Heterogeneity in Plasmodium falciparum whole sporozoite vaccine induced humoral immune responses and protection in African volunteers: The role of age, human pegivirus and human immunodeficiency virus co-infections.

Malaria, a vector borne disease caused by Plasmodium species remains a major public health problem especially in sub-Saharan Africa. In 2018 alone, there were an estimated 228 million clinical cases and 405,000 deaths attributed to malaria. New tools such as efficacious vaccines, better drugs and diagnostics are needed to supplement the current malaria control tools that rest mainly on vector control measures. Clinical trials in malaria naïve volunteers have demonstrated high level of sterile vaccine induced protection in healthy individuals immunized with live, metabolically active, irradiation attenuated purified sporozoites (PfSPZ Vaccine) or live non-attenuated purified sporozoites given under chloroquine chemoprophylaxis (PfSPZ-CVac). Immunogenicity and protective efficacy against malaria induced by these whole sporozoite based vaccines varies widely between European/US versus African volunteers. Interestingly, variations in protection and immunogenicity of these malaria vaccines have been observed among African volunteers residing in different malaria endemic regions of East and West Africa. These different outcomes could be linked to levels of malaria pre-exposure and co-infections at the time of vaccination. Until recently safety, tolerability, immunogenicity and protective efficacy of PfSPZ Vaccine in immunocompromised populations including individuals infected with the Human Immunodeficiency Virus (HIV) was unknown. Given the high geographical overlap of HIV and malaria, an effective malaria vaccine deployed in this population could possibly support the long-term objective of regional elimination of malaria by using mass vaccination. Furthermore, the role of asymptomatic, under researched, highly prevalent viruses like Human Pegivirus (HPgV-1) circulating in Sub-Saharan Africa on malaria pathogenesis and vaccination outcomes remains elusive. The aims of this PhD thesis include: 1) Evaluate the safety, tolerability, efficacy, and immunogenicity of the PfSPZ Vaccine in different populations and age groups residing in malaria endemic countries. 2) Compare the safety, tolerability, efficacy, and immunogenicity of two different whole sporozoite based vaccination approaches, PfSPZ Vaccine and PfSPZ-CVac in Equatorial Guinean adults. 3) The unbiased assessment of PfSPZ Vaccine induced humoral immunity using protein microarrays probed with serum samples of HIV positive and HIV negative volunteers to understand immune status before vaccination, immuno-dominance of vaccine induced antibody targets, and distinct antibody profiles that might be associated with vaccine-induced protection. 4) Investigate the interaction of chronic HPgV-1 co-infection on PfSPZ Vaccine induced humoral immunity and protection against homologous CHMI. These aims are structured around 6 manuscripts presented in this PhD thesis. Manuscript 1-3: Safety, immunogenicity, and efficacy of radiation attenuated whole sporozoite vaccine (PfSPZ Vaccine) in African populations of different ages In this chapter, we include the outcomes of clinical trials conducted in Bagamoyo, Tanzania. These trials for the first time i) evaluated the safety, immunogenicity and efficacy against homologous CHMI of irradiation attenuated purified Plasmodium falciparum sporozoites (PfSPZ Vaccine) in adult volunteers; ii) tested increasing dosages of PfSPZ Vaccine in different age groups including adults, adolescents, children and infants. We found PfSPZ vaccine to be safe and well tolerated and that vaccine inoculation by intravenous inoculation is well accepted even in younger age groups. Protective efficacy varied in the different trials leading to the identification of a vaccine regimen of 9x105PfSPZ per dose as suitable for further development. PfSPZ Vaccine induce immune responses, both cellular and humoral, were age dependent with infants mounting no measurable malaria specific cellular immunity in peripheral blood. Surprisingly, compared to other age groups, older children and adolescents mounted higher cellular and humoral immune responses. These findings are relevant for