CD4⁺ T cell–independent DNA vaccination against opportunistic infections

Depletion or dysfunction of CD4⁺ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4⁺ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8⁺ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4⁺ T cell–independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19⁺ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

Patients with defects in CD4⁺ T cell number and function, whether due to HIV infection, malignancy, or other immunosuppression, are an increasing risk group in modern medicine (1, 2). For example, despite current strategies to treat HIV infection and its complications, Pneumocystis (PC) pneumonia remains a common clinical problem (1). In a recent epidemiological study performed after the initiation of highly active antiretroviral therapy (HAART), the incidence of PC infection has declined; however, the rate of decline has been greater for other AIDS opportunistic infections such as Toxoplasma or CMV infection (1). Since subpopulations of HIV-infected patients remain at risk despite HAART (1, 3, 4), and as there is an increasing patient population on immunosuppressive medical regimens, there is need to develop CD4⁺ T cell–independent therapeutic strategies to prevent infection (2). We and others have previously shown that bone marrow–derived DCs can be genetically modified to express CD40 ligand (CD40L) (5, 6), which leads to DC activation, and when pulsed with PC antigens, elicit significant anti-PC antibody titers in CD4-defeicent mice as well as conferring protection in SCID mice upon adoptive transfer of immune serum (7). A drawback of this technology is the scalability of a DC approach. However, a potential strength of the DC-based technology was that the protective humoral antibody response was restricted to a few PC antigens (7). Based on these data, we proposed 2 hypotheses: first, that the scalability of the CD4-independent DC approach could be improved by incorporation of CD40L in a DNA vaccine or DNA/adenovirus prime-boost vaccination strategy that would result in antigen-specific immunity in CD4-deficient mice; and second, that serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs could be used to identify antigens from PC that would be beneficial when placed in the CD40L DNA prime-boost vaccination protocol.

Here we show, using the model antigen chicken OVA, that the addition of CD40L in both the prime and boost phase of vaccination in CD4-depleted mice results in antibody responses similar to those in CD4-replete mice. Moreover, using 1D and 2D gel electrophoresis of immunoprecipitated PC antigens (using serum from CD40L-DC vaccinated mice), we were able to identify immunodominant epitopes of PC. Kexin, an antigen identified by aminoterminal sequencing and tandem mass spectroscopy (8), which has been reported to be on the surface of PC (9), was used to validate DNA vaccination in CD4-deficient hosts.

These studies demonstrate, for the first time to our knowledge, a scalable, therapeutic vaccine strategy in a CD4-deficient mouse model against a CD4 T lymphocyte–dependent pathogen, PC pneumonia, using a defined PC antigen. The results of these studies show promise for advances in CD4-independent vaccines in high-risk hosts with defective CD4⁺ T lymphocyte function.

Evaluation of a prime-boost vaccine approach to achieve CD4-independent vaccination. Based on the fact that CD40L modification of murine bone marrow–derived DCs can result in CD4-indepdendent B cell class switching in vitro and protective antibody responses in vivo, we examined whether this could be exploited for in vivo vaccination approaches. We chose prime-boost vaccination (Figure 1A) with DNA vaccines and adenovirus vectors as a platform to test this due to the relative ease of plasmid DNA manipulation and the efficacy of this approach in eliciting both humoral and T cell responses (10). We chose OVA as a model antigen due to the availability of reagents to assess both humoral and T cell responses efficiently in vivo. Initially, CD4-depleted (with GK1.5) or CD4-replete (treated with rat IgG2b or PBS as a control) mice (C57BL/6) were vaccinated with 3 doses, 3 weeks apart, with a plasmid encoding the chicken OVA cDNA (pOVA) or pOVA/CD40L, and anti-OVA antibodies were measured 14 days after each vaccination (Figure 1A). CD4-replete mice developed both anti-OVA IgG2a (Figure 1B) and anti-OVA IgG1 (Figure 1C) with either pOVA or pOVA/CD40L, whether PBS or rat IgG2b was used as the control. In contrast, CD4-depleted mice failed to mount an antibody responses to pOVA alone. However, vaccination with pOVA/CD40L resulted in levels of anti-OVA IgG2a and IgG1 comparable to those in CD4-replete mice. Mice vaccinated with empty vector or pCD40L alone failed to mount an anti-OVA response (data not shown).

