Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity.

Our goal is to develop a vaccine that sustainably prevents Plasmodium falciparum (Pf) malaria in ≥80% of recipients. Pf sporozoites (PfSPZ) administered by mosquito bites are the only immunogens shown to induce such protection in humans. Such protection is thought to be mediated by CD8(+) T cells in the liver that secrete interferon-γ (IFN-γ). We report that purified irradiated PfSPZ administered to 80 volunteers by needle inoculation in the skin was safe, but suboptimally immunogenic and protective. Animal studies demonstrated that intravenous immunization was critical for inducing a high frequency of PfSPZ-specific CD8(+), IFN-γ-producing T cells in the liver (nonhuman primates, mice) and conferring protection (mice). Our results suggest that intravenous administration of this vaccine will lead to the prevention of infection with Pf malaria.

A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge.

The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-gamma ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-gamma ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF.

One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

Mannan-binding lectin enhances susceptibility to visceral leishmaniasis.

Levels of the serum opsonin mannan-binding lectin (MBL) were directly correlated with the probability of developing visceral leishmaniasis. Monocytes infected with MBL-opsonized Leishmania chagasi promastigotes secreted higher levels of tumor necrosis factor alpha and interleukin-6 than cells infected with nonopsonized parasites. Our findings indicate that MBL can modulate the clinical outcome of infection with L. chagasi and the function of infected macrophages.

Safety, tolerability and humoral immune responses after intramuscular administration of a malaria DNA vaccine to healthy adult volunteers.

DNA-based vaccines are considered to be potentially revolutionary due to their ease of production, low cost, long shelf life, lack of requirement for a cold chain and ability to induce good T-cell responses. Twenty healthy adult volunteers were enrolled in a Phase I safety and tolerability clinical study of a DNA vaccine encoding a malaria antigen. Volunteers received 3 intramuscular injections of one of four different dosages (20, 100, 500 and 2500 microg) of the Plasmodium falciparum circumsporozoite protein (PfCSP) plasmid DNA at monthly intervals and were followed for up to twelve months. Local reactogenicity and systemic symptoms were few and mild. There were no severe or serious adverse events, clinically significant biochemical or hematologic changes, or detectable anti-dsDNA antibodies. Despite induction of excellent CTL responses, intramuscular DNA vaccination via needle injection failed to induce detectable antigen-specific antibodies in any of the volunteers.

Secretion of a new basal layer of tectorial membrane following gentamicin-induced hair cell loss.

Scanning electron microscopy (SEM) and video-enhanced DIC light microscopy were used to assess morphological changes in the chick tectorial membrane (TM) following gentamicin-induced hair cell loss. Gentamicin was administered (100 mg/kg/day for 3 days) and isolated and in-situ TMs were examined in both fixed and unfixed preparations at days 5 and 10 after the initial injection. Although this protocol induced hair cell damage extending up to 75% of the length of the basilar papilla, there was no apparent damage to the TM itself. However, the ejection of damaged hair cells appeared to sever the filamentous attachments between the TM and the apical surface of the basilar papilla. In SEM preparations this detachment caused the TM to shrink back toward the superior edge. Interestingly, despite the lack of TM damage, gentamicin treatment did reveal the secretion of a new basal layer of TM. Secretion of this new basal layer had begun by day 5 and it was well organized by day 10. This new layer formed attachments to both the recovering basilar papilla and the overlying original TM, a step thought to be necessary for the restoration of auditory function in the regenerating cochlea.

Rheumatic fever: autoantibodies against a variety of cardiac, nuclear, and streptococcal antigens.

OBJECTIVE: To measure antibody titres to cardiac, nuclear, and streptococcal antigens in different groups of rheumatic fever (n = 60) and control subjects (n = 80) with the aim of identifying cross reactive antigens of potential laboratory diagnostic value. METHODS: Enzyme linked immunosorbent assays (ELISA), immunocytochemical, and electrophoretic techniques were used to measure titres of antibodies to a variety of cardiac, nuclear, and streptococcal antigens in seven groups comprising patients with rheumatic fever and control subjects. RESULTS: Increased concentrations of antibodies to several streptococcal and cardiac antigens, in addition to increased IgA and IgG levels, were noted in sera from patients with acute rheumatic fever and chronic rheumatic heart disease. Autoantibodies to nuclear antigens were evident in three rheumatic fever sera. CONCLUSION: Although we were unable to identify any unique cross reactivity between cardiac and streptococcal antigens, these results demonstrate that there is an exaggerated humoral response to several cardiac, nuclear and streptococcal antigens in patients with rheumatic fever.

