towards a vaccine for cryptococcus neoformans: principles and caveats

M I N I R E V I E W

Towardsavaccine forCryptococcusneoformans:principlesand caveatsKausik Datta1 & Liise-anne Pirofski1,2

1Department of Microbiology and Immunology, and 2Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

Correspondence: Liise-anne Pirofski,

Division of Infectious Diseases, Albert Einstein

College of Medicine, 1300 Morris Park

Avenue, Room 709 Forchheimer Bldg, Bronx,

NY 10461, USA. Tel.: 11 718 430 2372;

fax 11 718 430 2291;

e-mail: [email protected]

Received 3 October 2005; revised 8 November

2005; accepted 10 November 2005.

First published online 3 April 2006.

doi:10.1111/j.1567-1364.2006.00073.x

Editor: Stuart Levitz

Keywords

Cryptococcus neoformans ; antibody-mediated

immunity; vaccine

Abstract

In the Damage-response framework of microbial pathogenesis, infectious diseases

are one outcome of a host-microorganism interaction in a susceptible host. In

cryptococcal disease, damage to the host is caused by Cryptococcus neoformans

virulence determinants, the nature of the host response, or both. Further, the

disease may be acute or reactivated from a latent state. Hence, a vaccine for C.

neoformans would need to prevent disease resulting from either acute or

reactivated infection. The evidence to support the development of a vaccine for

C. neoformans that induces antibody-mediated immunity is discussed herein.

Introduction

In around the last hundred years, our capability to diagnose,

treat and prevent various infectious diseases has been

markedly improved through advances in medical research

and therapy, including the introduction of antimicrobial

chemotherapeutic agents. Paradoxically, however, there has

been an unprecedented increase in the number of patients

that are vulnerable to a wide array of infectious diseases. The

responsibility for this rests, in part, on the introduction and

therapeutic usage of anti-neoplastic chemotherapy and

organ and bone-marrow/stem cell transplantation to treat

malignancies, which result in or require suppression of

normal immunity. In addition, the advent of intensive-care

units and widespread use of intravascular catheters and

broad spectrum antibacterials has led to the growth of a

population of patients with impaired barrier immunity.

These individuals are immunosuppressed by virtue of hav-

ing disrupted integument, mucosal surfaces and/or replace-

ment of commensal microbiota with hospital-acquired

microorganisms that are more resistant to antibiotics.

Further contributing to host vulnerability has been, since

the early 1980s, the HIV pandemic. HIV infection has

profoundly affected the global population regardless of age,

sex, ethnicity and geographical barriers, resulting in large

numbers of immunocompromised individuals.

The immunocompromised host is an individual with

impaired immunity, including acquired immunodeficiency.

Such individuals are at increased risk for many infectious

diseases, depending on the nature of their immune defects.

The relatively sudden and increasing occurrence of infectious

diseases in immunocompromised hosts caused by microor-

ganisms formerly considered to be non-pathogens and/or

those rarely seen in individuals with normal immunity

provides clear evidence that infectious diseases are but one

outcome of host–pathogen interaction, and that they can

only occur in a susceptible host (Casadevall & Pirofski, 1999).

This concept was put forth in the Damage-response frame-

work of microbial pathogenesis. The Damage-response fra-

mework regards host damage as the common denominator of

any host-microorganism interaction. However, damage that

occurs during host-microorganism interaction reflects a

complex interplay of microorganism-induced damage, host-

induced damage, or both (Casadevall & Pirofski, 1999). The

pathogen classification system proposed by the Damage-

response framework plots the degree of host damage as a

function of the host immune response, which makes it

possible to dispense with terms such as pathogen, non-

FEMS Yeast Res 6 (2006) 525–536 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

pathogen and opportunistic pathogen, and to characterize

any microorganism within a unified theory of microbial

pathogenesis. The use of this schema allows discrimination

of pathogens that cause damage to hosts with normal or weak

immune responses from those that cause damage to hosts

with weak, as well as strong (excessive) immune responses. By

focusing on host damage as an outcome of host–pathogen

interactions, it is possible to approach rational vaccine design

by identifying targets that can minimize or repair host

damage, while avoiding the production of inflammatory

mediators, which, despite reducing the microbial burden,

could have a detrimental effect.

This article will discuss the prospects for developing a

vaccine for Cryptococcus neoformans from the vantage point

of how such a vaccine might work by inducing antibody-

mediated immunity. Although this article will focus on

antibody-mediated immunity, we would like to remind the

reader that vaccines that could induce cell-mediated im-

munity are also under development, and that it is likely that

a successful vaccine could promote collaboration between

antibody- and cell-mediated immunity.

Challenges to designing a vaccine forcryptococcal disease

The pathogen classes, as defined by the Damage-response

framework, depict the relative ability of a pathogen to cause

damage based on the host immune response. Cryptococcus

neoformans, along with other microorganisms that possess a

polysaccharide capsule and induce similar types of clinical

syndromes (Streptococcus pneumoniae, Haemophilus influ-

enzae and Neisseria meningitidis), were originally put in

Class 2, because they were not thought to induce damage in

the setting of a strong immune response (Casadevall &

Pirofski, 1999, 2003b). However, recent studies have re-

vealed that C. neoformans can induce an exuberant inflam-

matory response that results in disease in HIV-infected

patients on highly active anti-retroviral therapy (HAART)

(Jenny-Avital & Abadi, 2002) and in solid organ transplant

recipients after immune reconstitution (Singh et al., 2005).

