Antibody immunity and natural resistance to cryptococcosis.

The encapsulated fungus Cryptococcus neoformans (Cn) causes cryptococcal meningitis (CM). There are ~180,000 deaths per year worldwide attributed to CM, which is the most common cause of meningitis in adults with HIV in sub-Saharan Africa. HIV infection with advanced immunodeficiency is the most important predisposing risk factor for CM, highlighting the critical role that T cell mediated immunity plays in disease prevention. Numerous studies in the past decade demonstrate that antibody immunity also plays a role in resistance to CM, although its role has taken more time to establish. In mice, B cells reduce early dissemination from lungs to brain, and naïve mouse IgM can enhance fungal containment in the lungs. In concert with these findings, human studies show that patients with CM have lower IgM memory B cell levels and/or different serological profiles than controls. In this article, we review recent data on the role that B cells and/or antibody-based immunity play in host defense against Cn and natural resistance to CM.

Mycophenolate mofetil: effects on cellular immune subsets, infectious complications, and antimicrobial activity.

Mycophenolate mofetil (MMF) is one of the most frequently used immunosuppressive drugs in solid organ transplant recipients. MMF is an inhibitor of inosine-5'-monophosphate, and is able to preferentially inhibit B-cell and T-cell function. The immunosuppressive abilities of MMF have made it one of the most successful anti-rejection drugs in transplant patients, but patients also appear to have increased susceptibility to infections, specifically cytomegalovirus and BK virus. Despite its association with an increased risk of infection, MMF has also exhibited antimicrobial activity against pathogens including hepatitis C, Pneumocystis jirovecii, and human immunodeficiency virus. A thorough understanding of the functions of MMF on the immune system and interaction with infectious pathogens could be helpful in implementing preventative strategies against opportunistic infections in transplant patients.

Insights into mechanisms of antibody-mediated immunity from studies with Cryptococcus neoformans.

At first glance Cryptococcus neoformans appears an unlikely microbe to provide a new understanding of mechanisms of antibody-mediated immunity (AMI), because it is a facultative intracellular fungal pathogen for which the role of naturally acquired AMI in host defense is uncertain. However, numerous studies have now established that certain antibodies (Abs) against C. neoformans are protective in certain hosts. Studies with Abs to C. neoformans have provided new insights into AMI and generated new precedents with implications for other pathogens. The following concepts have emerged: 1) susceptibility to C. neoformans may be related to qualitative and quantitative aspects of the Ab response; 2) protective monoclonal Abs can be generated against pathogens even when the role of humoral immunity is uncertain; 3) Abs to C. neoformans mediate protection by immunomodulatory effects, thereby linking Ab efficacy to the overall host immune response; 4) Ab efficacy is critically dependent on fine specificity, which in turn is affected by immunoglobulin variable region usage, somatic mutation and constant region usage; 5) the efficacy of passive Ab therapy is a function of Ab dose and infecting innoculum, with lack of efficacy at the extremes of Ab concentration; 6) Ab-mediated toxicity resulting from antigen-Ab complex-induced release of platelet activating factor is isotype dependent. Observations with C. neoformans have stimulated a reappraisal of the role of humoral immunity for other pathogens and highlighted the limitations in current methods of assessing the role of Ab in host defense.

Feasibility of radioimmunotherapy of experimental pneumococcal infection.

Streptococcus pneumoniae is an important cause of community-acquired pneumonia, meningitis, and bacteremia. The problem of pneumococcal disease is exacerbated by increasing drug resistance. Furthermore, patients with impaired immunity are at high risk for invasive pneumococcal infections. Thus, there is an urgent need for new approaches to antimicrobial therapy. Antibody therapies take advantage of the specificity and high affinity of the antigen-antibody interaction to deliver antibacterial compounds to a site of infection in the form of naked or conjugated antibodies. We have recently established that radioimmunotherapy (RIT) can be used to treat experimental fungal infections in mice. In the present study, we investigated the feasibility of applying a RIT approach to the treatment of S. pneumoniae infection by evaluating the susceptibility of S. pneumoniae to radiolabeled antibody in vitro and in an animal infection model. For the specific antibody carrier, we used human monoclonal antibody D11, which binds to pneumococcal capsular polysaccharide 8. We have selected the alpha particle emitter (213)Bi as the radionuclide for conjugation to the antibody. Incubation of serotype 8 S. pneumoniae with (213)Bi-D11 resulted in dose-dependent killing of bacteria. RIT of S. pneumoniae infection in C57BL/6 mice showed that 60% more mice survived in the (213)Bi-D11-treated group (80 micro Ci) than in the untreated group (P < 0.01). The treatment did not cause hematological toxicity, as demonstrated by platelet counts. This feasibility study establishes that RIT can be applied to the treatment of bacterial infections.