further optimization of PfSPZ vaccine regimen that might need to be adapted to different age groups to optimize vaccine induced protection. As an extension of these trials, we have compared PfSPZ Vaccine safety, immunogenicity and efficacy in HIV positive versus HIV negative volunteers. We observed marked differences in PfSPZ Vaccine induced efficacy between HIV positive (0%) and HIV negative individuals (80%) undergoing homologous CHMI(Manuscript in preparation). Manuscript 4: Immunogenicity and protective efficacy of radiation-attenuated and chemo-attenuated PfSPZ vaccines in Equatoguinean adults This work describes the outcome of a first time side-by-side comparison of two whole sporozoite based vaccine approaches (PfSPZ Vaccine and PfSPZCVac) in malaria pre-exposed individuals of Equatorial Guinea. We evaluated PfSPZ Vaccine dosages (2.7 X106) given three times at 8-week interval and PfSPZ CVac dose (1X105) given three times at 4 weeks interval. Homologous CHMI was employed for assessment of vaccine efficacy. Both approaches were safe and well tolerated in malaria pre-exposed individuals but the immunogenicity and protective efficacy differed. Vaccine efficacy was lower in the PfSPZ Vaccine group (27%) compared to the PfSPZ CVac group (55%), despite induction of about 2.9 times higher antibody titres against the circumsporozoite protein in the PfSPZ Vaccine group prior to CHMI. These results highlight the potential involvement of different protective immune mechanisms induced by each of the two whole sporozoite vaccines approaches and the effect of malaria pre-exposure on pfSPZ CVac vaccine induced efficacy in comparison to malaria naïve volunteers. We show that induction of high antibody titres against the circumsporozoite protein does not correlate with protection since no difference was observed between CHMI protected and non-protected volunteers. Manuscript 5: HIV-1 positive and HIV-1 negative Tanzanian adults undergoing whole irradiation attenuated Plasmodium falciparum sporozoite vaccination mount antibody responses targeting the circumsporozoite protein and merozoite surface protein 5 In this manuscript, we investigated antibody profiles binding to 262 pre-selected antigens of Pf before and after vaccination as well as after homologus CHMI. We aimed to identify antibody profiles that might explain the observed poor vaccine induced protection in HIV positive individuals. We found a lower - albeit not statistically significant - antigen breadth in HIV positive volunteers at baseline before first vaccine inoculation. Immunization with PfSPZ Vaccine induced IgG and IgM isotypes specific for the Merozoite surface protein 5 (PfMSP 5) and the circumsporozoite protein (PfCSP) regardless of HIV infection status. Interestingly, volunteers displayed a highly personalized IgG and IgM immune profiles targeting Pf antigens before vaccination and these remained unchanged after PfSPZ vaccination confirming our previous results of antigenic imprinting in malaria. Manuscript 6: Role of Pegivirus infections in whole Plasmodium falciparum sporozoite vaccine induced humoral immunity and controlled human malaria infections in African volunteers In this study, we wanted to understand the role of human pegivirus infections in East and Western African adult volunteers and its impact on PfSPZ Vaccine induced humoral immune responses and homologous CHMI. We found HPgV-1 to be highly prevalent in our volunteers (29.2%) with circulating genotypes 1, 2 and 5 as described in other African settings. HPgV-1 infection did not alter PfSPZ vaccine induced antibody responses and parasite multiplication rates during CHMI. However, a higher proportion of individuals were protected against homologous CHMI that had ongoing, active human pegivirus infections. Significantly higher serum concentrations of IL-2 and IL-17A were measured in HPgV-1 positive volunteers likely indicating chronic activation of the immune system. CHMI was safe and well tolerated in HPgV-1 positive individuals since the viremia did not change upon acute asexual blood stage parasitemia. These results highlight the potential impact of chronic, asymptomatic viral infections on PfSPZ vaccine efficacy that needs confirmation in larger cohorts and in field studies of naturally occurring malaria infections