Hence, damage in the setting of C. neoformans infection is

associated with either a weak immune response, such as in

HIV-infected individuals with T-cell deficiency, or an exu-

berant inflammatory response, such as in HIV-infected

individuals or solid organ transplant recipients after recon-

stitution. The major mechanism of damage in the setting of

weak immune responses is often microorganism-mediated.

For C. neoformans, the microbial component is most likely

the capsular polysaccharide, of which the glucuronoxylo-

mannan (GXM) constituent has a wide array of deleterious

effects (Casadevall & Pirofski, 2005). In contrast, the major

mechanism of damage in the setting of a strong immune

response is likely to be host-mediated, such as by immune

complexes and/or Th1-type inflammatory responses. In

light of the discovery that cryptococcosis can occur after

immune-reconstitution, C. neoformans may be more appro-

priately classified as a Class 4 pathogen (Fig. 1).

Does a vaccine for C. neoformans make sense?

The interaction between the human host and C. neoformans

is likely to begin in childhood (Spitzer et al., 1993; Goldman

et al., 2001). Infection is believed to be asymptomatic and

the majority of cases of cryptococcal disease occur in adults

with underlying immune defects. Several lines of evidence

suggest that the outcome of C. neoformans infection is a

latent state, and that the occurrence of disease represents

reactivation (Spitzer et al., 1993; Garcia-Hermoso et al.,

1999). Hence, a vaccine for C. neoformans would have to

prevent reactivation and/or be effective in the setting of

established disease. Such a vaccine could be considered a

therapeutic vaccine. At present, only the rabies vaccine,

which is used after infection in conjunction with antibody,

is used as a therapeutic vaccine. However, the success of a

varicella-zoster vaccine in preventing herpes zoster in older

adults (Oxman et al., 2005) provides proof of the principle

that a vaccine can prevent an infectious disease caused by

Fig. 1. The basic Damage-response curve depicting Cryptococcus neo-

formans as a Class 4 pathogen. In the basic Damage-response curve

(broken line), host-pathogen interactions are depicted as the amount or

degree of host damage as a function of the nature of the host response

(Casadevall & Pirofski, 2003b, 2004). The U shape of the basic curve

demonstrates that host damage can occur in the setting of either weak

responses, often caused by microbial factors, or strong responses, often

caused by the inflammatory response. In depicting C. neoformans, the

components of the basic curve can be used to demonstrate that either

immunodeficiency, a weak response often associated with a high fungal

burden, or reconstitution, a strong response often associated with an

exuberant cell-mediated response and inflammation, can result in host

damage, whereas normal responses are generally not associated with

measurable host damage.

FEMS Yeast Res 6 (2006) 525–536c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

526 K. Datta & L. Pirofski

reactivation of latent microorganisms and raises confidence

that a similar vaccine for C. neoformans is feasible. Crypto-

coccal disease can also reflect the outcome of acute infection

(Nosanchuk et al., 2000). Therefore, the challenge of devel-

oping a vaccine for C. neoformans is that, ideally, it should

prevent reactivation as well as acute disease. As cryptococcal

disease can occur in the setting of both weak and strong

immune responses, a vaccine that enhances the immune

response could be deleterious by enhancing strong re-

sponses, but a vaccine that dampens strong responses could

be deleterious by being unable to stimulate weak responses.

As such, the present understanding of C. neoformans patho-

genesis suggests that different vaccine antigens could be

required depending on the immune status of the individual

to be vaccinated.

The human antibody response to naturalcryptococcal infection

Although a defined role for antibody-mediated immunity

(AMI) in resistance or susceptibility to human cryptococ-

cosis has not been identified, available data suggest that

quantitative and qualitative differences in the antibody

repertoires of individuals who are at high risk, compared to

those who are not, could translate into an additional risk

factor for disease. Available data suggest that antibody

deficiency and/or repertoire defects, along with impaired

cellular immune mechanisms, could lead to a higher fungal

burden and/or host damage from GXM and other crypto-

coccal virulence factors. The vast majority of patients who

develop cryptococcal disease have impaired immunity, most

notably HIV-associated T-cell immunodeficiency. The cen-

tral importance of intact CD41 T-cell mediated immunity

in resistance to cryptococcosis is incontrovertible. However,

susceptibility to HIV-associated cryptococcosis must de-

pend on additional factors, as the incidence of disease is

markedly less than that of HIV-induced CD41 T-cell

deficiency (Currie & Casadevall, 1994). Further, the ubiquity

of C. neoformans in the environment and strong evidence for

latency in humans (Spitzer et al., 1993; Garcia-Hermoso

et al., 1999) suggest that de novo infection alone does not

cause sufficient damage to result in disease. Most impor-

tantly, although C. neoformans can exploit host and/or

environmental factors to enhance virulence, this cannot

explain why only some HIV-infected individuals develop

disease, whereas others with similar immunological profiles

do not, or why certain healthy individuals develop disease

(Casadevall & Perfect, 1998; Banerjee et al., 2001).