Immune therapy for infectious diseases at the dawn of the 21st century: the past, present and future role of antibody therapy, therapeutic vaccination and biological response modifiers.

In the last decades of the 20th century, infectious diseases have re-emerged as a significant public health problem in the developed world. However, the available anti-infective armamentarium has proven to be alarmingly insufficient to combat many of the microbes that cause these diseases, such as drug resistant microbes, microbes for which therapy is not available or ineffective because of underlying host immune impairment, and microbes that only cause disease in the setting of impaired immunity but are not pathogens in normal individuals. Hence, there is an urgent need for new approaches to the treatment of infectious diseases that can increase the efficacy of anti-infective therapy and bolster the immune response to microbial agents in immunocompromised hosts, circumvent rising rates of antimicrobial drug resistance and be rapidly developed to fight emerging epidemics. Immune therapy, which encompasses pathogen-specific and non-pathogen specific modalities designed to augment or restore host immunity against disease causing microbes, are poised to play an important part in modern anti-infective therapy. Our growing understanding of host-microbe interaction and mechanisms of protective immunity have allowed for an increasingly rational approach to the design of immune based therapeutic modalities. As part of this effort, it is important to remember that the origin of modern anti-infective therapy was serum therapy, a pathogen-specific immune therapeutic modality. In this paper, we review the historical underpinnings and present and future applications of immune therapy for infectious diseases in light of current challenges to the field.

Structure-function relationships for human antibodies to pneumococcal capsular polysaccharide from transgenic mice with human immunoglobulin Loci.

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae, human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures, epitope specificities, and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7, 1A2, and 7C5) revealed that they use two different V(H)3 genes (A7 and 1A2 both use V3-15) and three different V(kappa) gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V(H)-D junction, whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10- and 1- micro g doses of A7 and 7C5 were protective, while only a 10- micro g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB(-/-)) or classical (C4(-/-)) complement pathway, but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure, specificity, and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy.

Polysaccharides, mimotopes and vaccines for fungal and encapsulated pathogens.

Vaccination is a rational alternative to treatment for Cryptococcus neoformans infections, as these infections are currently intractable in immunocompromised (including HIV-infected) individuals. Vaccines composed of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), the key C. neoformans virulence factor, elicit protective antibodies in mice, although deleterious antibodies can also be induced. By contrast, polysaccharides are poor immunogens in HIV-infected humans and others with B-cell defects. Peptide mimotopes of GXM can induce protective immunity to C. neoformans in mice, however, our knowledge of the mechanisms of mimotope-induced protection is incomplete and further work is needed if polysaccharide- or mimotope-based vaccines are to be used to manage C. neoformans infection.

Adjunctive immune therapy for fungal infections.

Fungal infections in immunocompromised patients can pose difficult problems in clinical management, because the available antifungal chemotherapy is often unable to eradicate the infection in these people. Hence, the use of immune modulating therapy to augment impaired host immune responses--and thus enhance the efficacy of antifungal drugs--is a reasonable approach to improve the prognosis of fungal infections. Advances in biotechnology have produced a variety of biological response modifiers with the potential to serve as adjunctive immune therapy for the treatment of fungal infections, including cytokines, monoclonal antibodies, and cell growth factors. In recent years, immune-modulating therapies have been studied in an effort to define their potential use for the treatment of fungal infections. Much of the available information on the use of this approach is encouraging and invites further investigation--with the caveats that the information is mostly anecdotal and that immune-modulating therapy occasionally has produced adverse effects.

Host-pathogen interactions: the attributes of virulence.