Red blood cell indices and\ud Prevalence of Hemoglobinopathies and Glucose 6 Phosphate Dehydrogenase Deficiencies in Male Tanzanian Residents of Dar es Salaam

Hemoglobinopathies, disorders of hemoglobin structure and production, are one of the most common monogenic disorders in humans. Glucose 6 phosphate dehydrogenase deficiency (G6PD) is an inherited enzymopathy resulting in increased oxygen stress susceptibility of red blood cells. The distributions of these genetic traits in populations living in tropical and subtropical regions where malaria has been or is still present are thought to result from survival advantage against severe life threatening malaria disease. 384 male Tanzanian volunteers residing in Dar es Salaam were typed for G6PD, sickle cell disease and α-thalassemia. The most prominent red blood cell polymorphism was heterozygous α+-thalassemia (37.8%), followed by the G6PD(A) deficiency (16.4%), heterozygous sickle cell trait (15.9%), G6PD(A-) deficiency (13.5%) and homozygous α+-thalassemia (5.2%). 35%, 45%, 17% and 3% of these volunteers were carriers of wild type gene loci, one, two or three of these hemoglobinopathies, respectively. We find that using a cut off value of 28.6 pg. for mean corpuscular hemoglobin (MCH), heterozygous α+-thalassemia can be predicted with a sensitivity of 84% and specificity of 72% in this male population. All subjects carrying homozygous α+-thalassemia were identified based on their MCH value < 28.6 pg

Mixed Th1 and Th2 Mycobacterium tuberculosis-specific CD4 T cell responses in patients with active pulmonary tuberculosis from Tanzania.

Mycobacterium tuberculosis (Mtb) and helminth infections elicit antagonistic immune effector functions and are co-endemic in several regions of the world. We therefore hypothesized that helminth infection may influence Mtb-specific T-cell immune responses. We evaluated the cytokine profile of Mtb-specific T cells in 72 individuals with pulmonary TB disease recruited from two Sub-Saharan regions with high and moderate helminth burden i.e. 55 from Tanzania (TZ) and 17 from South Africa (SA), respectively. We showed that Mtb-specific CD4 T-cell functional profile of TB patients from Tanzania are primarily composed of polyfunctional Th1 and Th2 cells, associated with increased expression of Gata-3 and reduced expression of T-bet in memory CD4 T cells. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by single IFN-γ and dual IFN-γ/TNF-α and associated with TB-induced systemic inflammation and elevated serum levels of type I IFNs. Of note, the proportion of patients with Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ. It is likely that the underlying helminth infection and possibly genetic and other unknown environmental factors may have caused the induction of mixed Th1/Th2 Mtb-specific CD4 T cell responses in patients from TZ. Taken together, these results indicate that the generation of Mtb-specific CD4 and CD8 T cell responses may be substantially influenced by environmental factors in vivo. These observations may have major impact in the identification of immune biomarkers of disease status and correlates of protection

Los extranjeros en el marco de la normativa laboral de la Unión Europea

La acción de la Unión Europea en la gestión de flujos migratorios ha estado, en los últimos años, encaminada a conseguir la armonización de las políticas de inmigración de sus Estados miembros, tarea que hasta ahora ha sido ardua y complicada, pues ello significa atravesar las fronteras de la soberanía nacional de cada país, y es este el factor preponderante por el cual actualmente no existe una política europea común en este ámbito. En este sentido, la política de inmigración tiene su piedra angular en la admisión de inmigrantes económicos y por tanto es necesario abordarla a nivel europeo en el contexto del desarrollo progresivo de una política común de inmigración coherente, una responsabilidad que reside en la cooperación de todos los Estados miembros. Así, el presente trabajo es un acercamiento a los instrumentos jurídicos existentes a nivel de la UE y a la situación actual.Graduado o Graduada en Relaciones Laborales y Recursos Humanos por la Universidad Pública de NavarraLan Harremanetan eta Giza Baliabideetan Graduatua Nafarroako Unibertsitate Publikoa

Red blood cell indices and prevalence of hemoglobinopathies and glucose 6 phosphate dehydrogenase deficiencies in male Tanzanian residents of Dar es Salaam