The dogma in cryptococcal research has been that AMI is

not important for natural resistance to cryptococcosis,

particularly as GXM is a poor immunogen and is believed

to suppress the immune response (Casadevall et al., 2002;

Casadevall & Pirofski, 2005). However, several lines of

evidence suggest a more prominent role for AMI in host

defense against cryptococcosis. First, immunocompetent

individuals are highly resistant to cryptococcosis, and most,

if not all, have antibodies to GXM in their serum (discussed

in Casadevall & Pirofski, 2005). Patients with immunoglo-

bulin defects, such as hypogammaglobulinemia and X-

linked hyper-IgM syndromes, are at an increased risk for

cryptococcosis; although these syndromes are characterized

by defects in acquired, T-cell dependent antibody responses,

they also feature central defects in the B-cell repertoire

(discussed in Subramaniam et al., 2005). Similarly, in the

pre-AIDS era, patients with the highest risk for cryptococ-

cosis often had B-cell defects in addition to impaired cellular

immunity, such as those with HIV-infection, B-cell or

hematologic malignancies. Second, at least four indepen-

dent groups have shown that passive administration of

antibodies to GXM or active vaccination with cryptococcal

antigens or mimetics can protect mice from a lethal crypto-

coccal challenge (see Fleuridor et al., 2001; Casadevall et al.,

2002; Maitta et al., 2004c). Third, HIV-infected individuals,

who are at high risk for cryptococcal disease, have been

shown to have quantitative and qualitative differences in

their non-specific and GXM-reactive antibody repertoires

compared to HIV-uninfected individuals, who are at very

low risk for disease (Fleuridor et al., 1999; Subramaniam

et al., 2005).

Isotype expression of naturally occurring humanGXM-reactive antibodies

GXM- or capsular polysaccharide-reactive antibodies have

been identified in human sera by a number of different

investigators (Dromer et al., 1988a; Houpt et al., 1994;

DeShaw & Pirofski, 1995; Fleuridor et al., 1999; Goldman

et al., 2001; Subramaniam et al., 2005). Subramaniam et al.

found that HIV-infected African subjects had significantly

lower levels of GXM-reactive IgM, but higher levels of total

IgM than did HIV-uninfected subjects (Subramaniam et al.,

2005). As HIV-associated hypergammaglobulinemia is char-

acterized by an expanded population of naı̈ve and a reduced

population of memory B cells (discussed in Subramaniam

et al., 2005), this could reflect HIV-associated perturbations

in the memory B cell repertoire (De et al., 2001; Chong et al.,

2004). Reductions in the IgM memory compartment have

been implicated in susceptibility to S. pneumoniae (Kruetz-

mann et al., 2003). Naturally occurring IgM is required for

resistance to a number of pathogens (Brown et al., 2002;

Alugupalli et al., 2003; Diamond et al., 2003). Hence, certain

subsets of specific IgM could contribute to resistance to C.

neoformans. In support of this possibility, human GXM-

reactive IgM mediated protection against experimental

cryptococcosis (Fleuridor et al., 1998; Maitta et al., 2004a,

c). Clinical history influenced the level of GXM-reactive


527Towards a vaccine for Cryptococcus neoformans

antibody in HIV-infected Africans (Subramaniam et al.,

2005). HIV-infected Africans with a history of pneumonia

had higher, whereas those with a history of herpes zoster had

lower, levels of GXM-reactive antibodies. These findings

suggest that variability in clinical history could be one factor

contributing to the difficulty in identifying disease-specific

antibody profiles in patients with cryptococcal disease.

Fleuridor et al. (1999) showed that GXM-reactive IgG

levels increased among HIV-infected subjects as CD41 T-

cell counts declined. Together with data from other studies

showing higher levels of GXM- or C. neoformans protein-

reactive IgG among HIV-infected subjects (DeShaw &

Pirofski, 1995; Chen et al., 1999; Subramaniam et al.,

2005), these observations suggest such antibodies could

have been elicited by a new infection and/or the reactivation

of C. neoformans. Further, high levels of GXM-reactive IgG

could be disease-enhancing, perhaps as a clinical correlate of

the prozone-like phenomenon observed in mouse models,

whereby high amounts of antibody abrogate successful AMI

(Taborda & Casadevall, 2001; Taborda et al., 2003; Maitta

et al., 2004a). Along the same lines, the apparent inefficacy

of AMI in HIV-infected individuals resembles the finding

that GXM induces protective and non-protective antibodies

in mice, and that the efficacy of AMI can depend on intact

cellular immunity (Casadevall & Pirofski, 2005). Antifungals

and/or immune reconstitution may reduce the fungal bur-

den in patients with cryptococcosis who are on HAART or

recovering from organ transplantation. However, the pre-

sence of non-protective and/or high antibody levels could

render AMI ineffective and enhance detrimental Th1-type,

cell-mediated inflammation. Further work is required to

determine whether, and if so, which, human antibody types

depend on intact cellular immunity for their biological

activity, and whether such antibodies are effective or in-

effective in the setting of immunodeficiency and/or recon-

stitution-associated cryptococcosis.