Quantitative nuclear morphometry, Markovian texture descriptors, and DNA content captured on a CAS-200 Image analysis system, combined with PCNA and HER-2/neu immunohistochemistry for prediction of prostate cancer progression.

One hundred and twenty-four localized prostate cancer patients operated on at Johns Hopkins Hospital (JHH) since 1975 were identified. The sample was optimized for evaluation of prostate cancer progression. Based upon accurate clinical histories, these radical prostatectomy patients included 50 progressors and 74 non-progressors using appearance of serum PSA as an indication of recurrence (mean follow-up = 8.6 +/- 1.8 years, range 7-15 years). All patients included in the study had no involvement of their seminal vesicles or lymph nodes at the time of prostatectomy. Average time to progression was 3.6 +/- 2 years, range of 1-8 years. Using paraffin-embedded specimens, several five micron sections were cut and placed on Probe-On slides; one slide was H&E-stained and the other was Feulgen-stained. The H&E and Feulgen-stained slides were screened and "dotted" by pathologists at JHH and CytoDynostics, Inc. A CAS-200 Image analysis system (Cell Image Systems, Elmhurst, IL) equipped with a Cell Measurement Program version 1.2 beta, was used to capture the Feulgen-stained images and to perform the calculations. From the "dotted" areas, 150 cancer cells were selected for measurement of DNA content and 27 nuclear morphometric shape and size factors, including 21 Markovian chromatin texture variables. Additional sections were used for immunochemistry staining with an alkaline phosphatase streptavidin-biotin complex stain to detect and quantitate cancer cells binding monoclonal antibodies directed against proliferating cell nuclear antigen (PCNA) and HER-2/neu antigen. All data were entered into a statistical program (STATA) for further analysis and univariate and multivariate statistical analysis was performed using logistic regression and its stepwise variant. The biomarkers of greatest utility to detect progressors when analyzed univariately included post-operative Gleason score (p = < 0.0001), HER-2/neu antigenicity (p = 0.0147), CAS-200 DNA ploidy (p = 0.008), and twelve Markovian nuclear texture and shape features (p = < 0.0001), whereas PCNA (p = 0.160) failed. The optimal set of nuclear morphometry progression tumor features were selected using backward stepwise logistic regression estimate analysis which drops variables due to collinearity. Although post-operative Gleason score is a strong univariate predictor of progression, DNA ploidy and HER-2/neu contributed significantly to further stratification of higher risk groups within the low Gleason score subpopulation. The best Markovian features combined with post-operative Gleason score generated sensitivity = 90%, specificity = 96%, positive predictive value = 94%, negative predictive value = 93% and the area under the receiver operator curve was 0.975.

Polymerase chain reaction evidence for human immunodeficiency virus 1 neutralization by passive immunization in patients with AIDS and AIDS-related complex.

We tried to assess the long-term safety and potential efficacy of passive immunization in AIDS-related-complex (ARC) and AIDS patients. We also wanted to establish whether hyperimmune plasma from healthy human immunodeficiency virus 1 (HIV-1)-infected individuals clears the cell-free virus from circulation. Using the polymerase chain reaction (PCR), we were able to provide conclusive evidence that hyperimmune plasma is effective and maintains long-term neutralization of viremia. Using the cell test, we found that in most patients the total antibody level was maintained; in one of the ARC patients, it actually increased 8-fold and has remained at that level for nearly 2 years. The CD4+ cell count decreased in the AIDS patients but was stable in the ARC patient. Clinically, there was an initial improvement in all patients, but five of six of the advanced/terminal AIDS patients had died by month 17. Our studies suggest that passive immunization may be safe in ARC and AIDS patients. It reduces HIV-1 viremia to levels undetectable even by PCR. To advanced/terminal patients, the benefit is of limited duration, while to ARC patients it may be long-term. Therefore, passive immunization should start early in the disease.