RESUMO
OBJECTIVES: Explore pediatric staff experiences administering the second influenza vaccine dose. STUDY DESIGN: Qualitative focus groups/interviews. METHODS: As part of the National Institutes of Health-funded Flu2Text randomized control trial of text message reminders for second influenza vaccine dose, we conducted seven focus groups and four individual interviews (n = 39 participants total) with clinicians and staff from participating practices from the American Academy of Pediatrics' Pediatric Research in Office Settings (PROS) Network. Of 37 participating practices, 10 were selected through stratified sampling of practices with highest (n = 5) and lowest (n = 5) randomized controlled trial effect sizes. A semi-structured discussion guide included questions that addressed parental, practice, and health system barriers/facilitators to second influenza vaccine dose administration. Using the Systems Model of Clinical Preventive Care as a conceptual framework, two investigators independently coded transcripts (Κ = 0.86, high agreement) with NVivo 12 Plus. Coding inconsistencies were resolved by consensus. RESULTS: Clinicians/staff reported that administering the second influenza vaccine dose in a season was more complex than other childhood vaccines. They highlighted parental uncertainty about the need for the second dose and the difficulty and inconvenience of bringing children back to the office as important barriers. Caregiver-staff relationships were perceived as helpful in getting children vaccinated with their second dose and vaccine reminders were seen as important cues-to-action. CONCLUSIONS: Ensuring receipt of two doses of the influenza vaccine in a given season presents unique challenges. Themes identified provide a framework for understanding opportunities to bolster second dose receipt, including explaining why two doses are needed, offering flexible hours for vaccination, and sending vaccine reminders.
RESUMO
In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.
Assuntos
Antígenos de Protozoários/imunologia , Macaca mulatta/imunologia , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Vacinas Antimaláricas/efeitos adversos , Proteína 1 de Superfície de Merozoito/efeitos adversosRESUMO
Several lines of evidence suggest that targeting pre-erythrocytic-stage parasites for malaria vaccine development can provide sterile immunity. The objectives of this study were (i) to evaluate preclinically the safety and immunogenicity of a new recombinant pre-erythrocytic-stage antigen, liver-stage antigen 1 (LSA1), in nonhuman primates; and (ii) to investigate the potential for immune interference between LSA1 and the leading malaria vaccine candidate, RTS,S, by comparing the immune responses after single-antigen vaccination to responses after simultaneous administration of both antigens at separate sites. Using a rhesus monkey model, we found that LSA1 formulated with the GlaxoSmithKline proprietary adjuvant system AS01B (LSA1/AS01B) was safe and immunogenic, inducing high titers of antigen-specific antibody and CD4+ T-cell responses, as monitored by the production of interleukin-2 and gamma interferon, using intracellular cytokine staining. RTS,S/AS01B vaccination was well tolerated and demonstrated robust antibody and moderate CD4+ T-cell responses to circumsporozoite protein (CSP) and HBsAg. Positive CD8+ T-cell responses to HBsAg were detected, whereas the responses to CSP and LSA1 were negligible. For both LSA1/AS01B and RTS,S/AS01B, no statistically significant differences were observed between individual and concurrent administration in the magnitude or duration of antibody and T-cell responses. Our results revealed that both pre-erythrocytic-stage antigens were safe and immunogenic, administered either separately or simultaneously to rhesus monkeys, and that no significant immune cross interference occurred with concurrent separate-site administration. The comparison of the profiles of immune responses induced by separate-site and single-site vaccinations with LSA1 and RTS,S warrants further investigation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Protozoários/imunologia , Lipídeo A/análogos & derivados , Macaca mulatta/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Saponinas/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Lipídeo A/administração & dosagem , Lipídeo A/farmacologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Saponinas/administração & dosagem , Fatores de TempoRESUMO
The Plasmodium falciparum circumsporozoite (PfCS) protein (aa 19-405) has been cloned and expressed in E. coli. The protein was purified in a two-step process that was rapid and reproducible. E. coli cells were grown to a high density before induction for 1 h. Cells were disrupted by high pressure microfluidization and the total bacterial protein solubilized in 6 M Gu-HCl. The protein was refolded while bound to Ni-NTA agarose by exchange of 6 M Gu-HCl for 8 M urea and then slow removal of the urea. The eluted protein was further purified on Q Sepharose Fast Flow using conditions developed to remove E. coli proteins and reduce endotoxin (to 10 EU/50 microg). Yield was 20 mg of PfCS protein from 10 g of wet cell paste. The final protein product bound to HepG2 liver cells in culture and inhibited the invasion of those cells by sporozoites in an ISI assay greater than 80% over control cultures when used at 10 microg/ml.