Idiotype expression of naturally occurringhuman GXM-reactive antibodies

The predominant human immunoglobulin gene family used

in antibodies to GXM and other capsular polysaccharides is

VH3 (Pirofski, 2001), which comprises about half of the

circulating VH repertoire in adult peripheral B cells (Chang

et al., 2004). Fleuridor et al. found that HIV-infected

subjects who subsequently developed cryptococcosis had

lower levels of antibodies expressing VH3 than did those

who did not (Fleuridor et al., 1999). The finding of

decreased VH3 expression among HIV-infected subjects

(Fleuridor et al., 1999; Chang et al., 2004) was consistent

with the phenomenon of HIV-associated depletion of B cells

and antibodies expressing VH3, which is thought to be

mediated by binding of HIV gp120 to variable region

epitopes of VH3-positive gene products (Chang et al.,

2000). In contrast to studies by Fleuridor et al. (1999) and

Abadi et al. (1998), which examined cohorts in the United

States, Subramaniam et al. (2005) found that VH3 antibody

levels were elevated in HIV-infected Africans; however, the

level depended on clinical history. Among those who devel-

oped cryptococcal disease there was a trend towards higher

VH3 levels. In contrast, those who developed cryptococcal

disease, but did not have a history of pneumonia, had a

trend towards lower VH3 levels. This finding was similar to

the finding that HIV-infected children with a history of

invasive pneumococcal disease had increased B-cell IgG VH3

expression (Chang et al., 2004). GXM-reactive antibodies

from African subjects did not manifest substantial cross-

reactivity with pneumococcal capsular polysaccharides.

Hence, the Subramaniam study raised the intriguing possi-

bility that the African subjects could have had cryptococcal,

rather than what was believed to be pneumococcal, pneu-

monia. More information is required to answer this ques-

tion. Nonetheless, the fact that a human VH3-positive GXM-

reactive MAb that was protective against experimental

cryptococcosis was ineffective when too much or too little

MAb was administered (Taborda & Casadevall, 2001; Maitta

et al., 2004a) suggests that altered levels of VH3 antibodies

could influence susceptibility and resistance to HIV-asso-

ciated cryptococcal disease.

The rationale for a vaccine forC. neoformans

Of relevance to a vaccine for C. neoformans is that many

effective vaccines do not prevent infection. The central

mechanism by which vaccines mediate protection is by

preventing disease. While the precise mechanisms of vaccine

efficacy remain to be determined for most available vaccines,

their efficacy depends on inducing immune responses that

can reduce the host damage that results from the host-

microorganism interaction that occurs after an infection

below the threshold for disease. This shifts the basic Da-

mage-response curve (Fig. 1) to a point at which there is less

host damage. Currently licensed vaccines depend on AMI

to prevent disease. A role for AMI against C. neoformans

was initially difficult to establish because of limitations in

available antibody reagents and animal models; however,

the advent and development of MAbs to C. neoformans

antigens led to studies that established that AMI could be

effective against cryptococcal disease (Casadevall & Pirofski,

2005). It was rapidly recognized that mechanisms of anti-

body efficacy against C. neoformans included classical anti-

body functions as well as novel and unexpected functions

ranging from direct antimicrobial effects to counteracting

cryptococcal virulence factors to modulation of the host

inflammatory response (Casadevall & Pirofski, 2004, 2005).



Many lines of evidence support the feasibility of a vaccine

for C. neoformans that would work by inducing AMI.

Antibodies to GXM have a variety of functions and mechan-

isms of action, including that they promote phagocytosis,

alter the inflammatory response to C. neoformans to the

benefit of the host, prevent release of the capsular poly-

saccharide and reduce serum GXM levels. Antibodies to

cryptococcal melanin (Nosanchuk et al., 1998) and crypto-

coccal proteins (Rodrigues et al., 2000) inhibit fungal

growth, and antibodies to melanin have been shown to

prolong survival (Rosas et al., 2001). Hence, AMI to C.

neoformans is not limited to the classical antibody functions,

complement activation and opsonization (Casadevall &

Pirofski, 2003a, 2004). As such, AMI has great potential to

enhance host immunity to C. neoformans by combating

different pathways and mechanisms of cryptococcal viru-

lence. Further, the proven efficacy of AMI against experi-

mental cryptococcal infection (Casadevall & Pirofski, 2005)

and the use of a mouse MAb to GXM in a clinical trial in

patients with HIV-associated cryptococcosis (Larsen et al.,

2005) provide a clear rationale for the development of

vaccines that elicit antibodies to GXM.

Candidate antigens for a vaccine for C.neoformans

Capsular polysaccharide

Cryptococcus neoformans is unique among fungi in having a

polysaccharide capsule as its main virulence determinant.

