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Infection and Immunity, April 2006, p. 2446-2448, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2446-2448.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Active Immunization against Pneumocystis carinii with a Recombinant P. carinii Antigen

Jesse Wells,1 Constantine G. Haidaris,1 Terry W. Wright,1,2 and Francis Gigliotti1,2*

Departments of Microbiology and Immunology,1 Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York 146422

Received 29 December 2005/ Returned for modification 31 December 2005/ Accepted 11 January 2006


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ABSTRACT
 
Mice immunized with recombinant mouse Pneumocystis carinii antigen A12-thiredoxin fusion protein developed an antibody response that recognized P. carinii antigens, as determined by Western blotting and immunofluorescence analysis. Compared to mice immunized with thioredoxin alone, mice immunized with A12-thioredoxin had significantly reduced lung P. carinii burdens after CD4+ T-cell depletion and challenge with P. carinii.


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TEXT
 
An infection caused by Pneumocystis carinii, P. carinii pneumonia (PCP), remains an important cause of morbidity and mortality in immunocompromised hosts. Although there are drug treatments for P. carinii pneumonia, poor compliance, adverse side effects, and recurrent infections remain a problem (12). Therefore, other treatments and strategies to prevent PCP merit further investigation.

Immunization of BALB/c mice with intact P. carinii cells provides protection against PCP (5, 9, 14). Since P. carinii cannot be satisfactorily cultivated, development of a subunit vaccine is essential. However, there are no convincing data showing that a protective immune response has been elicited after active immunization with an isolated antigen of P. carinii.

Here we describe active immunization of mice against P. carinii with a recombinant P. carinii antigen that we designated A12 (13). A12, which exhibits homology to P. carinii Kex1 (7, 13), was selected because it is recognized by a monoclonal antibody that has been shown to provide passive prophylaxis against development of PCP (3).

Immunization with recombinant A12 produces an antibody response to native mouse P. carinii. To investigate the immunogenicity of a recombinant fusion protein encoded by a portion of P. carinii cDNA clone A12, a PCR amplicon encoding the first 142 amino acids of the A12 polypeptide (GenBank accession no. AY371664) was cloned into pBAD:THIO, a thioredoxin six-His fusion vector (Invitrogen, Carlsbad, CA). A121-142:THIO and a thioredoxin control were expressed in Escherichia coli and purified under denaturing conditions by metal ion affinity chromatography. BALB/c mice were immunized subcutaneously three times at 4-week intervals with 25 µg of either A121-142:THIO or thioredoxin in TiterMax gold adjuvant (Sigma, St. Louis, MO). Two experiments were performed using the same lots of recombinant A12, control antigen, and adjuvant. P. carinii-specific serum antibody responses were determined by immunofluorescence analysis and Western blotting of P. carinii-infected and normal mouse lung homogenates (2, 3, 5). Serum antibodies obtained after the third immunization (and prior to infection) from A121-142:THIO-immunized mice bound to the surface of P. carinii cysts in an immunofluorescence analysis up to a 1:400 dilution (Fig. 1A), while sera from control mice did not recognize P. carinii cysts at dilutions as low as 1:25 (data not shown). Therefore, the antibody to recombinant A12 was capable of recognizing native P. carinii antigens.


Figure 1
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  		FIG. 1. Immunization with recombinant A12 fusion protein produces a specific antibody response to native P. carinii antigens. (A) Immunofluorescence assay showing P. carinii cysts in P. carinii-infected mouse lung homogenates using different dilutions of pooled anti-P. carinii A121-142:THIO antisera and goat anti-mouse immunoglobulin G:BODIPY-Fl secondary antibody. (B to E) Western blots for P. carinii isolated from infected mouse lungs and normal lung homogenates probed with A121-142:THIO preimmune sera (B), A121-142:THIO postimmune sera (C), thioredoxin preimmune sera (D), and thioredoxin postimmune sera (E). All sera were diluted 1:250. Lane 1, P. carinii-infected mouse lung homogenate; lane 2, normal lung homogenate; lane 3, molecular mass markers.

In Western blots with P. carinii-infected mouse lung homogenates and noninfected mouse lungs, anti-A121-142:THIO immune serum antibodies detected several bands in the range from 50 to 120 kDa in P. carinii-infected lung homogenates (Fig. 1C, lane 1). The major bands detected were at Mrs of 65,000 and 120,000. Anti-A12 sera from a pilot study with six mice and from both of the immunized cohorts described in this report gave similar Western blot banding profiles. The A121-142:THIO postimmune sera did not show enhanced binding to any antigens in lanes containing an equal amount of uninfected mouse lung homogenate (Fig. 1C, lane 2). Preimmune sera from the same mice did not recognize P. carinii antigens (Fig. 1B, lane 1). The thioredoxin fusion partner did not induce antibody reactivity to P. carinii (Fig. 1D and E, lane 1).

The recognition of multiple bands by the A121-142:THIO immune sera may have been due to epitopes shared by A12 and other P. carinii antigens, proteolytic processing, or degradation. Cross-reactivity to a number of shared epitopes in different P. carinii antigens has been demonstrated with anti-mouse P. carinii monoclonal antibody 4F11, which is capable of recognizing at least two P. carinii antigens, A12 and Kex1 (13). It may be beneficial that immunization with A12 produces a response to a number of P. carinii antigens, as this may enhance the repertoire of T-cell responses and/or the opsonic capacity of the immune sera.