Hemoglobinopathies, disorders of hemoglobin structure and production, are one of the most common monogenic disorders in humans. Glucose 6 phosphate dehydrogenase deficiency (G6PD) is an inherited enzymopathy resulting in increased oxygen stress susceptibility of red blood cells. The distributions of these genetic traits in populations living in tropical and subtropical regions where malaria has been or is still present are thought to result from survival advantage against severe life threatening malaria disease. 384 male Tanzanian volunteers residing in Dar es Salaam were typed for G6PD, sickle cell disease and α-thalassemia. The most prominent red blood cell polymorphism was heterozygous α(+)-thalassemia (37.8%), followed by the G6PD(A) deficiency (16.4%), heterozygous sickle cell trait (15.9%), G6PD(A-) deficiency (13.5%) and homozygous α(+)-thalassemia (5.2%). 35%, 45%, 17% and 3% of these volunteers were carriers of wild type gene loci, one, two or three of these hemoglobinopathies, respectively. We find that using a cut off value of 28.6 pg. for mean corpuscular hemoglobin (MCH), heterozygous α(+)-thalassemia can be predicted with a sensitivity of 84% and specificity of 72% in this male population. All subjects carrying homozygous α(+)-thalassemia were identified based on their MCH value < 28.6 pg

Immune system development varies according to age, location, and anemia in African children

Children from low- and middle-income countries, where there is a high incidence of infectious disease, have the greatest need for the protection afforded by vaccination, but vaccines often show reduced efficacy in these populations. An improved understanding of how age, infection, nutrition, and genetics influence immune ontogeny and function is key to informing vaccine design for this at-risk population. We sought to identify factors that shape immune development in children under 5 years of age from Tanzania and Mozambique by detailed immunophenotyping of longitudinal blood samples collected during the RTS,S malaria vaccine phase 3 trial. In these cohorts, the composition of the immune system is dynamically transformed during the first years of life, and this was further influenced by geographical location, with some immune cell types showing an altered rate of development in Tanzanian children compared to Dutch children enrolled in the Generation R population-based cohort study. High-titer antibody responses to the RTS,S/AS01E vaccine were associated with an activated immune profile at the time of vaccination, including an increased frequency of antibody-secreting plasmablasts and follicular helper T cells. Anemic children had lower frequencies of recent thymic emigrant T cells, isotype-switched memory B cells, and plasmablasts; modulating iron bioavailability in vitro could recapitulate the B cell defects observed in anemic children. Our findings demonstrate that the composition of the immune system in children varies according to age, geographical location, and anemia status

Demographic and clinical data.

<p>Demographic and clinical data.</p

<i>Mtb</i>-specific CD4 T cells from TB patients from TZ have mixed Th1/Th2 cytokine profile.

<p>(<b>A</b>) Representative flow cytometry profile of <i>Mtb</i>-specific CD4 T cells producing IFN-γ, IL-4/5/13, TNF-α and/or IL-2 of one TB patient from SA (#08), one TB patients from TZ (#60062) and one <i>Mtb</i>/helminth co-infected patient from TZ (#60031). Cytokine profiles of CD4 T cells stimulated with SEB (positive control) or left unstimulated (negative control) are also shown. (<b>B</b>) Percentage of <i>Mtb</i>-specific CD4 T cells producing TNF-α, IFN-γ, IL-2 or IL-4/5/13 of TB patients from SA (n = 17), TB patients from TZ (n = 25) and <i>Mtb</i>/helminth co-infected patients from TZ (n = 30). (<b>C</b>) Proportion of <i>Mtb</i>-specific CD4 T-cell responses producing IFN-γ, IL-4/5/13, TNF-α and/or IL-2 of TB patients from SA (n = 17), TB patients from TZ (n = 25) and <i>Mtb</i>/helminth co-infected patients from TZ (n = 30). All the possible combinations of the responses are shown on the x axis and the percentage of the functionally distinct cell populations within the <i>Mtb</i>-specific CD4 T-cell populations are shown on the y axis. Responses are grouped and color-coded on the basis of the number of functions. The pie chart summarizes the data, and each slice corresponds to the fraction of <i>Mtb</i>-specific CD4 T cell response with a given number of functions within the responding CD4 T-cell population. Bars correspond to the fractions of different functionally distinct CD4 T-cell populations within the total CD4 T cells. Red arcs correspond to IL-4/5/13-producing CD4 T-cell populations. (<b>D</b>) Levels of IFN-γ, TNF-α, IL-10, IL-2, IL-4, IL-5, IL-13, IL-17A and IL-17F produced in <i>Mtb</i>-stimulated culture supernatants of TB patients from SA (n = 12), TB patients from TZ (n = 21) and <i>Mtb</i>/helminth co-infected patients from TZ (n = 29) assessed by luminex assay. TB patients were color coded (<b>B</b> and <b>D</b>); TB patients from SA, blue; TB patients from TZ, red and <i>Mtb</i>/helminth co-infected patients, green. Red stars indicate statistical significance. Statistical significance (* = <i>P</i><0.05) was calculated using one way Anova Kruskal-Wallis test followed by a Mann-Whitney test (<b>B</b> and <b>D</b>). Statistical analyses of the global cytokine profiles (pie charts, <b>C</b>) were performed by partial permutation tests using the SPICE software as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005817#pntd.0005817.ref097" target="_blank">97</a>].</p