GXM, which is the most well-studied capsular polysaccharide

determinant, is a logical vaccine antigen because it is a target

of the host response and there are highly successful pre-

cedents for polysaccharide-based vaccines for encapsulated

pathogens. Although purified polysaccharides can be poorly

immunogenic, conjugation to a protein carrier can markedly

increase their immunogenicity. The development of conju-

gate vaccines, which convert poorly immunogenic T-inde-

pendent antigens to T-dependent antigens (Pirofski, 2001),

was a landmark advance. Conjugate vaccines consisting of

the capsular polysaccharides of Str. pneumoniae or Haemo-

philus influenzae type b (Hib) markedly enhanced the im-

munogenicity of the relevant polysaccharide in infants and

young children. The introduction of the Hib vaccines in the

mid-1980s led to the rapid eradication of Hib meningitis

(Robbins et al., 1996), and use of the pneumococcal con-

jugate vaccine, introduced in 2000, led to a marked reduction

in the incidence of invasive pneumococcal disease in children

(Black et al., 2000). However, as the efficacy of pneumococcal

vaccines against pneumonia is less pronounced (see Burns

et al., 2005), the efficacy of polysaccharide vaccine-induced

antibodies against different outcomes of infection could

differ. For example, non-opsonic IgM protected against

pneumococcal pneumonia through immunomodulatory ef-

fects (Burns et al., 2005), and different types of carbohydrate

vaccine-induced antibodies mediated optimal protection

against systemic and mucosal candidiasis (Cassone et al.,

2005). Hence, a vaccine that prevents disease caused by acute

C. neoformans infection might not work against reactivation

disease, and vice versa. Of concern for a polysaccharide-based

vaccine for C. neoformans is that polysaccharide-based vac-

cines are less immunogenic in individuals with HIV infec-

tion, B-cell defects, and in the elderly (Pirofski, 2001). VH3

immunoglobulin repertoire defects in HIV-infected indivi-

duals could translate into an impaired ability to respond to a

GXM-based vaccine, as described for a pneumococcal vac-

cine (Abadi et al., 1998; Chang et al., 2000). However, the

observation that the VH3 response to a pneumococcal

vaccine was reconstituted in patients on HAART (Subrama-

niam et al., 2005) suggests such a vaccine could hold promise

in the setting of immune reconstitution.

Defined antibodies to GXM can protect against experi-

mental cryptococcosis (Casadevall & Pirofski, 2005). The

efficacy of these antibodies is governed by a complex group

of antibody characteristics, including isotype, idiotype and

specificity (reviewed in Casadevall & Pirofski, 2005), in

addition to host factors such as the genetic background

(Dromer et al., 1988b) and immune status (Yuan et al.,

1997; Beenhouwer et al., 2001). Since a mechanism by which

these antibodies are believed to mediate protection is by

enhancing the efficacy of host effector cells against C. neofor-

mans, the induction of such antibodies by a vaccine could, in

principle, augment host antifungal mechanisms through

immunomodulation. Therefore, a vaccine that induces anti-

bodies to GXM makes sense. However, GXM suffers from

important limitations as a vaccine antigen: it is a poor

immunogen and T-cell-independent type 2 antigen that does

not induce affinity maturation, class switching or memory T-

cells (Pirofski, 2001). Although this problem was ameliorated

by conjugation to a protein carrier, (Devi et al., 1991;

Casadevall et al., 1992, 2002; Devi, 1996), such a vaccine,

GXM-tetanus toxoid (GXM-TT), induced non-protective

and deleterious, in addition to protective, antibodies to

GXM in mice (Casadevall & Pirofski, 2005). Because the

nature of the GXM oligosaccharide epitopes that elicit

protective antibodies have not been fully identified, various

groups have focused on the identification of surrogate anti-

gens, which would be able to induce a response to GXM

epitopes that are recognized by protective antibodies.

Peptide mimotopes

The ability of small peptides to mimic the conformation of

carbohydrate antigens (Monzavi-Karabassi et al., 2002) has

led to efforts to identify peptides that mimic GXM epitopes

(see Pirofski, 2001). Peptide mimetics can be selected from



libraries consisting of phage-displayed random peptides by

panning the libraries with antibodies that bind to a native

antigen, such as GXM. Peptides that bind the selecting

antibody are referred to as mimetics; peptides that are able

to elicit antibodies that bind the native antigen when they

are used as immunogens are known as mimotopes. The

epitopes to which protective and non-protective antibodies

bind are often referred to as protective and non-protective

epitopes, respectively. The goal of mimotope-based immu-

nization is to elicit an antibody response to a protective

epitope on the native antigen. Hence, the efficacy of

mimotope-based vaccines depends upon the ability of

antibodies to and/or that cross-react with the native antigen

to prevent disease. The rationale for the development of a

mimotope-based vaccine for C. neoformans is that, in

principle, peptides that mimic defined GXM epitopes could

focus the GXM response on protective epitopes, rather than

on a mixture of protective and non-protective epitopes. In

addition, although the specificity of carbohydrate-reactive

and peptide mimotope-reactive antibodies can differ (Har-

ris et al., 1997), the use of a peptide, rather than poly-

saccharide, antigen might bypass the restriction inherent in

antibody responses to polysaccharides. Along these lines,

peptides are T-dependent antigens, which can be highly

immunogenic when conjugated to a protein carrier. Further,

peptides are biochemically defined molecules that can be

reliably produced in large amounts. The disadvantages of

peptide-based vaccines are that there is currently no such

vaccine in use in humans; focusing the response on a single

epitope could enhance the risk of escape variants; and the

induction of mimotope responses might be unpredictable if

the desired antibodies are conformation-dependent.

