Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Cost-effectiveness of introducing the 10-valent pneumococcal conjugate vaccine into the universal immunisation of infants in Brazil

Background Cost-effectiveness studies have been increasingly part of decision processes for incorporating new vaccines into the Brazilian National Immunisation Program. This study aimed to evaluate the cost-effectiveness of 10-valent pneumococcal conjugate vaccine (PCV10) in the universal childhood immunisation programme in Brazil. Methods A decision-tree analytical model based on the ProVac Initiative pneumococcus model was used, following 25 successive cohorts from birth until 5 years of age. Two strategies were compared: (1) status quo and (2) universal childhood immunisation programme with PCV10. Epidemiological and cost estimates for pneumococcal disease were based on National Health Information Systems and literature. A 'top-down' costing approach was employed. Costs are reported in 2004 Brazilian reals. Costs and benefits were discounted at 3%. Results 25 years after implementing the PCV10 immunisation programme, 10 226 deaths, 360 657 disability-adjusted life years (DALYs), 433 808 hospitalisations and 5 117 109 outpatient visits would be avoided. The cost of the immunisation programme would be R$10 674 478 765, and the expected savings on direct medical costs and family costs would be R$1 036 958 639 and R$209 919 404, respectively. This resulted in an incremental cost-effectiveness ratio of R$778 145/death avoided and R$22 066/DALY avoided from the society perspective. Conclusion The PCV10 universal infant immunisation programme is a cost-effective intervention (1-3 GDP per capita/DALY avoided). Owing to the uncertain burden of disease data, as well as unclear long-term vaccine effects, surveillance systems to monitor the long-term effects of this programme will be essential.

Glucocorticoid: Major Factor for Reduced Immunogenicity of 2009 Influenza A (H1N1) Vaccine in Patients with Juvenile Autoimmune Rheumatic Disease

Objective. To assess the immunogenicity and safety of non-adjuvanted influenza A H1N1/2009 vaccine in patients with juvenile autoimmune rheumatic disease (ARD) and healthy controls, because data are limited to the adult rheumatologic population. Method's. A total of 237 patients with juvenile ARD [juvenile systemic lupus erythematosus (JSLE), juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM), juvenile scleroderma, and vasculitis] and 91 healthy controls were vaccinated. Serology for anti-H1N1 was performed by hemagglutination inhibition assay. Seroprotection rate, seroconversion rate, and factor-increase in geometric mean titer (GMT) were calculated. Adverse events were evaluated. Results. Age was comparable in patients and controls (14.8 +/- 3.0 vs 14.6 +/- 3.7 years, respectively; p = 0.47). Three weeks after immunization, seroprotection rate (81.4% vs 95.6%; p = 0.0007), seroconversion rate (74.3 vs 95.6%; p < 0.0001), and the factor-increase in GMT (12.9 vs 20.3; p = 0.012) were significantly lower in patients with juvenile ARD versus controls. Subgroup analysis revealed reduced seroconversion rates in JSLE (p < 0.0001), JIA (p = 0.008), JDM (p = 0.025), and vasculitis (p = 0.017). Seroprotection (p < 0.0001) and GMT (p < 0.0001) were decreased only in JSLE. Glucocorticoid use and lymphopenia were associated with lower seroconversion rates (60.4 vs 82.9%; p = 0.0001; and 55.6 vs 77.2%; p = 0.012). Multivariate logistic regression including diseases, lymphopenia, glucocorticoid, and immunosuppressants demonstrated that only glucocorticoid use (p = 0.012) remained significant. Conclusion. This is the largest study to demonstrate a reduced but adequate immune response to H1N1 vaccine in patients with juvenile ARD. It identified current glucocorticoid use as the major factor for decreased antibody production. The short-term safety results support its routine recommendation for patients with juvenile ARD. ClinicalTrials.gov; NCT01151644. (First Release Nov 15 2011; J Rheumatol 2012;39:167-73; doi:10.3899/jrheum.110721)

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8(+) T-Cell Response: Reversal by Adenoviral Vaccine

MHC class la-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T-cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8(+) T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

A phase I clinical trial of a new 5-valent rotavirus vaccine

We conducted a phase I, double-blind, placebo-controlled trial to evaluate a new 5-valent oral rotavirus vaccine’s safety and immunogenicity profiles. Subjects were randomly assigned to receive 3 orally administered doses of a live-attenuated human-bovine (UK) reassortant rotavirus vaccine, containing five viral antigens (G1, G2, G3, G4 and G9), or a placebo. The frequency and severity of adverse events were assessed. Immunogenicity was evaluated by the titers of anti-rotavirus IgA and the presence of neutralizing antibodies anti-rotavirus. No severe adverse events were observed. There was no difference in the frequency of mild adverse events between experimental and control groups. The proportion of seroconversion was consistently higher in the vaccine group, for all serotypes, after each one of the doses. The 5-valent vaccine has shown a good profile of safety and immunogenicity in this small sample of adult volunteers.