Immunization with recombinant P. carinii A12 significantly reduces the organism burden in CD4+ T-cell-depleted mice. To determine whether the immune response to recombinant A121-142:THIO reduced the organism burden, groups of A121-142:THIO- and thioredoxin-immunized mice were depleted of CD4+ T cells and challenged by cohousing them with P. carinii-infected source mice. Six weeks following the third immunization, mice were depleted of CD4+ T cells by twice-weekly administration of 250 µg anti-CD4 monoclonal antibody (clone GK 1.5; ATCC, Manassas, VA). To simulate natural exposure to P. carinii, the immunized mice were exposed to P. carinii-infected SCID mice 1 day following the second administration of monoclonal antibody GK1.5 for 5 days. Two cohorts of seven recombinant P. carinii antigen-immunized mice and six control-immunized mice were studied. The mice were sacrificed 6 weeks after termination of cohousing, and the P. carinii burden was determined by real-time PCR quantification of the single-copy kex1 gene as described previously (4). Because the outcomes of the two experiments were identical, the results were combined for statistical analysis. As shown in Table 1, only 4 of the 14 (29%) A121-142:THIO-immunized mice contained detectable P. carinii, whereas 8 of the 10 (80%) thioredoxin-immunized mice contained detectable P. carinii ({chi}2 = 6.17; P ≤ 0.025). (Two control mice died during the retroorbital bleeding procedure before they were exposed to P. carinii.) The organism burden was also analyzed. Using a conservative assumption (9.99 x 103 P. carinii copies in the lungs of mice without PCR-detectable P. carinii [the limit of detection for this assay was 104 kex1 copies per mouse lung]), there was an approximately 1-log reduction (P ≤ 0.005) in the average P. carinii burden in mice immunized with A121-142:THIO compared to mice immunized with thioredoxin. Since organisms were not detected by PCR in a majority of the A121-142:THIO-immunized mice, we also used a censored regression method of statistical analysis (SAS PROC LIFETEST) with the assumption that the distribution of the undetectable values was log normal between 102 and 104. This increased the P value to 0.0005. A more sensitive qualitative PCR assay targeting the multicopy P. carinii gene, gpA, was positive for the mice with infections that were not detectable by the quantitative real-time assay. Thus, we did not achieve sterilizing immunity.


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  		TABLE 1. Effect of immunization on P. carinii burden

In the second experiment, mice were tagged and sacrificed so we could compare the serum antibody response after the final boost, which was determined by an enzyme-linked immunosorbent assay (ELISA) as previously described (5), and the P. carinii count for each mouse. Sera were diluted 1:50. The background optical density in the ELISA in which uninfected mouse lungs were used was subtracted from the optical density obtained using P. carinii-infected lungs. In the A121-142:THIO-immunized group, the resulting optical density values were 0.02 and 0.03 for the two mice with detectable P. carinii and 0.07, 0.14, 0.24, 0.59, and 0.62 for the five mice with undetectable P. carinii. Thus, higher ELISA values appeared to be associated with lower organism burdens. There was no detectable optical density above the background level for any of the four control thioredoxin-immunized mice. A limitation of this ELISA format for the purposes of this experiment was that we were unable to determine the amount of "A12" that was bound to the plate.

The degree of protection observed in our study was not as pronounced as the degree of protection achieved by immunization with P. carinii cells (5). It may be possible to achieve greater protection by varying the dose and adjuvant, by immunizing with a larger portion of the A12 molecule, or by using a combination of A12 and additional P. carinii antigens. Previous attempts to elicit protective immunity against PCP with recombinant or purified native P. carinii antigens were unsuccessful unless the immunized mice were also cohoused with P. carinii-infected mice, which confounds the analysis of the vaccine effect (2, 6, 10, 11).

Zheng et al. have recently published results similar to those described here, which were obtained by using DNA immunization with the gene for P. carinii Kex1 (15). Because kexins are generally intracellular molecules (1), the success of vaccination with Kex1 is somewhat surprising and interesting. Alignment of the primary amino acid sequence of the immunogen A121-142 with the primary amino acid sequence of mouse P. carinii kexin revealed 54% identity and 67% similarity in a region where 137 amino acids overlap. A comparison of the cross-reactive antibody responses to the two antigens using either the A12 polypeptide or kexin protein as the immunogen may help to more precisely define critical protective epitopes that can be exploited for vaccine development. Given our results, further defining the antigenic relationship between A12, Kex1, and possibly P. carinii protease 1 (7, 8) discussed above is necessary.


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ACKNOWLEDGMENTS
 
We thank Margaret Chovaniec, Stephanie Campbell, and Victoria Houseknecht for technical assistance.

This work was supported by NIH grants RO1 AI23302 and NIAID 5T32AI07362.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Box 690, 601 Elmwood Ave., Rochester, NY 14642. Phone: (585) 275-5944. Fax: (585) 273-1104. E-mail: Francis_Gigliotti{at}urmc.rochester.edu. Back

Editor: A. Casadevall


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Infection and Immunity, April 2006, p. 2446-2448, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2446-2448.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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  • Casadevall, A., Pirofski, L.-a. (2007). Antibody-Mediated Protection through Cross-Reactivity Introduces a Fungal Heresy into Immunological Dogma. Infect. Immun. 75: 5074-5078 [Full Text]  

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