Virulence is one of a number of possible outcomes of host-microbe interaction. As such, microbial virulence is dependent on host factors, as exemplified by the pathogenicity of avirulent microbes in immunocompromised hosts and the lack of pathogenicity of virulent pathogens in immune hosts. Pathogen-centered views of virulence assert that pathogens are distinguished from nonpathogens by their expression of virulence factors. Although this concept appears to apply to certain microbes that cause disease in normal hosts, it does not apply to most microbes that cause disease primarily in immunocompromised hosts. The study of virulence is fraught with the paradox that virulence, despite being a microbial characteristic, can only be expressed in a susceptible host. Thus, the question "What is a pathogen?" begs the question, "What is the outcome of the host-microbe interaction?" We propose that host damage provides a common denominator that translates into the different outcomes of host-microbe interaction.

Vaccination against infectious disease following hematopoietic stem cell transplantation.

Patients undergoing hematopoietic stem cell transplantation (HSCT) experience a prolonged period of dysfunctional immunity associated with an increased risk of bacterial and viral infections. Effective approaches toward vaccinating patients against common pathogens are being explored but are limited by poor levels of responsiveness. Relevant studies examining the nature of reconstitution of cellular and humoral immunity and its impact on vaccination strategies against infectious pathogens are reviewed. Following transplantation, deficiencies in cellular immunity are characterized by the inversion of CD4/CD8 ratios, a decreased proliferative response to mitogens, and the development of anergy to recall antigens as measured by delayed-type hypersensitivity testing. The impact on humoral immunity consists of decreased levels of circulating immunoglobulin, impaired immunoglobulin class switching, and a loss of complexity in immunoglobulin gene rearrangement patterns. In this setting, a loss of protective immunity has been demonstrated against viral and bacterial pathogens previously targeted by childhood vaccination. Infections due to encapsulated bacterial organisms such as Streptococcus pneumoniae and Haemophilus influenzae type B remain prevalent even in the late posttransplantation period. The efficacy of vaccination following HSCT is influenced by the time elapsed since transplantation, the nature of the hematopoietic graft, the use of serial immunization, and the presence of graft-versus-host disease. Strategies to enhance vaccine efficacy include pretransplantation immunization of the stem cell donor and the use of cytokine adjuvants.

A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection.

Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.

A pneumococcal capsular polysaccharide vaccine induces a repertoire shift with increased VH3 expression in peripheral B cells from human immunodeficiency virus (HIV)-uninfected but not HIV-infected persons.

The molecular mechanism of pneumococcal vaccine failure in human immunodeficiency virus (HIV)-infected persons is not fully understood. A polymerase chain reaction ELISA was used to determine the proportion of peripheral IgG, IgA, and IgM CD19-positive B cells expressing 6 immunoglobulin heavy-chain variable region (VH) subgroups before and 7 days after pneumococcal vaccination of 12 HIV-infected and 12 HIV-uninfected subjects. Significant postvaccination increases in the expression of the VH3 subgroup by IgG and IgA and a greater serologic response to vaccination were observed in the HIV-uninfected group. In contrast, the HIV-infected group had reduced prevaccination IgG VH3 and a postvaccination increase in IgG VH5. These results demonstrate that pneumococcal vaccination changes the pattern of B cell VH gene expression and support the concept that aberrant VH3 expression may translate into a poor antipneumococcal response in the setting of HIV infection.

Production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci.

Infections with Streptococcus pneumoniae remain a significant cause of morbidity and mortality. To gain insight into structure-function relationships for human antibodies to pneumococcal capsular polysaccharide (PPS), we studied the response of transgenic mice reconstituted with human immunoglobulin loci, XenoMouse, to PPS antigens in a pneumococcal vaccine. Enzyme-linked immunosorbent assays of sera from mice vaccinated with a 23-valent pneumococcal vaccine revealed that they produced serotype-specific human antibodies, with the greatest response being to the PPS of serotype 3 (PPS 3). Molecular sequence analysis of three monoclonal antibodies (MAbs) to PPS 3 generated from lymphoid cells from mice vaccinated with a 23-valent pneumococcal vaccine or a PPS 3-bovine serum albumin conjugate revealed that they all used heavy-chain immunoglobulin genes from the V(H)3 family, two expressed light chain genes from the human Vkappa1 family, and one expressed a mouse lambda light chain. The protective efficacy of the two MAbs was examined in mice. A 10-microgram dose of both, and a 1-microgram dose of one, significantly prolonged survival from a lethal serotype 3 infection in CBA/N mice. Our data show that XenoMouse mice produced protective, serotype-specific human antibodies to PPS 3, and they lend support to the proposal that these animals represent a useful model to study the human antibody response to PPS antigens.