Exome analysis of HIV patients submitted to dendritic cells therapeutic vaccine reveals an association of CNOT1 gene with response to the treatment

INTRODUCTION: With the aim of searching genetic factors associated with the response to an immune treatment based on autologous monocyte-derived dendritic cells pulsed with autologous inactivated HIV, we performed exome analysis by screening more than 240,000 putative functional exonic variants in 18 HIV-positive Brazilian patients that underwent the immune treatment. METHODS: Exome analysis has been performed using the ILLUMINA Infinium HumanExome BeadChip. zCall algorithm allowed us to recall rare variants. Quality control and SNP-centred analysis were done with GenABEL R package. An in-house implementation of the Wang method permitted gene-centred analysis. RESULTS: CCR4-NOT transcription complex, subunit 1 (CNOT1) gene (16q21), showed the strongest association with the modification of the response to the therapeutic vaccine (p=0.00075). CNOT1 SNP rs7188697 A/G was significantly associated with DC treatment response. The presence of a G allele indicated poor response to the therapeutic vaccine (p=0.0031; OR=33.00; CI=1.74-624.66), and the SNP behaved in a dominant model (A/A vs. A/G+G/G p=0.0009; OR=107.66; 95% CI=3.85-3013.31), being the A/G genotype present only in weak/transient responders, conferring susceptibility to poor response to the immune treatment. DISCUSSION: CNOT1 is known to be involved in the control of mRNA deadenylation and mRNA decay. Moreover, CNOT1 has been recently described as being involved in the regulation of inflammatory processes mediated by tristetraprolin (TTP). The TTP-CCR4-NOT complex (CNOT1 in the CCR4-NOT complex is the binding site for TTP) has been reported as interfering with HIV replication, through post-transcriptional control. Therefore, we can hypothesize that genetic variation occurring in the CNOT1 gene could impair the TTP-CCR4-NOT complex, thus interfering with HIV replication and/or host immune response. CONCLUSIONS: Being aware that our findings are exclusive to the 18 patients studied with a need for replication, and that the genetic variant of CNOT1 gene, localized at intron 3, has no known functional effect, we propose a novel potential candidate locus for the modulation of the response to the immune treatment, and open a discussion on the necessity to consider the host genome as another potential variant to be evaluated when designing an immune therapy study

RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

In order to achieve host cell entry, the apicomplexan parasite Neospora caninum relies on the contents of distinct organelles, named micronemes, rhoptries and dense granules, which are secreted at defined timepoints during and after host cell entry. It was shown previously that a vaccine composed of a mixture of three recombinant antigens, corresponding to the two microneme antigens NcMIC1 and NcMIC3 and the rhoptry protein NcROP2, prevented disease and limited cerebral infection and transplacental transmission in mice. In this study, we selected predicted immunogenic domains of each of these proteins and created four different chimeric antigens, with the respective domains incorporated into these chimers in different orders. Following vaccination, mice were challenged intraperitoneally with 2 × 10(6)N. caninum tachzyoites and were then carefully monitored for clinical symptoms during 4 weeks post-infection. Of the four chimeric antigens, only recNcMIC3-1-R provided complete protection against disease with 100% survivors, compared to 40-80% of survivors in the other groups. Serology did not show any clear differences in total IgG, IgG1 and IgG2a levels between the different treatment groups. Vaccination with all four chimeric variants generated an IL-4 biased cytokine expression, which then shifted to an IFN-γ-dominated response following experimental infection. Sera of recNcMIC3-1-R vaccinated mice reacted with each individual recombinant antigen, as well as with three distinct bands in Neospora extracts with similar Mr as NcMIC1, NcMIC3 and NcROP2, and exhibited distinct apical labeling in tachyzoites. These results suggest that recNcMIC3-1-R is an interesting chimeric vaccine candidate and should be followed up in subsequent studies in a fetal infection model.

Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection

The major route of transmission of Neospora caninum in cattle is transplacentally from an infected cow to its progeny. Therefore, a vaccine should be able to prevent both the horizontal transmission from contaminated food or water and the vertical transmission. We have previously shown that a chimeric vaccine composed of predicted immunogenic epitopes of NcMIC3, NcMIC1 and NcROP2 (recNcMIC3-1-R) significantly reduced the cerebral infection in BALB/c mice. In this study, mice were first vaccinated, then mated and pregnant mice were challenged with 2×10(6)N. caninum tachyzoites at day 7-9 of pregnancy. Partial protection was only observed in the mice vaccinated with a tachyzoite crude protein extract but no protection against vertical transmission or cerebral infection in the dams was observed in the group vaccinated with recNcMIC3-1-R. Serological and cytokine analysis showed an overall lower cytokine level in sera associated with a dominant IL-4 expression and high IgG1 titers. Thus, the Th2-type immune response observed in the pregnant mice was not protective against experimental neosporosis, in contrary to the mixed Th1-/Th2-type immune response observed in the non-pregnant mouse model. These results demonstrate that the immunomodulation that occurs during pregnancy was not favorable for the protection against N. caninum infection conferred by vaccination with recNcMIC3-1-R.