GXM mimotopes selected by mouse and human antibodies

to GXM have been reported. Valadon et al. (1998) selected a

panel of mimetics with a high-affinity mouse IgG1 MAb that

revealed unexpected complexity among GXM-induced anti-

bodies (Nakouzi et al., 2001), but elicited a low mimotope

(GXM) antibody response in mice, despite a high level of

peptide-reactive antibodies. Higher-affinity peptides reactive

with the same selecting MAb produced in mice a robust

mimotope response focused on antibodies reactive with pro-

tective epitopes, but only after first priming with GXM-TT

(Beenhouwer et al., 2002). Zhang et al. identified a decapeptide

mimetic (P13) of GXM, from the same phage display library

used with the mouse MAb, with a protective human IgM

GXM-reactive MAb (Zhang et al., 1997). Interestingly, P13

appeared to mimic a biologically relevant epitope, because it

inhibited the GXM-binding of serum antibodies from HIV-

uninfected, but not HIV-infected, individuals (Zhang et al.,

1997; Fleuridor et al., 1999). This finding suggested that the

specificity of GXM-reactive antibodies from individuals who

were at risk from cryptococcal disease was different from those

who were not, and was consistent with the concept that HIV-

infected individuals may have a hole in their GXM-reactive

antibody repertoire (Pirofski, 2001). Immunization of mice

with P13 conjugated to TT or BSA induced a mimotope

response, which prolonged survival after a lethal cryptococcal

challenge, as did passive administration of immune serum

from these P13-conjugate-vaccinated mice to naı̈ve mice

(Fleuridor et al., 2001). Given that GXM-TT induced deleter-

ious and non-protective antibodies (Casadevall & Pirofski,

2005), a GXM mimotope-based vaccine may hold promise for

focusing the response on protective epitopes.

Other C. neoformans antigens

Proteins or peptides hold promise as vaccine antigens,

because they are biochemically defined and able to induce

T-dependent responses. The mannoprotein component of

C. neoformans promotes strong Th1-type immunity (Vec-

chiarelli, 2000). Mannoprotein immunization of mice re-

sulted in an enhanced Th1-type cytokine response in the

brain (Mansour et al., 2004). The ability of mannoprotein to

enhance the host antifungal inflammatory response depends

on IL-12 (Pietrella et al., 2004) and T cells (Mansour et al.,

2004). A mannoprotein-based vaccine could be used to

augment Th1-type immunity in individuals with weak im-

mune responses to C. neoformans, with the caveat that such

individuals may not be able to respond to an immunogen

that requires intact T-cell immunity. The use of a manno-

protein-based vaccine in individuals with a strong immune

response could run the risk of inducing a deleterious

inflammatory response. Nonetheless, mannoproteins could

potentially be used as adjuvants with other vaccine antigens

to enhance cellular immunity to C. neoformans.

Other antigens that induce protective responses to C.

neoformans include the C. neoformans culture filtrate anti-

gen (CneF), a heterogeneous mixture of relatively high

molecular weight polysaccharides and proteoglycans. CneF

elicited delayed-type hypersensitivity and CD41T cell acti-

vation in mice (Murphy, 1993), and CneF-immunized mice

manifested prolonged survival and a lower fungal burden

than controls (Murphy et al., 1998). However, CneF is a

heterogeneous culture filtrate, which contains GXM and has

mannoprotein as a major constituent. As such, it is not a

suitable vaccine antigen. Other cryptococcal proteins that

could hold promise as vaccine antigens include other

mannoprotein chitin deacetylases (Biondo et al., 2002),

although further preclinical development is required to

assess their feasibility as antigens for humans.

The human antibody response toGXM-based vaccines

The mouse response to GXM-based vaccines has been

reviewed extensively (Casadevall et al., 2002, 2005), and the

reader is referred to the foregoing articles on the topic.



Information on the human response to Cryptococcus neofor-

mans vaccines is largely limited to studies that were

performed in human immunoglobulin transgenic mice.

However, the serum response to the investigational vaccine,

GXM-TT, was examined in human volunteers. The GXM

moiety in this vaccine was from a serotype-A C. neoformans

strain (NIH 371). This study revealed that, like the response

to other capsular polysaccharide vaccines, the antibody

response was predominantly IgG2 (Zhong & Pirofski,

1996). Immune sera from the volunteers contained anti-

bodies that expressed VH3 determinants (Fleuridor et al.,

1998) and promoted complement-independent phagocyto-

sis by human polymorphonuclear neutrophils (PMNs)

(Zhong & Pirofski, 1996). Two GXM-reactive IgM MAbs

were produced from circulating B cells of one vaccinated

volunteer. These MAbs used VH3 genes (Pirofski et al.,

1995), and one, 2E9, promoted phagocytosis and the killing

of C. neoformans by human PMNs (Zhong & Pirofski, 1998)

and prolonged the survival of mice with experimental

cryptococcosis (Fleuridor et al., 1998).

Human GXM-reactive MAbs were produced from human

immunoglobulin transgenic mice that were vaccinated with

a GXM-diphtheria toxoid (GXM-DT) conjugate (Maitta

et al., 2004a). These transgenic mice express human IgM,

IgG2 and kappa immunoglobulin genes (Mendez et al.,

1997). The GXM-DT conjugate used the GXM from a

serotype-D C. neoformans strain (ATCC24067). The MAbs

produced from these mice were fully human IgMs that used

the same kappa light chain. Two of the MAbs, G14 and G19,

used the VH6 gene element and one, G15, used a VH3 gene

element. Epitope specificity of G15 differed from that of the

other MAbs and was the only MAb that protected against

the C. neoformans challenge, although it manifested a

prozone-like effect when given in high amounts. VH6-

derived MAbs were not protective. Although they have

different specificity, human VH3-derived and mouse GXM-

reactive MAbs share a common CDR2 motif: -I---G----

YY--SV-G (Maitta et al., 2004a). This motif is found in six

germline VH3 genes; V3-07, V3-11, V3-30.3, V3-30.5 and

V3-33. The human GXM-reactive MAbs were germline,

whereas the mouse MAbs were somatically mutated. This

suggests that combinatorial diversity may be sufficient to

produce antibodies to GXM in humans, which could con-

tribute to their high level of natural resistance to cryptococ-

cal disease. Since the GXM-reactive human MAbs used the

same light-chain gene, their unique specificity could be a

function of VH gene use and/or CDR3 structure. The

influence of VH gene use on antibody specificity requires

the study of more antibodies. Nonetheless, the difference in

the GXM specificity of antibodies from HIV-infected and

HIV-uninfected individuals (Zhang et al., 1997; Fleuridor

et al., 1999) suggests that VH3 repertoire defects could

contribute to the apparent lack of efficacy of AMI in HIV-

infected individuals, despite their having high antibody

levels. As immunocompetent individuals are largely resis-

tant to cryptococcal disease while maintaining C. neofor-

mans in a latent state, it is reasonable to hypothesize that

naturally occurring antibodies with the characteristics of

protective MAbs could contribute to resistance. As such,

aberrant VH gene use, such as that observed in response to a

pneumococcal vaccine (Chang et al., 2000), by antibodies

with different specificity could contribute to disease suscept-

ibility in HIV-infected and other individuals with antibody

repertoire defects.

The human antibody response to peptidemimotope-based vaccines

The human antibody response to conjugates consisting of

the GXM mimotope P13 and TT or DT was examined in

human immunoglobulin transgenic mice expressing human

IgG2 and IgG4 (Maitta et al., 2004b). Vaccination with both

conjugates elicited antibodies that bound to the GXMs of C.

neoformans serotype A (H99 and SB4) and D (ATCC 24067)

strains (Maitta et al., 2004b). This pattern of reactivity,

which is similar to that described for mouse and human

MAbs produced from GXM-protein conjugate-vaccinated

mice, demonstrated that peptide mimotopes of GXM can

induce antibodies that react with multiple GXM epitopes.

However, GXM-reactive serum antibodies from P13 con-

jugate-immunized mice did not manifest cross-reactivity

with P13, and vice versa (Maitta et al., 2004b). In view of the

fact that a decamer peptide can assume multiple conforma-

tions, the lack of mimetic-mimotope cross-reactivity sug-

gests that GXM- and P13-reactive antibodies are likely to be

induced by different conformations of the peptide. The

availability of GXM oligosaccharides and new approaches

to epitope mapping will help unravel this and other ques-

tions on the role of cross-reactivity in mimotope-induced

responses to capsular polysaccharides.

P13-TT and P13-DT both induced human IgM and IgG

to P13 and GXM in human immunoglobulin transgenic

mice that expressed human IgG2 (Maitta et al., 2004b).

Conjugate-elicited antibodies expressed VH3 determinants

that were also expressed by GXM-TT-elicited antibodies

(Fleuridor et al., 1998) and that were found among naturally

occurring antibodies to GXM (Fleuridor et al., 1999). VH3

gene use by GXM-reactive antibodies in the natural reper-

toire, as well as those induced by GXM- and P13-protein

conjugates, suggests that conformational constraints, rather

than the molecular form of the antigen, govern binding to

certain GXM determinants. Hence, peptide surrogates

might not overcome the poor response to capsular poly-

saccharides observed in HIV-infected and other individuals,

if the response depends on components of the antibody

repertoire that are depleted or disregulated. This concept



requires further investigation, but if it is validated, it would

provide a compelling reason to consider passive antibody

therapy and/or approaches to antibody repertoire reconsti-

tution or replacement in certain immunocompromised

patients. One caveat regarding the immunogenicity of P13

conjugates in human immunoglobulin transgenic mice is

that they were administered with the oligodeoxynucleotide

CpGs, which was used as an adjuvant (Maitta et al., 2004b).

CpG-treated mice manifested a GXM-reactive antibody

response, including an anamnestic IgM response following

a C. neoformans challenge. As CpGs enhance innate and

Th1-type immunity (Ashkar & Rosenthal, 2002) and can

have direct antifungal effects (Miyagi et al., 2005), their

ability to enhance immunity to C. neoformans, perhaps by

enhancing AMI, deserves further consideration.

The survival of P13-DT-vaccinated human immunoglo-

bulin transgenic mice that were challenged and rechallenged

with C. neoformans was prolonged compared to control

mice, whereas that of P13-TT-vaccinated mice was not

(Maitta et al., 2004b). Analysis of the serum antibody

response revealed that P13-DT primed for a C. neoformans-

induced IgG anamnestic response to GXM, whereas P13-TT

primed for an IgM response. Hence, in the model under

investigation, the nature of the antibody response and

whether or not it was protective was a function of the

protein carrier. Carrier-specific differences have been shown

to influence the response to other conjugate vaccines. May

et al. found that BALB/c mice immunized with a GXM

peptide mimotope conjugated to glutaraldehyde-treated

keyhole limpet hemocyanin (KLH) developed high levels

of antibodies to GXM (May et al., 2003). However, control

peptides similarly conjugated to KLH also elicited GXM-

reactive antibodies that bound to a non-protective epitope,

as did glutaraldehyde-treated KLH and unmodified KLH.