Quantitative and qualitative differences in the serum antibody profiles of human immunodeficiency virus-infected persons with and without Cryptococcus neoformans meningitis.

The importance of humoral immunity for resistance to Cryptococcus neoformans is uncertain. A case-controlled study of the human antibody response to C. neoformans comparing the serum antibody profiles of human immunodeficiency virus (HIV)-infected persons who did (HIV+/CM+) or did not (HIV-infected controls) develop cryptococcal meningitis (CM) and HIV-uninfected persons with samples obtained from the Multicenter AIDS Cohort Study was performed. Total immunoglobulin concentrations were determined, and the specificity, isotype, and idiotype expression of antibodies to C. neoformans capsular glucuronoxylomannan were analyzed by ELISA. Compared with the HIV+/CM+ group, the HIV-infected control group had significantly lower levels of total IgM, IgA, and antibodies expressing a certain VH3 determinant. The HIV-infected control group manifested an increase in immunoglobulin levels with a decrease in CD4 lymphocytes. The findings suggest a possible association between reduced expression of certain immunoglobulin subsets and HIV-associated CM.

Antibodies reactive with the cryptococcal capsular polysaccharide glucuronoxylomannan are present in sera from children with and without human immunodeficiency virus infection.

Antibodies reactive with the cryptococcal polysaccharide glucuronoxylomannan (GXM) are present in sera from both human immunodeficiency virus (HIV)-uninfected and -infected adults. However, the prevalence of these antibodies in US children is unknown. An antigen-capture ELISA was used to determine the presence, serotype specificity, isotype, and IgG subclass distribution of antibodies to GXM in sera from 27 HIV-uninfected and 34 HIV-infected children. The children were of similar age and socioeconomic background. IgG and IgM to GXM were present in sera from all children, although HIV-uninfected children had significantly higher titers. HIV-uninfected children had IgG1 and IgG2 to GXM and significantly greater IgG2 than in HIV-infected children. These findings of type-specific antibodies to GXM in early childhood suggest that exposure to or subclinical infection with Cryptococcus neoformans may be an early childhood event.

Molecular and functional characteristics of a protective human monoclonal antibody to serotype 8 Streptococcus pneumoniae capsular polysaccharide.

The structural characteristics and biological activity of human antibodies that are reactive with the capsular polysaccharides of most serotypes of Streptococcus pneumoniae, including serotype 8, are unknown. This paper describes the generation, molecular structure, and protective efficacy of a human monoclonal antibody (MAb) reactive with the capsular polysaccharide of serotype 8 Streptococcus pneumoniae. We generated the immunoglobulin M(kappa) [IgM(kappa)] MAb D11 by Epstein-Barr virus transformation of peripheral lymphocytes from a Pneumovax recipient. Nucleic acid sequence analysis revealed that MAb D11 uses V3-15/V(H)3 and A20/V(kappa) gene segments with evidence of somatic mutation. In vitro studies revealed MAb D11-dependent complement deposition on the capsule of serotype 8 organisms via either the classical or the alternative complement pathway. In vivo, MAb D11 prolonged the survival of both normal and C4-deficient mice with lethal serotype 8 S. pneumoniae infection. Our findings demonstrate that a serotype-specific human IgM with certain structural and functional characteristics was protective in mice lacking a functional classical complement pathway and show that alternative complement pathway activation is an important determinant of pneumococcal protection.

Antibody response to Cryptococcus neoformans proteins in rodents and humans.

The prevalence and specificity of serum antibodies to Cryptococcus neoformans proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to C. neoformans, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with C. neoformans proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with C. neoformans proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with C. neoformans proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with C. neoformans proteins. The results are consistent with the view that C. neoformans infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis.