Acellular and "low" pertussis vaccines: adverse events and the role of mutations

Objective: to discuss the current PAHO recommendation that does not support the substitution of traditional cellular DTP vaccine by acellular DTP, and the role of mutations, in humans, as the main cause of rare adverse events, such as epileptic-like convulsions, triggered by pertussis vaccine. Data review: the main components related to toxic effects of cellular pertussis vaccines are the lipopolysaccharide of bacterial cell wall and pertussis toxin. The removal of part of lipopolysaccharide layer has allowed the creation of a safer cellular pertussis vaccine, with costs comparable to the traditional cellular vaccine, and which may be a substitute for the acellular vaccine. Conclusion: The new methodology introduced by Instituto Butantan allows for the development of a new safer pertussis vaccine with low LPS content (Plow), and the use of the lipopolysaccharide obtained in the process in the production of monophosphoryl lipid A. This component has shown potent adjuvant effect when administered together with influenza inactivated vaccine, making possible to reduce the antigen dose, enhancing the production capacity and lowering costs.

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Prophylactic and Therapeutic Vaccination Using Dendritic Cells Primed with Peptide 10 Derived from the 43-Kilodalton Glycoprotein of Paracoccidioides brasiliensis

Vaccination with peptide 10 (P10), derived from the Paracoccidioides brasiliensis glycoprotein 43 (gp43), induces a Th1 response that protects mice in an intratracheal P. brasiliensis infection model. Combining P10 with complete Freund's adjuvant (CFA) or other adjuvants further increases the peptide's antifungal effect. Since dendritic cells (DCs) are up to 1,000-fold more efficient at activating T cells than CFA, we examined the impact of P10-primed bone-marrow-derived DC vaccination in mice. Splenocytes from mice immunized with P10 were stimulated in vitro with P10 or P10-primed DCs. T cell proliferation was significantly increased in the presence of P10-primed DCs compared to the peptide. The protective efficacy of P10-primed DCs was studied in an intratracheal P. brasiliensis model in BALB/c mice. Administration of P10-primed DCs prior to (via subcutaneous vaccination) or weeks after (via either subcutaneous or intravenous injection) P. brasiliensis infection decreased pulmonary damage and significantly reduced fungal burdens. The protective response mediated by the injection of primed DCs was characterized mainly by an increased production of gamma interferon (IFN-gamma) and interleukin 12 (IL-12) and a reduction in IL-10 and IL-4 compared to those of infected mice that received saline or unprimed DCs. Hence, our data demonstrate the potential of P10-primed DCs as a vaccine capable of both the rapid protection against the development of serious paracoccidioidomycosis or the treatment of established P. brasiliensis disease.

Low Sensitivity of NS1 Protein Tests Evidenced during a Dengue Type 2 Virus Outbreak in Santos, Brazil, in 2010

In 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.

Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC

Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.

Pandemic unadjuvanted influenza A (H1N1) vaccine in dermatomyositis and polymyositis: Immunogenicity independent of therapy and no harmful effect in disease

The goal of the present study was to evaluate the influence of the influenza A H1N1/2009 vaccine on dermatomyositis/polymyositis (DM/PM) disease parameters and the potential deleterious effect of therapy on immune response. Thirty-seven DM and 21 PM patients (Bohan and Peter's criteria) were gender- and age-matched to 116 healthy controls. Seroprotection, seroconversion, the geometric mean titers (GMTs) and the factor increase (FI) in the GMTs were calculated. Disease safety was determined from a muscle enzyme analysis and the DM/PM scores [patient's visual analog scale (VAS), physician's VAS, manual muscle strength (MMT-8)] evaluated pre- and post-vaccination. The mean age (43.1 +/- 9.9 vs. 43.8 +/- 8.4 years, p = 0.607) and gender distribution (p = 1.00) were comparable between the patients and controls. After 21 days, seroconversion (p = 0.394), seroprotection (p = 0.08), GMT (p = 0.573) and the FI in the GMT (p = 0.496) were similar in both groups. The disease and muscle parameters remained stable throughout the study, including the creatine kinase (p = 0.20) and aldolase levels (p = 0.98), the physicians' VAS (p = 1.00), the patients' VAS (p = 1.00) and the MMT-8 (p = 1.00). Regarding the influence of treatment, the seroconversion rates were comparable between the controls and patients undergoing treatment with glucocorticoid (GC) (p = 0.969), GC >0.5 mg/kg/day (p = 0.395) and GC + immunosuppressors (p = 0.285). Vaccine-related adverse events were mild and similar in the DM/PM and control groups (p > 0.05). Our data support the administration of the pandemic influenza A H1N1/2009 vaccination in DM/PM, as we found no short-term harmful effects related to the disease itself and adequate immunogenicity in spite of therapy. Further studies are necessary to identify any long-term adverse effects in patients with these diseases.(c) 2012 Elsevier Ltd. All rights reserved.