On the other hand, the response to an experimental

conjugate for another microbe was enhanced by the ability

of the protein carrier to induce a microbe-specific response

(Batzloff et al., 2003). These observations illustrate that

current knowledge is insufficient to predict the correct

conjugation partner for polysaccharide- and/or polysacchar-

ide mimotope-based vaccines. Although the mechanisms of

mimotope efficacy remain uncertain, available data show

that it is not a straightforward function of the induced

mimotope titer at the time of challenge. Further studies are

needed to identify useful surrogates for mimotope efficacy.

Nonetheless, the mimotope approach is a rationale-based

approach to reverse antigen discovery, which can reveal

unique protective epitopes through the use of defined anti-

body reagents (Pirofski, 2001).

Efforts to identify surrogates for vaccine and antibody

efficacy against C. neoformans have been very difficult.

Despite the knowledge gained from studies with immune

sera, the functional mediator(s) of protection in such

polyclonal reagents can be difficult to discern (Casadevall &

Pirofski, 2004, 2005). In addition, assessments of the degree

of protection afforded by polyclonal sera are hampered

by its complexity and the possibility that the activity of

protective antibodies, which may be present in limiting

amount, can be obscured or diminished by non-protective

antibodies (Casadevall & Pirofski, 2004, 2005). An advan-

tage of using sera from human immunoglobulin transgenic

mice is that, as it only contains IgM and one IgG subclass, its

complexity is reduced. Maitta et al. (2004c) administered

heat-treated immune sera from P13-conjugate and adjuvant

(CpG)-immunized human immunoglobulin transgenic

mice to BALB/c mice prior to systemic C. neoformans

challenge in order to examine the efficacy of mimotope-

induced human antibodies. Early sera taken from P13-DT-

vaccinated mice 7 days after primary vaccination prolonged

the survival of naı̈ve BALB/c mice, whereas sera from P13-

TT- and CpG-vaccinated mice did not. The functional

mediator of protection in these sera was almost certainly

GXM-reactive IgM, since the sera contained no specific IgG

and the absorption of the sera abrogated its efficacy. In

contrast, each of the late sera taken 30 days after vaccination

from P13-DT-, P13-TT- and CpG-vaccinated mice pro-

longed the survival of naı̈ve BALB/c mice. The functional

mediator of protection in these sera was uncertain, because

they each contained specific IgG and IgM. A notable aspect

of these studies was that late sera from P13-TT-vaccinated

mice were protective, despite the fact that P13-TT vaccina-

tion did not protect immune mice against a C. neoformans

challenge (Maitta et al., 2004c). This underscored findings

from studies with mouse MAbs (Casadevall & Pirofski,

2005) that, rather than being a fixed characteristic, the

efficacy of AMI against C. neoformans differed as a function

of the immune status of the host. Naı̈ve hosts, albeit of a

different mouse strain, were protected, but immunized hosts

were not. Another interesting finding was that sera from

CpG-vaccinated mice contained a specific antibody that was

protective. The ability of CpGs to enhance Th1-type im-

munity is well documented; however, the passive transfer

studies suggested they may also stimulate production of

specific antibody. Although this could occur through Toll-

like Receptor-9 signaling, more studies are needed to

determine the mechanism by which this occurs.

Outlook for the development of a vaccinefor C. neoformans

Available information from studies of cryptococcal patho-

genesis in small animal models provides clear evidence in

support of the feasibility of a vaccine for C. neoformans. The

ability of defined antibodies to protect against experimental

cryptococcal disease, the existence of protective antigens and

the variety of mechanisms by which antibody can mediate



protection provide a strong rationale for the development of

a vaccine that induces AMI. Based on our current under-

standing of the virulence mechanisms of C. neoformans,

including that cryptococcal disease can occur in the setting

of either T-cell immunodeficiency or a reconstituted T-cell

milieu, the challenge of developing a vaccine for C. neofor-

mans will be to identify vaccine antigens that augment the

host response in the setting of immunodeficiency, while

dampening the response in the setting of reconstitution

(and/or other states of enhanced cell-mediated immunity).

Notably, the GXM and mannoproteins dampen and stimu-

late cell-mediated immune responses, respectively. The anti-

body responses to GXM among susceptible and resistant

individuals are different, whereby susceptible individuals

often have less GXM-reactive IgM, but more GXM-reactive

IgG. The contribution of this type of antibody profile to

cryptococcal pathogenesis has not been established. How-

ever, high antibody amounts can enhance experimental

cryptococcosis, certain IgMs are effective against experi-

mental cryptococcosis and innate immunity to encapsulated

pathogens can depend on IgM (Brown et al., 2002). Hence,

it is logical to hypothesize that a vaccine for C. neoformans

should induce a balanced immune response and promote

cooperative interactions between innate and cell-mediated

immunity. The variety of mechanisms of antibody action

against C. neoformans (Casadevall & Pirofski, 2003a, 2004,

2005) suggests it should be possible to induce immunosti-

mulatory and immunoregulatory responses (Fig. 2). As

such, the challenges that lie ahead are to identify targets that

induce such antibodies, and to determine whether or not

one or more than one vaccine will be needed to protect

against acute and reactivation disease, and protect indivi-

duals with different immune status.

Acknowledgements

This work was supported by National Institutes of Health

grants R01AI035370, R01AI045459, and R01AI044374 (LP).

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