977. CD4+ T Cell Independent DNA Vaccination Against Pneumocystis Carinii in Mice

Abstract

Host defenses are profoundly compromised in HIV infection and other immunocompromised states due to progressive depletion of CD4+ T-lymphocytes. Deficient CD4+ T lymphocytes impair vaccination approaches to prevent opportunistic infection. CD40 ligand (CD40L) is expressed on activated CD4+ T cells and is critical for host defense against Pneumocystis carinii (PC) as well as bacterial pneumonia. Our lab has demonstrated that 25 kDa and 55 kDa antigens of PC generate antigen specific B-cell responses in dendritic cell (DC) vaccination. Using 2-D gel electrophoresis and N-terminal sequencing, we have shown that one of these antigens has a high degree of homology to Kexin, a protease encoded in the PC genome. Therefore we investigated a CD4+ T-cell independent CD40L DNA vaccine approach to a prototypic AIDS-defining infection, PC pneumonia. Plasmids encoding the control antigen, OVA (pACCOVA) , CD40L (pACCCD40L), OVA plus CD40L (pACCOVA-CD40L), Kexin (pACCKexin) and Kexin plus CD40L(pACCKexin-CD40L) driven by the CMV promoter were injected intramuscularly into control or CD4-depleted BALB/c mice 3 times. At specific time points, sera was collected and assayed for anti-OVA and anti-PC antibodies by ELISA. Antigen specific CD8 T cells response were analyzed by IFN-g secretion and dimer OVA-antigen specific positive cells in splenocytes. For adoptive transfer experiments, sera and B cells from vaccinated mice were transferred to na|[iuml]|ve severe combined immunodeficient (SCID) mice followed by challenge with PC. To asses the affect of adoptively transferred sera or cells on PC infection, SCID mice were sacrificed at 28 days and PC burden was determined by real-time PCR. ACCOVA alone resulted in the induction of anti-OVA IgG1 and IgG2a in control mice but this response was significantly attenuated in CD4- depleted mice. Co-administration of pACCCD40L with pACCOVA resulted in similar levels of anti-OVA IgG1 and IgG2a despite the absence of CD4+ T -cells. pACCKexin-CD40L significantly induced a higher titer of anti-PC (IgG1 and IgG2a) then pACCKexin alone. Moreover, the induced anti-PC Ab showed significant opsonic-mediated PC killing in an in vitro killing assay. SCID mice that received either sera or CD19+ splenocytes from pACCKexin + pACCCD40L were significantly protected from PC challenge compared to the OVA or pACCKexin alone control groups. These studies show the promise of proteomic screening to identify antigens for vaccination and the addition of CD40L in DNA vaccine protocols enhances antibody responses in CD4-defecint hosts. Taken together, these data support the rationale for CD4-independent antigen specific antigen DNA vaccines against HIV-related opportunistic infection.