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-1(19) and PvMSP-3 alpha(359-798)) and their relationship with hematological features in malaria patients from the Brazilian Amazon

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-1(19) and PvMSP-3 alpha(359-798). Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-1(19) was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3 alpha(359-798). Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3 alpha), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3 alpha(359-798) during natural infection. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Paracoccidioidomycosis vaccine

Paracoccidioidomycosis is a granulomatous pulmonary infection that is generally controlled by chemotherapy. The efficacy of treatment, however, is limited by the status of the host immune response. The inhibition of a Th-2 immunity or the stimulation of Th-1 cytokines generally increases the efficacy of antifungal drugs.(1) This has been achieved by immunization with an internal peptide of the major diagnostic antigen gp43 of Paracoccidioides brasiliensis. Peptide 10 (QTLIAIHTLAIRYAN) elicits an IFN-gamma rich Th-1 immune response that protects against experimental intratracheal infection by this fungus. The combination of chemotherapy with P10 immunization showed additive protective effect even after 30 d of infection or in anergic mice, rendering in general, increased production of IL-12 and IFN-gamma and reduction of IL-4 and IL-10. Immunotherapy with P10 even in the absence of simultaneous chemotherapy has been effective using various protocols, adjuvants, nanoparticles, P10-primed dendritic cells, and especially a combination of plasmids encoding the P10 minigene and IL-12. Gene therapy, in a long-term infection protocol succeeded in the virtual elimination of the fungus, preserving the lung structure, free from immunopathological side effects.

Immunogenicity of a meningococcal serogroup C conjugate vaccine in HIV-infected children, adolescents, and young adults

Children and adolescents infected with HIV typically have a lower response to immunization than do those in the general population. In most developed countries, meningococcal serogroup C conjugate vaccine is one of the recommended vaccines for such individuals. However, there have been no studies evaluating the antibody response to this vaccine in HIV-infected children, adolescents or young adults. In this study, we evaluated that response using serum bactericidal antibody (SBA) and enzyme-linked immunosorbent assay, comparing HIV-infected with non-HIV-infected patients, as well as analysing the occurrence of side effects. In non-responders, we assessed the antibody response to revaccination. This clinical trial involved 92 patients between 10 and 20 years of age: 43 HIV-infected patients (HIV+ group) and 49 non-HIV-infected patients (HIV- group). After one dose of the vaccine, 72.1% of the HIV+ group patients and 100% of the HIV- group patients were considered protected. Of the HIV+ group patients who received a second dose of the vaccine, only 40% acquired protection. Overall, 81.4% of the HIV+ group patients acquired protection (after one or two doses of the vaccine). Side effects occurred in 16.3% and 44% of the HIV+ group and HIV- group patients, respectively. Therefore, the meningococcal serogroup C conjugate vaccine proved to be safe and effective for use in HIV-infected children, adolescents, and young adults, although their antibody response was weaker than that shown by non-HIV-infected patients. This indicates the need to discuss changes to the immunization schedule for children, adolescents, and young adults infected with HIV, in order to ensure more effective protection against meningococcal disease. (c) 2012 Elsevier Ltd. All rights reserved.

Bacillus atrophaeus inactivated spores as a potential adjuvant for veterinary rabies vaccine

Rabies is a viral encephalitis, nearly always fatal, but preventable through vaccines. Rabid animal bite is the prime transmission act, while veterinary vaccination is one of the best strategies for rabies general prevention. Aluminum compounds and saponin are the commercial adjuvants used for this vaccine nowadays. Nevertheless, aluminum compounds can provoke undesired side effects and saponin has a narrow activity range without toxicity. B. atrophaeus inactivated spores (BAIS), with or without saponin, were then used as an alternative to boost the inactivated rabies virus response. BAIS was as effective as saponin in augmenting antibody titers, but combination of both adjuvants doubled the titers raised by them individually. The combined adjuvant formulation maintained viability for 21 months when stored at 4-8 degrees C. Overall, BAIS was demonstrated as a viable alternative to commercial adjuvants, while its combination with saponin resulted in even higher vaccine potency with good stability. (C) 2012 Elsevier Ltd. All rights reserved.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS = 0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (FST) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants. (C) 2011 Elsevier Ltd. All rights reserved.

HIV-1 induces NALP3-inflammasome expression and interleukin-1 beta secretion in dendritic cells from healthy individuals but not from HIV-positive patients

Objective: NALP3-inflammasome is an innate mechanism, alternative to type-1 interferon, which is able to recognize nucleic acids and viruses in the cytoplasm and to induce pro-inflammatory response. Here, we hypothesized the involvement of inflammasome in the early defense against HIV-1 and in the full maturation of dendritic cells: for this, we evaluated the response of dendritic cells pulsed with HIV-1 in terms of inflammasome activation in healthy donors. Moreover, inflammasome response to HIV was evaluated in HIV-infected individuals. Design and methods: Monocyte-derived dendritic cells isolated from 20 healthy individuals (HC-DC) and 20 HIV-1-infected patients (HIV-DC) were pulsed with alditrithiol-2-inactivated HIV-1. We then analyzed inflammasome genes expression and interleukin-1 beta (IL-1 beta) secretion. Results: In HC-DC, HIV-1 induced higher NLRP3/NALP3 mRNA expression compared with other inflammasome genes such as NALP1/NLRP1 or IPAF/NLRC4 (P < 0.001). This augmented expression was accompanied by CASP1-increased and IL1B-increased mRNA levels and by a significant increment of IL-1b secretion (P < 0.05). Otherwise, HIV-1 failed to activate inflammasome and cytokine production in HIV-DC. HIV-DC showed an increased NLRP3/NALP3 basal expression, suggesting a chronic inflammatory profile of patients' immune cells. Conclusion: HIV-1 was able to induce a NALP3-inflammasome response in healthy individuals, indicating that this inflammasome could play a role in the first steps of HIV-1 infection; the consequent inflammatory process may be important for directing host immune response against the virus and/or disease progression. HIV-DC seemed to be chronically activated, but unresponsive against pathogens. Our findings could be of interest considering the ongoing research about dendritic cell manipulation and therapeutic strategies for AIDS involving dendritic cell-based immune-vaccines. (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins