Christopher E. Taylor

Human polymicrobial infections

Summary Context Polymicrobial diseases, caused by combinations of viruses, bacteria, fungi, and parasites, are being recognised with increasing frequency. In these infections, the presence of one micro-organism generates a niche for other pathogenic micro-organisms to colonise, one micro-organism predisposes the host to colonisation by other micro-organisms, or two or more non-pathogenic micro-organisms together cause disease. Starting point Recently, Gili Regev-Yochay (JAMA 2004; 292: 716–20) and Debby Bogaert (Lancet 2004; 363: 1871–72), and their colleagues, suggested another interaction: microbial interference—the ability of Streptococcus pneumoniae carriage to protect against Staphylococcus aureus carriage, and the inverse effect of pneumococcal conjugate vaccination on the increased carriage of Staph aureus and Staph-aureus-related disease. Strep pneumoniae carriage protected against Staph aureus carriage, and the bacterial interference could be disrupted by vaccinating children with pneumococcal conjugate vaccines that reduced nasopharyngeal carriage of vaccine-type Strep pneumoniae. Where next The medical community is recognising the significance of polymicrobial diseases and the major types of microbial community interactions associated with human health and disease. Many traditional therapies are just starting to take into account the polymicrobial cause of diseases and the repercussions of treatment and prevention.

T Cells Regulate the IgM Antibody Response of BALB/c Mice to Dextran B1355

The IgM antibody response of BALB/c mice to bacterial (Leuconostoc) dextran B1355 is influenced in a positive and negative manner by regulatory CD4+ and CD8+ T cells, respectively. Treatment with concanavalin A (ConA) at the time of immunization or 2 days later caused suppression and enhancement of the antibody response, respectively. Priming of mice with a sub-immunogenic dose of dextran resulted in profound suppression upon subsequent immunization 3 days later. None of these effects were demonstrable in athymic mice. Transfer of T cells from mice primed 18 h previously with a subimmunogenic dose of dextran suppressed the antibody response in immunized recipients; such suppression was abolished by the treatment of transferred cells with anti Thy 1.2 or anti Lyt 2.2 (CD8) antibody in the presence of complement. By contrast, the transfer of T cells from mice, which had been given an immunogenic dose of dextran 4 days previously, increased the antibody response in immunized recipients; such enhancement was abolished by treating transferred cells with anti Thy 1.2 or anti L3T4 (CD4) antibody in the presence of complement. These findings indicate that the immune response to dextran B1355 is regulated by CD4+ T-amplifier cells (Ta cells) and by CD8+ T-suppressor cells (Ts cells) which are activated during the course of a normal antibody response.

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG1 antibody response was noted after the administration of Con A whereas profound enhancement of IgG2a antibody response was noted after PHA was given.

Immunosuppressive Effects Induced by the Polysaccharide Moiety of Some Bacterial Lipopolysaccharides

The immunomodulatory properties of several lipopolysaccharides (LPS) derived from clinical isolates of Pseudomonas aeruginosa, Branhamella catarrhalis, and Bordetella pertussis were evaluated for their capacity to influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III), which is known to be regulated by suppressor and amplifier T cells (Ts and Ta, respectively). The administration of LPS, two days after immunization resulted in a significant increase in the antibody response. Such enhancement may be due mainly to the ability of the lipid A moiety of LPS to abolish the negative effects of activated Ts, thereby enabling Ta function to be more fully expressed; however, B cell mitogenicity of the LPS molecule also may be involved. By contrast, treatment with LPS at the time of immunization with SSS-III induces significant suppression of the SSS-III-specific antibody response; such suppression is not induced by LPS or lipid A derived from Escherichia coli and Salmonella minnesota, and is independent of the capacity of LPS to activate B cells polyclonally, an activity generally attributed to the lipid A fraction of LPS. Studies conducted with the LPS of P. aeruginosa indicated that the suppression induced is T cell dependent and mediated by the polysaccharide (PS) fraction of LPS; it appears to be due-at least in part-to the capacity of PS to expand or increase the size of the precursor pool of Ts, activated in response to SSS-III. The significance of these findings to the pathogenesis of certain gram-negative infections is discussed.

Bacterial Polysaccharides, Endotoxins, and Immunomodulation

These studies show that at least some--though certainly not all--of the adjuvant effects of LPS and its derivatives can be attributed to its ability to eliminate the inhibitory effects of Ts which are activated during the course of a normal immune response. The ability of nontoxic MPL to act in this fashion suggests that it can be used as a safe and acceptable alternative to Freunds complete adjuvant to increase the immunogenicity of poorly immunogenic antigens. More important, the ability of MPL to eliminate the expression of Ts activity, without adversely influencing other T cell functions (e.g., Th, Ta, and Tc activity) makes its use as an adjuvant even more promising since it can then permit those T cell functions to be expressed in a much more efficient manner. Obviously, this would have great significance for the development of tumor immunity.