Memory CD4 T cells of TB patients from TZ harbor increased Gata-3 and reduced T-bet expression.

<p>(<b>A</b>) Representative flow cytometry profile of memory (CD45RA^-) CD4 T cells (red dots) isolated from one TB patient from SA (#03), one TB patient (#60037) and one <i>Mtb</i>/helminth co-infected patient (#60057) from TZ expressing Gata-3 and/or T-bet. Gates were set using naïve (CD45RA⁺) CD4 T cells and CD8 T cells from each individual. Gata-3 and T-bet expression of individual CD8 T-cell are also shown (black dots). Percentage of memory (CD45RA^-) CD4 T cells isolated from TB patients from SA (n = 12), TB patients (n = 14) and <i>Mtb</i>/helminth co-infected patients (n = 13) from TZ expressing Gata-3 (<b>B</b>) or T-bet^high (<b>C</b>). (<b>D</b>) Correlation between the percentage of IFN-γ-producing <i>Mtb</i>-specific CD4 T cells and the percentage of memory CD4 T cells expressing T-bet^high of TB patients from SA (n = 12), TB (n = 14) and <i>Mtb</i>/helminth co-infected patients from TZ (n = 13). (<b>E</b>) Correlation between the percentage of IL-4/5/13-producing <i>Mtb</i>-specific CD4 T cells and the percentage of memory CD4 T cells expressing Gata-3 of TB patients from SA (n = 12), TB (n = 14) and <i>Mtb</i>/helminth co-infected patients from TZ (n = 13). TB patients were color coded (<b>B</b>-<b>E</b>); TB patients from SA, blue; TB patients from TZ, red and <i>Mtb</i>/helminth co-infected patients, green. Red stars indicate statistical significance. Statistical significance (* = <i>P</i><0.05) was calculated using one way Anova Kruskal-Wallis test followed by a Mann-Whitney test (<b>B</b> and <b>C</b>) or Spearman rank test for correlation (<b>D</b> and <b>E</b>).</p

Reduced proportion of patients with detectable <i>Mtb</i>-specific CD8 T cells in <i>Mtb</i>/helminth co-infected patients.

<p>(<b>A</b>) Proportion of TB patients with detectable <i>Mtb</i>-specific CD8 T cells of TB and <i>Mtb</i>/helminth patients from TZ (n = 12 and 7, respectively). (<b>B</b>) Percentage of <i>Mtb</i>-specific CD8 T-cell responses producing TNF-α, IFN-γ, IL-2 and/or perforin of TB patients (n = 12) and <i>Mtb</i>/helminth patients (n = 7) from TZ. TB patients were color coded; TB patients from TZ, red and <i>Mtb</i>/helminth co-infected patients, green. (<b>C</b>) Functional profile of <i>Mtb</i>-specific CD8 T cells of TB patients and <i>Mtb</i>/helminth co-infected patients from TZ. Red and green circles represent the proportion of <i>Mtb</i>-specific CD8 T cells producing TNF-α, IFN-γ, IL-2 and/or perforin of TB and <i>Mtb</i>/helminth patients from TZ, respectively (<b>C</b>). Red arcs identify perforin producing cell populations (<b>C</b>). Red stars indicate statistical significance (* = <i>P</i><0.05). Statistical significance (<i>P</i><0.05) was calculated using Chi square test (<b>A</b>) and Mann-Whitney test (<b>B</b>). Statistical analyses of the global cytokine profiles (pie charts, <b>C</b>) were performed by partial permutation tests using the SPICE software as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005817#pntd.0005817.ref097" target="_blank">97</a>].</p