy. Using the increase in size, we determined the agglutination index. The cutoff value was set by measuring a series of non-agglutinating antibodies. With this method, we show that not only anti-polysaccharide capsule antibodies are able to induce agglutination but that also anti-PspA protein antibodies have agglutinating capabilities. In conclusion, we have described and validated a novel method to quantify pneumococcal agglutination, which can be used to screen sera from murine or human vaccination studies, in a high-throughput manner.

Aptamers for respiratory syncytial virus detection

The identification of the infectious agents is pivotal for appropriate care of patients with viral diseases. Current viral diagnostics rely on selective detection of viral nucleic acid or protein components. In general, detection of proteins rather than nucleic acids is technically more suitable for rapid tests. However, protein-based virus identification methods depend on antibodies limiting the practical applicability of these approaches. Aptamers rival antibodies in target selectivity and binding affinity, and excel in terms of robustness and cost of synthesis. Although aptamers have been generated for virus identification in laboratory settings, their introduction into routine virus diagnostics has not been realized, yet. Here, we demonstrate that the rationally designed SELEX protocol can be applied on whole virus to select aptamers, which can potentially be applied for viral diagnostics. This approach does not require purified virus protein or complicated virus purification. The presented data also illustrate that corroborating the functionality of aptamers with various approaches is essential to pinpoint the most appropriate aptamer amongst the panel of candidates obtained by the selection. Our protocol yielded aptamers capable of detecting respiratory syncytial virus (RSV), an important pathogen causing severe disease especially in young infants, at clinically relevant concentrations in complex matrices.

Non-encapsulated Streptococcus pneumoniae, vaccination as a measure to interfere with horizontal gene transfer

The polysaccharide capsule is the most important virulence factor of Streptococcus pneumoniae, however it is neither a requirement for persistent carriage nor for transmission. It has been postulated that reduced or complete lack of capsule expression facilitates colonization of the nasopharynx as a result of enhanced binding to epithelial cells. On the other hand, the large investment of energy in the synthesis of a polysaccharide capsule protect the pneumococci against complement mediated killing. Furthermore, a capsule might prevent agglutination in a mucus rich environment avoiding clearance from the nasopharyngeal cavity. An important group of pneumococci is serologically nontypeable (NT) due to the lack of a capsule, therefore called non-encapsulated Streptococcus pneumoniae (NESp). They are divided in 2 groups: group I, which contains a conventional but defective cps locus as a consequence of different types of mutations causing capsule gene inactivation and group II, which completely lacks a cps locus. The latter group consists of 3 ‘null capsule clades’ (NCC), classified based on the presence of 3 genes pspK (or nspA), aliC (or aliB-like ORF1) and aliD (or aliB-like ORF2): NCC1 (pspKC, aliC¡, aliD¡), NCC2 (pspK¡, aliCC, aliDC), NCC3 (pspK¡, aliC¡, aliDC). The pspK, aliC and aliD genes have replaced the capsule locus, and recombination of flanking homologous genes (dexB and aliA) has the potential to transfer this gene cluster between encapsulated and non-encapsulated strains. It has been shown that group II NESp can colonize mouse nasopharynges as well as capsulated pneumococci, which implies that they have been able to compensate for the lack of a capsule enabling persistent carriage. The current pneumococcal vaccines are made of purified conjugated (10 or 13 valent) or non-conjugated (23 valent) polysaccharide capsules, which evidently do not protect against NESp. Vaccination with a limited number of serotypes leads to an increased pressure on a specific subset of pneumococci that opens an environmental niche that NESp strains are able to exploit. This might explain why an increase in NESp prevalence has been observed following the introduction of pneumococcal conjugate vaccines. Asymptomatic carriage rates of NESp have been measured, ranging from 4% to 19%. 8 Generally, NESp cause mainly non-invasive pneumococcal diseases such as otitis media and infectious conjunctivitis. In rare cases, infection with NESp can also lead to invasive pneumococcal disease, although mainly in immunodeficient patients. Nevertheless, the total number of clinical cases caused by NESp might be underestimated since serotyping is not routinely performed. A potentially more serious concern is the role of NESp in the spread of antibiotic resistance and virulence genes. The pneumococcus exploits different mechanisms to mediate horizontal gene transfer and recombination, including transformation, the use of mobile elements and transducing phages. Despite the great advantages of having a capsule, providing resistance to clearance during colonization, it strongly hampers DNA uptake limiting the capability of transformation-mediated adaptation. In vitro studies have shown that capsule-negative mutants acquire genes through recombination at a higher frequency than the isogenic encapsulated strains. 10 In the largest pneumococcal sequencing study thus far, with more than 3000 isolates, it was found that NESp showed the highest frequencies of receipt and donation of recombined DNA fragments, confirming that they are a potential major reservoir of genetic diversity for the wider population. The model proposed by Andam and Hanage explains how frequent loss and subsequent gain

Development of Endotoxin Tolerance Does Not Influence the Response to a Challenge with the Mucosal Live-Attenuated Influenza Vaccine in Humans In Vivo

Introduction The effects of bacterial infections on the response to subsequent viral infections are largely unknown. This is important to elucidate to increase insight into the pathophysiology of bacterial and viral co-infections, and to assess whether bacterial infections may influence the course of viral infections. Methods Healthy male subjects received either bacterial endotoxin [Escherichia coli-derived lipopolysaccharide (LPS), 2 ng/kg, n = 15] or placebo (n = 15) intravenously, followed by intranasal Fluenz (live-attenuated influenza vaccine) 1 week later. Results LPS administration resulted in increased plasma cytokine levels and development of endotoxin tolerance in vivo and ex vivo, illustrated by attenuated cytokine production upon rechallenge with LPS. Following Fluenz administration, infectivity for the Fluenz A/B strains was similar between the LPS–Fluenz and placebo–Fluenz groups (13/15 subjects in both groups). Also, the Fluenz-induced increase in temperature and IL-6, G-CSF and IP-10 concentrations in nasal wash were similar between both groups. Conclusion While endotoxemia profoundly attenuates the immune response upon a second LPS challenge, it does not influence the Fluenz-induced immune response. These results suggest immune suppression after bacterial infection does not alter the response to a subsequent viral infection.

Antibiotic resistance of Streptococcus pneumoniae colonising the nasopharynx of HIV-exposed Tanzanian infants.

To determine antibiotic susceptibility of colonising pneumococcal serotypes in HIV‐exposed infants before the introduction of the 13‐valent pneumococcal conjugate vaccine (PCV13), because HIV‐exposed infants are at increased risk of invasive pneumococcal infections.