Passive administration of mAbs elicited by M2-HSA was not protective and there was no difference in the survival of mice immunized with M2-HSA and HSA. for the identification of GXM PT2977 epitopes. Keywords:Antibody, Cryptococcus neoformans, Rabbit Polyclonal to SEPT7 Immune response, Vaccine == Introduction == Cryptococcus neoformansis a yeast-like fungus that is a major cause of meningoencephalitis individuals with impaired immunity, such as patients with AIDS [1]. Cryptococcosis is almost always fatal without therapy, yet the available therapies remain unsatisfactory, since current treatment requires prolonged administration of antifungal drugs and even with treatment there is high mortality and morbidity PT2977 [1]. Other populations at risk include organ transplant recipients, individuals receiving immunosuppressive therapy, and occasional children with hyper IgM syndrome. Studies in rats have raised the possibility that the spectrum ofC. neoformans-related disease may extent to the immunologically normal population where primary infection is often asymptomatic and lifelong [2]. Rats infected withC. neoformansare asymptomatic but manifest immune dysregulation in the lungs that translates into hyper-reactive airways, leading to the suggestion that cryptococcal infection may be a contributing factor to the pathogenesis of asthma [2]. Given the problem of cryptococcosis in immunosuppressed populations and PT2977 the possibility that this infection is a co-factor in pulmonary disease of normal individuals there is considerable interest in development of an effective vaccine [3]. Studies in laboratory animals show that it is possible to protect against experimentalC. neoformansinfection with vaccines that elicit antibody or cell-mediated immunity [38]. Given that this fungus is encased in a polysaccharide capsule that is a major virulence determinant, that polysaccharide epitopes can be found in fungal spores [9], that the capsular polysaccharide can elicit protective antibodies [10], there is considerable effort to generate a suitable vaccine that targets the capsule. TheC. neoformanscapsular polysaccharide is composed of two major components, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). GXM is a large molecular weight polymer that comprises most of the mass of the capsule [11]. All polysaccharide vaccine-related work forC. neoformansto date has focused on GXM and a conjugate vaccine composed of GXM linked to tetanus toxoid was shown to elicit a protective antibody response in mice and humans [12;13]. However, a prior vaccine composed of total polysaccharide conjugated to protein was immunogenic but not protective [14;15]. GXM is structurally complex [16], and studies using monoclonal antibodies (mAbs) generated from mice vaccinated with GXM-containing vaccines have shown that this molecule can elicit protective, non-protective and even disease-enhancing antibodies [17;18]. Furthermore, the capsular polysaccharide has potent immunosuppressive properties raising concern as to its suitability as a vaccine component [1921]. Consequently, interest in vaccines that elicit antibodies to GXM has evolved to focus on the suitability of peptide mimotopes of GXM and oligosaccharide representing discrete domains of polysaccharide structure [2225]. An alternative approach is to synthesize oligosaccharides representing the major structural motifs of GXM to focus the response on a few epitopes. The recent report that oligosaccharide-based vaccines can protect againstC. albicansinfection provide encouragement for the development of this strategy against cryptococcosis [26]. The basic structural unit of GXM is a tri-mannose repeat with a glucuronic acid residue in the first mannose [11]. This structure is further modified in individual strains by the addition of xylose substitutions on the mannan backbone. Cherniak and collaborators defined six triads known as M1-M2 that are found in various proportions in GXM from the various serotypes [27]. M2 is the most common triad in serotype A GXM PT2977 [27]. Given that serotype A strains are the most common clinical isolates and that serotype A GXM can elicit protective antibodies, a heptasaccharide oligosaccharide representing the M2 structural motif was chemically synthesized, conjugated to human serum albumin (HSA) and used to immunize mice. A preliminary study revealed that the M2-HSA motif was immunogenic but, surprisingly, elicited antibodies that produced a punctuate immunofluorescence pattern on theC. neoformanscapsule reminiscent of that associated with non-protective mAbs [28]. To investigate the functional efficacy of antibodies elicited by M2-HSA we generated mAb and challenged immunized mice withC. neoformansinfection. The results indicate that M2 elicits a non-protective antibody response. == Materials and methods == == C. neoformans and Glucuronoxylomannan == Strain 24067 (Serotype D) was obtained from the American Type Tissue Collection (Rockville, MD). GXM was produced from culture supernatants with minor modifications of standard protocols. Strain 24067 was used in all immunofluorescence, phagocytosis and challenge experiments. Strain H99 (Serotype A) was obtained from Dr. John Perfect (Duke University, NC), Strain NIH 3939 (Serotype B) was obtained from Dr. Kwong.
Passive administration of mAbs elicited by M2-HSA was not protective and there was no difference in the survival of mice immunized with M2-HSA and HSA