Effects of Interferon Gamma on the Antibody Response to Pseudomonas aeruginosa Lipopolysaccharide in Mice

Different strains of mice were examined for the capacity to produce an Ig subclass-specific antibody response to purified Pseudomonas aeruginosa lipopolysaccharide (PALPS). With the exception of the AKR strain, the predominant isotype for most of the strains tested was IgG3 whereas the least frequent isotype expressed was either IgG2b or IgG1. AKR mice were unique in that the predominant isotype produced was IgG2a, rather than IgG3; however, the administration of anti-interferon gamma antibody, at the time of immunization with PALPS caused a substantial decrease in the IgG2a antibody response. Selected B10 congenic strains were used to assess the relationship between the antibody responses and the major histocompatibility complex (MHC) genes. Here, the isotype-patterns for the antibody responses were essentially the same regardless of the MHC haplotype. Interestingly, an increase in IgG2a, with a concomitant decrease in IgM and IgG1 antibody was noted when C3H mice were given interferon gamma at the time of immunization. These studies indicate that, in general, the antibody response to PALPS consists of IgG3 antibody as the predominant isotype, and that the antibody response can be modified by interferon gamma.

Effects of IL-4 depletion on the antibody response to Pseudomonas aeruginosa lipopolysaccharide in mice.

These studies were done to examine the role of interleukin-4 (IL-4) in the generation of isotype specific antibody responses of mice to Pseudomonas aeruginosa lipopolysaccharide (PALPS) by neutralization of IL-4 in vivo using anti-IL-4 antibody (11B11). We found that the administration of anti-IL-4 antibody (11B11) 24 h before immunization with PALPS resulted in a decreased PALPS-specific antibody response for all isotypes examined (IgM, IgG1, IgG2a, IgG2b, IgG3). By contrast, we observed that the non-antigen-specific (polyclonal) IgM response of mice following treatment with 11B11 antibody and PALPS was increased while the polyclonal responses for the other isotypes were unaffected. When mice were given recombinant IL-10 at the time of immunization with PALPS there was a decrease in the PALPS-specific antibody response but an increase in the polyclonal IgM, IgG2a, IgG2b, IgG3 response whereas the polyclonal IgG1 response was decreased by a five-fold margin. The results of these studies suggest that both the antigen-specific and the polyclonal response can be influenced in a different manner by IL-4 or by IL-10.

Antigen specific suppressor T cells respond to cytokines released by T cells.

These studies were conducted to examine the cytokine requirement for clonal expansion of regulatory T cells. It was observed that the in vivo administration of recombinant IL2 (rIL2), rIL5 or interferon (IFN) gamma at the time of immunization with pneumococcal polysaccharide Type III (SSS-III) resulted in substantial suppression of the antibody response in each case. Using our well established cell transfer system we found that such suppression of the antibody response could be transferred using 10-100-fold fewer primed spleen cells providing these cells were treated in vitro with rIL2 before cell transfer; spleen cells from unimmunized mice or from mice primed with an unrelated antigen and then treated with rIL2 did not cause suppression of the antibody response to SSS-III, thereby eliminating the possibility of non-specific carry-over effects induced by rIL2. We also found that the in vivo administration of anti-IL2 receptor antibody inhibited the generation of Ts cells in vivo. Spleen cells from SSS-III primed animals treated with rIL4, rIL5 and IFN gamma--but not rIL6--likewise are able to transfer suppression of the antibody response with fewer cells than that required using primed cells not treated with cytokines. Thus, these studies indicate that Ts cell activity is greatly influenced by cytokines. The studies also suggest that these cytokines may be required during the activation and/or clonal expansion of Ts cells.

Workshop on Carbohydrate Moieties as Vaccine Candidates.