Immunization of Macaca fascicularis against experimental periodontitis using a vaccine containing cysteine proteases purified from Porphyromonas gingivalis

INTRODUCTION: Periodontitis is a common infectious disease to which Porphyromonas gingivalis has been closely linked, in which the attachment tissues of the teeth and their alveolar bone housing are destroyed. We conducted a study to determine if immunization using a purified antigen could alter the onset and progression of the disease. METHODS: Using the ligature-induced model of periodontitis in Macaca fascicularis, we immunized five animals with cysteine protease purified from P. gingivalis and used an additional five animals as controls. Alveolar bone loss was measured by digital subtraction radiography. RESULTS: Immunization induced high titers of specific immunoglobuin G serum antibodies that were opsonic. Total bacterial load, levels of P. gingivalis in subgingival plaque and levels of prostaglandin E(2) in gingival crevicular fluid were significantly reduced. Onset and progression of alveolar bone loss was inhibited by approximately 50%. No manifestations of toxicity were observed. CONCLUSIONS: Immunization using a purified protein antigen from P. gingivalis inhibits alveolar bone destruction in a ligature-induced periodontitis model in M. fascicularis.

NOVEL MECHANISMS OF ANTIGEN PROCESSING THAT ENHANCE BCG VACCINE EFFICACY

Mycobacterium tuberculosis, the causative agent of tuberculosis, is the most lethal single infectious agent afflicting man today causing 2 million deaths per year. The World Health Organization recommends a vaccine as the best option to prevent this disease. The current vaccine, BCG, has a variable efficacy and does not protect adults. It is known that BCG vaccine becomes sequestered in special phagosome compartments of macrophages that do not fuse with lysosomes. Since lysosome fusion is necessary for peptide production and T cell priming leading to protective TH1 immunity, we hypothesized that vaccine efficacy is reduced and occurs perhaps due to non-lysosome dependent mechanisms. We therefore proposed an in depth analysis of phagosome environment, and its proteome to unravel mechanisms of antigen processing and presentation. We initially discovered that three mechanisms of pH regulation including vacuolar proton ATPase, phagocyte oxidase and superoxide dismutase (SOD) secretion from BCG vaccine affect antigen processing within phagosomes. These studies led to the discovery that a mutant of BCG vaccine which lacked SOD was a better vaccine. Subsequently, the proteomic analysis of vaccine phagosomes led to the discovery of novel protease (γ-secretase) enriched on BCG vaccine phagosomes. We then demonstrated that these proteases generated a peptide from the BCG vaccine which was presented through the MHC-II pathway to T cells and induced a TH1 response. The specificity of antigen production from γ-secretase was confirmed through siRNA knockdown of the components of the protease namely, nicastrin, presenilin and APH, which led to a decrease in antigen presentation. We therefore conclude that, even though BCG phagosomes are sequestered and do not fuse with lysosomes to generate peptide antigens, there are complex and novel in situ mechanisms within phagosomes that are capable of generating an immune response. We conclude that TH1 immunity to BCG vaccine arises mostly due to non-lysosome dependent immune mechanisms of macrophages and dendritic cells.

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

A vaccine for HIV type 1: The antibody perspective

Antibodies that bind well to the envelope spikes of immunodeficiency viruses such as HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) can offer protection or benefit if present at appropriate concentrations before viral exposure. The challenge in antibody-based HIV-1 vaccine design is to elicit such antibodies to the viruses involved in transmission in humans (primary viruses). At least two major obstacles exist. The first is that very little of the envelope spike surface of primary viruses appears accessible for antibody binding (low antigenicity), probably because of oligomerization of the constituent proteins and a high degree of glycosylation of one of the proteins. The second is that the mature oligomer constituting the spikes appears to stimulate only weak antibody responses (low immunogenicity). Viral variation is another possible obstacle that appears to present fewer problems than anticipated. Vaccine design should focus on presentation of an intact mature oligomer, increasing the immunogenicity of the oligomer and learning from the antibodies available that potently neutralize primary viruses.

A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.

DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines.

Vaccination of two chimpanzees against hepatitis B virus (HBV) by intramuscular injection of plasmid DNA encoding the major and middle HBV envelope proteins induced group-, subtype- and preS2-specific antibodies. These were initially of IgM isotype, and then they were of IgG (predominantly IgGl) isotype. The chimpanzee injected with 2 mg of DNA attained >100 milli-international units/ml of anti-HBs antibody after one injection and 14,000 milli-international units/ml after four injections. A smaller dose (400 microg) induced lower and transient titers, but a strong anamnestic response occurred 1 year later. Comparison with responses in 23 chimpanzees receiving various antigen-based HBV vaccines suggests that the DNA approach is promising for prophylactic immunization against HBV.