Figure 1

Humoral responses after pOVA or pOVA/CD40L DNA vaccination. (A) Schema of prime-boost vaccination protocol. DNA vectors used were pOVA, pOVA/CD40L, pBUDCE4 (as empty vector), or pCD40L. Adenovirus boosting was carried out with AdOVA, AdOVA and AdCD40L, AdLuc, or AdLuc and AdCD40L. Anti-OVA IgG2a (B) and IgG1 (C) responses after 3 successive intramuscular DNA vaccinations with pOVA or pOVA/CD40L in CD4-depleted and CD4-replete mice n = 6–8 per group. All data are mean titer ± SEM. *P < 0.05 compared with control. A line denotes the limit of detection in the ELISA.

To examine the effect of DNA/adenovirus prime-boosting, mice were randomized to be boosted with an adenovirus encoding OVA (AdOVA) or firefly luciferase (AdLuc, as an irrelevant antigen) or the combination AdOVA/AdCD40L or AdLuc/AdCD40L. Mice boosted with the irrelevant antigen firefly luciferase, with or without CD40L, showed no increase in pre-boosting anti-OVA IgG1 (Figure 2A) or anti-OVA IgG2a titers (Figure 2B). In contrast, in both CD4-replete mice and in CD4-depleted mice, anti-OVA titers could be significantly boosted with the administration of AdOVA or AdOVA and AdCD40L (Figure 2, A and B). Moreover, in CD4-depleted mice, the addition of CD40L in the boosting regimen resulted in an additional increase in anti-OVA IgG1 and IgG2a that was not observed in CD4-replete mice, resulting in anti-OVA titers comparable to those in the CD4-replete group. Administration of AdOVA or AdOVA/CD40L alone to naive mice resulted in levels that were comparable to those in mice vaccinated with DNA alone, with detectable anti-OVA titers but with anti-OVA IgG1 and IgG2a titers less than 1:1,000 (see supplemental data; supplemental material available online with this article; doi:10.1172/JCI26306DS1).

Figure 2

Anti-OVA IgG1 (A) and IgG2a (B) responses after AdOVA or AdOVA/CD40L boosting after intramuscular DNA priming with pOVA or pOVA/CD40L in CD4-depleted and CD4-replete mice. n = 6–8 per group All data are mean titer ± SEM. *P < 0.05 compared with control; **P < 0.01 compared with control. A line denotes the limit of detection in the ELISA.

To investigate the ability of CD40L to induce CD4-independent CD8⁺ T cells, CD4-depleted or CD4-replete C57BL/6 mice underwent similar prime-boost vaccination with pOVA or pOVA/CD40L as described above, and SIINFEKL-specific (11) CD8⁺ T cells were analyzed after 3 DNA immunizations or after AdOVA or AdOVA/CD40L boosting (Figure 3). CD4-depleted mice primed with 3 doses of pOVA alone had 2% SIINFEKL dimer–positive cells in the spleen, which is near background. Moreover, there was no statistically significant increase in this population of CD8⁺ T cells in the spleen after AdOVA or AdOVA/CD40L boosting, although there was a trend toward a higher percentage of SIINFEKL-positive CD8 T cells after AdOVA boosting (Figure 3A). In contrast, CD4-depleted mice primed with pOVA/CD40L showed a significant increase in SIINFEKL-positive CD8 T cells after AdOVA or AdOVA/CD40L boosting, demonstrating the critical need to have CD40L in the priming vector (Figure 3A). Importantly, the percentage of SIINFEKL-positive cells in CD4-depleted mice was similar to that in CD4-replete mice undergoing a prime-boost protocol with either pOVA or pOVA/CD40L as the priming vector (Figure 3B). Furthermore, IFN-γ secretion by CD8⁺ T cells in CD4-depleted mice after CD40L prime-boosting was similar to that of CD4-replete mice (Figure 3C).

Figure 3

Antigen-specific CD8⁺ T cell responses. SIINFEKL-specific CD8⁺ T cell responses in CD4-depleted (A) and CD4-replete (B) mice after OVA and OVA/CD40L prime-boost regimens. n = 3–4 per group. *P < 0.05 compared with no-boost control. (C) IFN-γ secretion determined by ELISA in OVA-stimulated CD8⁺ T cells from both CD4-replete and CD4-depleted mice. n = 3–4 per group. *P < 0.05 compared with pOVA control. All data are mean values ± SEM.

Proteomic approach to identify protein antigens from PC. We have previously reported that PC antigen–pulsed, CD40L-modified DC-based vaccination results in an oligoclonal protective B cell response in CD4-defcient mice recognizing a 55-kDa antigen (7). This antibody was used in immunoprecipitations followed by 1D (Figure 4A) and 2D gel electrophoresis (Figure 4B). For 1D gels, bands were either transferred to nitrocellulose, stained with Ponceau S and subjected to N-terminal sequencing (12), or excised and subjected to enzyme digestion followed by TOF/TOF mass spectrometry (MS) (13). Spots from 2D gels were picked and underwent TOF/TOF MS analysis as well. Tandem MS and Edman degradation of the excised 55-kDa antigen revealed heavy-chain mouse IgG (as expected) but also a peptide, GSVYVFAS, with a high homology to Kexin from Saccharomyces cerevisiae, using the BLAST search algorithm for short, nearly exact matches in the yeast database. Based on these data, the full-length Kexin cDNA from PC muris was cloned as vaccine candidate.