Workshop on Carbohydrate Moieties as Vaccine Candidates Bethesda, MD, USA October 6-7, 2004 Whole microbes, microbial subunits and extracts, and peptide and protein antigens have been the focus of much vaccine research and development. While studies of peptide and protein antigens have been facilitated by the rapid advances in genomics and proteomics, studies of sugar chains, which are abundantly expressed on the outer surfaces of viral, bacterial, protozoan, and fungal pathogens and on the membranes of mammalian cells, have not kept pace with technologic advances. Polysaccharide-based vaccines have demonstrated efficacy in disease-prevention strategies, e.g., Haemophilus influenzae b and pneumococcal conjugate vaccines. However, our understanding of several aspects of polysaccharide vaccines is limited, and more knowledge is needed to allow greater development and deployment. The goals of this workshop were to examine the mechanisms involved in generating an appropriate immune response to selected carbohydrate antigens, highlight recent and novel advances, and discuss how this information could be used in the development of effective vaccines. The workshop participants included national and international research scientists and clinicians from the National Institutes of Health, the Food and Drug Administration, academia, and industry. The meeting was organized into 7 sessions on such topics as genetic and cellular mechanisms of carbohydrate immunity, carbohydrate antigens for vaccines, and new tools for studying carbohydrates. Understanding the mechanistic aspects of the genetic control and the cellular pathways of the immune response to bacterial carbohydrate antigens should provide insights into ways to enhance the immune response and thus facilitate vaccine development. Studies were also presented on novel molecules involved in the recognition of carbohydrate antigens such as specific intercellular adhesion molecule (ICAM)–grabbing nonintegrins, which are C-type lectins that show substantial expression in many tissues, and toll-like receptors, which function as pattern recognition receptors for conserved pathogen structures and serve as key links between innate and adaptive immunity. Investigations are ongoing to determine how these molecules function in bacterial clearance and in signaling innate and adaptive responses. A number of presentations were focused on the role of CD1 proteins, which present lipid antigens (e.g., from mycobacteria or Francisella tularensis, a potential weapon of bioterrorism) to T cells. The evidence that CD1-restricted T cells contribute to immunity against microbial infection includes the observation that CD1 is expressed at higher levels in lesions of tuberculoid leprosy in comparison to lepromatous leprosy. The design of optimal vaccines against such pathogens should include lipid and peptide antigens. Presentations from several invited experts emphasized the current challenges facing the development of vaccines for meningococcal meningitis. Given that group B meningococcal capsular polysaccharide is similar to host molecules, studies are ongoing to identify vaccine candidates that elicit protective antibody without eliciting autoantibodies. A licensed outer membrane vesicle vaccine was recently introduced for widespread use in New Zealand to control an epidemic. Regarding groups A and C polysaccharides, differences exist in age-related immune responses; for example, for unknown reasons group A polysaccharide is uniquely immunogenic in infants as young as 6 months of age and repeated doses elicit booster antibody responses, whereas group C is poorly immunogenic and repeated doses do not induce adequate responses. A continuing problem in vaccine research is devising methods to enhance the immune response and then ensuring that it is protective against disease; for instance, the use of CD40 agonist antibody plus antigen showed remarkably rapid immune response and protection in a murine model of anthrax. Equally challenging are efforts to develop combined and multivalent vaccines; several new approaches were discussed. For instance, a diphtheria, tetanus, pertussis, hepatitis B, H. influenzae b, meningococcal A and C combination vaccine has been tested in an open, randomized, controlled study. It induces groups A and C bactericidal antibody responses in infants and has reactogenicity profiles similar to meningococcal A and C conjugates given separately. Recent efforts in HIV vaccine research indicate that producing a multivalent envelope glycan conjugate vaccine to induce production of broad neutralizing antibodies is possible. New technologies such as carbohydrate microarrays, automated syntheses of oligosaccharides, and biophysical and computational methods for studying antigen-antibody interactions are now available for providing insight into the structure of and immune response to carbohydrate antigens. Additionally, peptide mimotopes may be used in carbohydrate technologies designed for proteins. An oligosaccharide synthesizer is now being used in the development of a number of vaccines, including those for malaria, leishmaniasis, HIV, tuberculosis, and leprosy. A Consortium for Functional Genomics (Web site available from http://www.functionalglycomics.org) has been established at the Scripps Research Institute and has developed a novel glycan array format that uses covalent coupling of glycans to glass slides. At the conclusion of the workshop, participants were asked to identify gaps in knowledge and resource needs. The gaps include 1) elucidating the mechanisms of immunity to and regulation of carbohydrate antigens in adults, children, and neonates and using opportunities (e.g., computer capacity) for modeling carbohydrate antigen-antibody interactions; 2) defining the molecular basis of enhanced immunogenicity with glycoconjugate vaccines and investigating the role of adjuvants; 3) examining the role of CD1-reactive T cells in the immune response to capsular polysaccharides; and 4) developing surrogates for in vivo immunity for the use of glycolipids as CD1-based vaccines in humans. Resource needs include the following: 1) enhanced availability of carbohydrate microarrays; 2) development of appropriate animal models and the availability of a transgenic mouse platform that allows generation of human antibodies; 3) development of tetramers and analytical chemistry to help in the identification of antigens, e.g., for the use of glycolipids as CD-1-based vaccines; and 4) enhanced good laboratory practice (GLP) resources to produce synthetic carbohydrate vaccines and GLP testing for vaccine candidate safety in animals and for production of good manufacturing practice vaccine candidates. As a result of the workshop, an open LISTSERV (GLYCOIMMUNOLOGY) has been established, and the Journal of Clinical Infectious Diseases will publish a review article. For additional information, contact ctaylor@niaid.nih.gov The workshop was sponsored by funds from the National Vaccine Program Office and the National Institute of Allergy and Infectious Diseases.

The influence of monophosphoryl lipid A (MPL) on erythrocyte autoantibody formation.

The onset and the amount of erythrocyte autoantibodies induced by the injection of C57BL/6N mice with rat red blood cells (RRBC) were hastened and increased, respectively, after the administration of monophosphoryl lipid A (MPL); this was not the case for similarly treated BALB/cAnN mice, which make a lower autoantibody response after immunization with RRBC. The transfer of spleen cells from donor C57BL/6N mice immunized with RRBC suppressed autoantibody formation in recipient mice subsequently immunized with RRBC; however, treatment with MPL prevented neither the induction nor the expression of such suppression. This suggests that the increased autoantibody response in RRBC-immunized C57BL/6N mice treated with MPL is not due to the inactivation of suppressor cell activity which, in other studies, was found to be extremely sensitive to MPL.