Assuntos
Escherichia coli/genética , Plasmodium falciparum/química , Proteínas de Protozoários/isolamento & purificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The Plasmodium vivax merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42) is a promising vaccine candidate antigen against the blood stage of the malarial parasite. We have developed a process for the production of this vaccine target, keeping in mind its use in human volunteers. A novel strain, Origami(DE3), of Escherichia coli with mutations in the glutathione and thioredoxin reductase genes yielded 60% more soluble PvMSP-1 p42 than the conventional E. coli BL21(DE3) strain. Recombinant PvMSP-1 p42 was purified to > or = 99% purity with a rapid two-step protocol designed for easy scaling up. The final product had a low endotoxin content and was stable in its lyophilized form. PvMSP-1 p42 was found to have the predicted primary and tertiary structures and consisted of a single conformer containing one free cysteine, as predicted. The product was recognized by conformational monoclonal antibodies against P. vivax MSP-1. Immunogenicity studies of PvMSP-1 p42 were carried out with two strains of mice and the adjuvants Montanide ISA51 and Montanide ISA720. Both formulations were found to induce high levels of immunoglobulin G1 (IgG1), IgG2b, and IgG2a antibodies along with low levels of IgG3. Lymphocytes from animals in all the PvMSP-1 p42-immunized groups showed proliferative responses upon stimulation with PvMSP-1 p42; the cytokines interleukin 2 (IL-2), gamma interferon, IL-4, and IL-10 were detected in the culture supernatants. These results indicate that PvMSP-1 p42 in combination with both of the adjuvants elicited cellular and humoral responses in mice.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Reagentes de Ligações Cruzadas , Dissulfetos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Ativação Linfocitária/imunologia , Manitol/análogos & derivados , Proteína 1 de Superfície de Merozoito/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ácidos Oleicos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinação , Vacinas Sintéticas/imunologiaAssuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Plasmodium vivax/metabolismo , Proteínas de Protozoários , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Baculoviridae/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/isolamento & purificação , Células Cultivadas , Sistema do Grupo Sanguíneo Duffy , Epitopos , Eritrócitos/metabolismo , Humanos , Immunoblotting , Insetos , Dados de Sequência Molecular , Plasmodium vivax/genética , Plasmodium vivax/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Vacinas Antimaláricas/genética , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Vacinas Sintéticas/genética , Sequência de Aminoácidos , Animais , Antígenos de Superfície/genética , Sequência de Bases , Primers do DNA/química , Genes de Protozoários , Vetores Genéticos , Células HeLa , Humanos , Macaca mulatta , Malária Falciparum/imunologia , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Vaccinia virusRESUMO
NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria.
Assuntos
Malária/prevenção & controle , Plasmodium berghei/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/farmacologia , Vacinas Sintéticas/farmacologia , Animais , Anticorpos Antiprotozoários/biossíntese , Humanos , Imunidade Celular , Malária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Vacinas Protozoárias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Subpopulações de Linfócitos T/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/farmacologia , Vacinas Sintéticas/genética , Vaccinia virus/genética , Vaccinia virus/imunologiaRESUMO
EBA-175, erythrocyte binding antigen 175, is a 175-kDa antigen of Plasmodium falciparum which has been shown to be involved in the recognition of erythrocytes by merozoites and may be involved in the process of erythrocyte invasion. Invasion of erythrocytes by Camp strain merozoites is inhibited by pre-treatment of red blood cells by EBA-175 from the heterologous strain, FCR-3. The sequence of the Camp strain has been published and we report here the sequence of the FCR-3 strain. The sequences are nearly identical except for a 423-bp segment in the FCR-3 strain, F-segment, that is not found in the Camp strain and a 342-bp segment, C-segment, present in the Camp strain but not in the FCR-3 strain. The locations of these two segments are different in Camp and FCR-3 EBA-175 genes and there is little DNA or amino acid sequence homology between them. The essentially dimorphic alleles, F-segment and C-segment, are conserved in all isolates examined to date. Evidence of genetic cross-over between the FCR-3 and the Camp EBA-175 genes was not observed in the analysis of a limited number of wild isolates. The continued study of the biological relevance of these sequence divergences in EBA-175 may further elucidate the sequence of events resulting in merozoite invasion of erythrocytes.