Figure 4

Proteomics of PC antigens. (A) Immunoprecipitation of PC antigens after DC-based vaccination in mice with 1 antigen identified at 55 kDa. N-terminal sequencing and tandem MS analysis of this antigen showed greater than 85% homology with Kexin. M denotes molecular weight marker. (B) Typhoon images of 2D gels of Cy5-labeled naive serum (left) or immunoprecipitated PC antigens (right). Proteins were separated within pH 5–8 for the first dimension. Boxes indicate proteins that were analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF/TOF mass spectrometry.

CD4-independent vaccination of PC infection using Kexin DNA vaccination. To further evaluate CD40L in CD4-independent vaccination regimen against an opportunist infection that is critically dependent on intact CD4⁺ T cell function, we investigated the full-length Kexin cDNA. We chose this antigen over others identified in the proteomic screen, such as major surface glycoprotein–A, due to the fact that Kexin shows greater conservation (see supplemental data) across PC species than major surface glycoprotein–A (14). Immunization with pKexin (schema depicted in Figure 5A) resulted in significant anti-PC IgG1 and IgG2a titers in CD4-replete mice, whereas titers were significantly lower in CD4-depleted mice (Figure 5B). In comparison, CD4-depleted mice immunized with pKexin/CD40L demonstrated significantly higher titers of anti-PC IgG1 compared with CD4-depleted mice immunized with pKexin alone (Figure 5B). Mice immunized with pCD40L or empty vector alone demonstrated no detectable anti-PC titer (data not shown). Moreover, the anti-PC titers observed after Kexin immunization were higher than those observed after DNA vaccination with OVA alone (Figure 1). To verify the CD4- independence of Kexin vaccination, we performed 3 rounds of pKexin versus pKexin/CD40L vaccination in wild-type or CD4^–/– mice. We observed that, similarly to what occurred in mice depleted with GK1.5, anti-PC IgG1 and IgG2a levels significantly increased in CD4^–/– mice when vaccinated with pKexin/CD40L as opposed to pKexin alone (Figure 5C).

Figure 5

Humoral responses after pKexin or pKexin/CD40L DNA vaccination. (A) Schema of PC Kexin vaccination protocol. DNA vectors used were pKexin, pKexin/CD40L, pBUDCE4 (as empty vector), or pCD40L. (B) Anti-PC IgG1 and IgG2a responses after pKexin or pKexin/CD40L after intramuscular DNA immunization in CD4-depleted and CD4-replete mice. n = 6–8. All data are mean titer ± SEM. *P < 0.05 compared with control. (C) Anti-PC IgG1 and IgG2a responses after pKexin or pKexin/CD40L after intramuscular DNA immunization in CD4^–/– and CD4^+/+ mice. n = 4–6. All data are mean titer ± SEM. **P < 0.05 compared with CD4^+/+ mice or pKexin/CD40L in CD4^–/– mice. A line denotes the limit of detection in the ELISA.

Since we observed significant anti-PC titers after Kexin DNA immunization in CD4-depleted mice, to determine whether this response was protective, we performed a PC challenge experiment. PC organism burden was assessed 28 days after challenge by quantitative PCR as well as by histological scoring, as previously described (7). Quantitative PCR analysis showed that mice immunized with pKexin compared with control mice, receiving pCD40L alone, had approximately one-half log lower PC-specific ribosomal RNA (rRNA) content in lung tissue (Figure 6A). Furthermore, mice immunized with pKexin/CD40L showed significantly lower organism burdens, with nearly a 3-log greater protection compared with control CD4-depleted mice. Of note, 10⁴ rRNA copy number is near the limit of detection of organisms by histological staining with Gomori methenamine-silver (GMS). Similar trends were observed when infection was also scored blindly on GMS-stained lung sections (7), with mice immunized with pKexin/CD40L showing the greatest protection (Figure 6B).

Figure 6

Protection against a primary PC challenge in CD4-depleted mice after DNA immunization. CD4-depleted mice were vaccinated with 3 rounds of control (pCD40L), pKexin, or pKexin/CD40L followed by challenge with PC. PC was quantified by quantitative real-time PCR (A) or by histology (B). n = 4–6. All data are mean values ± SEM. *P < 0.05, **P < 0.01 compared with control.

Kexin DNA vaccination results in protective antibody responses. Due to the fact that we observed protection in the primary challenge with PC, we investigated whether this was antibody mediated. We initially determined whether the antibody generated by pKexin/CD40L was capable of mediating opsonic killing of PC. Organisms were incubated with immune serum from pKexin/CD40L-vaccinated mice or control serum followed by incubation with peritoneal macrophages for 16 hours. Remaining viable PC was quantified by assessing the integrity of PC rRNA using real-time PCR as previously described (7, 15). Incubation with serum from pKexin-immunized mice showed opsonic killing, with an increase in macrophage-mediated killing of PC from 24% in naive mice to 38% in pKexin-immunized mice. However, mice immunized with pKexin/CD40L had the greatest increase in killing, to nearly 80%. This serum-mediated increase in killing was not attenuated by heat inactivation but was significantly abrogated by incubation with protein A and G, suggesting it was due to opsonic anti-PC immunoglobulin in the serum (Figure 7A).

Figure 7

Functionality and antigen specificity of serum after pKexin/CD40L vaccination in mice. (A) Opsonic activity of serum after DNA immunization with control, pKexin, or pKexin/CD40L vectors. (B) Passive transfer of serum or CD19⁺ splenocytes confers protection against a PC challenge in SCID mice. n = 4–6. All data are mean values ± SEM. *P < 0.05, **P < 0.01 compared with control. (C) Representative Western blot analysis of serum after Kexin vaccination against sonicated PC antigen. Neither naive serum nor serum from CD4-depleted mice with active PC infection gave any activity on Western blotting. Serum from mice undergoing 3 rounds of pKexin/CD40L vaccination (far right 2 lanes) reacted with a 105- and 55-kDa protein. (D) Representative immunofluorescent staining of mouse and monkey PC with anti-Kexin serum.

To further investigate whether antibody induction was critical to protection after pKexin/CD40L vaccination, we performed adoptive transfer experiments with serum or CD19⁺ splenocytes from pKexin/CD40L-vaccinated or control mice (CD40L vaccination alone) in SCID mice followed by PC challenge. SCID mice receiving either serum or CD19⁺ splenocytes from pKexin/CD40L-vaccinated mice showed significantly lower PC burdens by quantitative real-time PCR 28 days after PC challenge compared with control mice (Figure 7B), showing that both serum and B cells were capable of conferring protection against PC. Last, we investigated the antigen specificity of serum from mice given pKexin/CD40L compared with naive serum or serum from CD4-depleted mice for 28 days in a Western blot assay against sonicated PC antigen, as well as staining of mouse and monkey PC. Neither naive serum nor serum from CD4-depleted mice infected with PC for 28 days showed any immunoreactivity against sonicated PC antigen (Figure 7C), demonstrating the critical need for CD4 cells to generate antibody against PC. In contrast, serum from pKexin/CD40L-immunized mice demonstrated robust reactivity to a 105-kDa antigen (the predicted molecular weight of full-length Kexin) as well as to a 55-kDa antigen that migrates a similar distance as the antigen observed in mice immunized with PC-pulsed, CD40L-modified DCs (Figure 4) (7). Naive mouse serum failed to stain mouse or monkey PC (Figure 7D), whereas serum from mice vaccinated with pKexin/CD40L stained PC derived from either mouse (red staining) or monkey (yellow staining).

There is a critical need to develop CD4-indpendent vaccine approaches for infections, as the numbers of immunocompromised hosts are increasing (16). One strategy toward this end is to define the factors that mediate CD4⁺ T cell help and provide therapeutic replacement of these factors. Toward this end, we have previously demonstrated that overexpression of IFN-γ, a potent Th1 cytokine produced by CD4⁺ T cells, can result in eradication of PC in the absence of CD4⁺ T cell help (17), in part through the augmentation of IFN-γ–secreting type I (Tc1) CD8⁺ T cell response (18).

Another molecule expressed on activated CD4⁺ T cells that is critically important for costimulation and CD4⁺ T cell help is CD40L. CD40L is expressed on activated T cells and activates DCs to influence CD8⁺ cytotoxic T cell (19, 20) and B cell (21) immune responses. Kikuchi and colleagues have demonstrated that CD40L gene–modified DCs pulsed with Pseudomonas aeruginosa (PA) could stimulate naive B cells to produce anti-PA antibodies (22) and confer protection against PA challenge. Furthermore, we recently showed that CD40L-modifed DCs pulsed with PC could result in a protective antibody response in CD4-depleted mice and protect them against a PC challenge.

Although these studies demonstrate the proof-of-principle that CD40L is an excellent adjuvant to elicit CD4-independent immune responses, DC approaches suffer from significant issues of scalability. Second, vaccine approaches to organisms such as PC that have not been successfully adapted to large-scale ex vivo culture have been hampered by a lack of good candidate antigens. We sought to overcome these shortcomings by (a) using CD40L-modified DCs to identify vaccine candidates and (b) using CD40L in a prime-boost regimen to overcome the lack of immunogenicity of DNA vaccines in CD4-deficient hosts.

We have previously shown that IgG responses generated by AdCD40L-modifed DC technology were largely restricted to a 55-kDa antigen of PC that is protective upon adoptive transfer (7). To further define this and other antigens, we used a combined approach of 1D and 2D gel electrophoresis followed by transfer to nitrocellulose and N-terminal sequencing or spot excision, enzyme digestion, and tandem MS. N-terminal sequencing of 1 of the 55-kDa bands revealed a peptide sequence with a high degree of homology to Kexin, a furin-like protease (7). We also identified major surface glycoprotein–A (14); however, due to significant divergence in this protein across PC species, we focused on investigating Kexin. Furthermore, a monoclonal antibody to this antigen has shown protection in passive immunization experiments in mice (23).

Despite the identification of Kexin as a vaccine candidate, a platform was required to achieve a CD4-independent vaccine approach in vivo. We chose DNA vaccination, as DNA vaccines have been shown to traduce DCs in muscle (24, 25) but also allow for cross-presentation of antigens in muscle (26), and CD40L transduction would aid in DC maturation as well as potentially activation and class-switching of B cells (27). The addition of CD40L in OVA vaccination described here clearly allows for the generation of antigen-specific IgG1 and IgG2a independently of CD4⁺ T cells. Moreover, DNA vaccines, like certain replication-defective viral vectors, appear to have the important advantage of safety for use in immune-deficient individuals, unlike replicating vector systems. The antibody titer after DNA vaccination could be boosted significantly with recombinant adenovirus vectors encoding the same antigen. Whether direct transduction of DCs or cross-presentation of antigen by myocytes is occurring in vivo is unknown at present but is being investigated. The addition of CD40L in the priming regimen, along with eliciting the above-described humoral responses, also allowed for the generation of antigen-specific CD8⁺ T cells after adenovirus boosting. Based on these data, we investigated whether this approach could yield therapeutic vaccination against a prototypic AIDS-defining illness, PC pneumonia.

Toward this end, DNA immunization with Kexin/CD40L resulted in significant anti-PC antibody titers, which were protective in primary challenge experiments as well as in adoptive transfer experiments. It is important to note that the efficacy was greater in the primary challenge experiments compared with the adoptive transfer experiments, likely due to the dilution effect of serum transfer and the half-life of the antibody. In support of this, after transfer of immune serum with an anti-PC titer of greater than 1:1,500, the remaining anti-PC IgG1 titer 28 days later was 1:346. Moreover, Kexin/CD40L immunization resulted in antibody capable of mediating opsonic phagocytosis of PC, which may be critical for its therapeutic affect. Western blot analysis demonstrated that mice immunized with Kexin/CD40L recognized a protein of the predicted full-length Kexin (105 kDa) as well as a 55-kDa protein that may be a proteolytic cleavage product of Kexin in the sonicated PC antigen. As this was the same antigen lot used to pulse DCs, this lends further support for using antigen-pulsed DCs for epitope identification/prioritization.

We did not examine adenovirus boosting with PC, as antibody generation correlates well with protection in this model (7); however, based on the ability to induce a Tc1 CD8⁺ T cell response, prime boosting may be the preferred approach where a CD8⁺ T cell response — as, for example, against tumors (28, 29) or intracellular pathogens (30) — is critical. Ultimately a CD8⁺ T cell response, in addition to an antibody response, may also be important in PC, as there is evidence that polarized, IFN-γ–producing CD8⁺ T cells are effective in mediating host defenses against this pathogen (18). Although the safety and efficacy of this novel vaccine approach will need to be validated in other systems, the data presented here show promise in eliciting strong cellular and humoral responses in the absence of CD4⁺ T cells.

View Supplemental data

This work was supported by Public Health Service grants P01HL076100 and R01HL061271.

Nonstandard abbreviations used: CD40L, CD40 ligand; GMS, Gomori methenamine-silver; MS, mass spectrometry; PC, Pneumocystis; pOVA, plasmid encoding the chicken OVA cDNA; TOF, time-of-flight.

Conflict of interest: The authors have declared that no conflict of interest exists.

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