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Background Measles is a highly contagious viral disease. Vaccinated mothers transfer fewer antibodies during pregnancy, resulting in shortened infant immunity. Earlier primary vaccination might avert the gap in protection. Methods Healthy 5–7-month-old Danish infants were assigned in a 1:1 ratio to M-M-RVaxPro or placebo (solvent) in a double-blind, randomized trial between April 15, 2019 and November 1, 2021 (ClinicalTrials.gov NCT03780179, EudraCT 2016-001901-18). Eligibility criteria were birth weight >1000 g and gestational age ≥32 weeks. Immunogenicity was measured by plaque reduction neutralization test (PRNT) and IgG ELISA before intervention, four weeks after intervention and routine MMR. Reactogenicity data were collected for six weeks and measured by hazard ratios (HR). Findings 647 and 6540 infants participated in the immunogenicity and reactogenicity study, respectively; 87% and 99% completed follow-up. After early MMR, seroprotection rates (SPRs) were 47% (13%) in measles PRNT; 28% (2%), 57% (8%) in mumps and rubella IgG (placebo). For measles PRNT, geometric mean ratio was 4.3 (95% CI: 3.4–5.3) between randomization groups after intervention and 1.5 (95% CI: 1.3–1.9) after routine MMR. Reactogenicity was independent of randomization (HR, 1.0; 95% CI: 0.9–1.1). Severe adverse events occurred in 25 infants (HR, 1.8; 95% CI: 0.8–4.0); none deemed vaccine related. Interpretation Early MMR elicited low SPRs but did not negatively impact short-term responses to a subsequent MMR. MMR at 5–7 months was safe and not associated with higher rates of reactogenicity than placebo., Background: Measles is a highly contagious viral disease. Vaccinated mothers transfer fewer antibodies during pregnancy, resulting in shortened infant immunity. Earlier primary vaccination might avert the gap in protection. Methods: Healthy 5–7-month-old Danish infants were assigned in a 1:1 ratio to M-M-RVaxPro or placebo (solvent) in a double-blind, randomized trial between April 15, 2019 and November 1, 2021 (ClinicalTrials.gov NCT03780179, EudraCT 2016-001901-18). Eligibility criteria were birth weight >1000 g and gestational age ≥32 weeks. Immunogenicity was measured by plaque reduction neutralization test (PRNT) and IgG ELISA before intervention, four weeks after intervention and routine MMR. Reactogenicity data were collected for six weeks and measured by hazard ratios (HR). Findings: 647 and 6540 infants participated in the immunogenicity and reactogenicity study, respectively; 87% and 99% completed follow-up. After early MMR, seroprotection rates (SPRs) were 47% (13%) in measles PRNT; 28% (2%), 57% (8%) in mumps and rubella IgG (placebo). For measles PRNT, geometric mean ratio was 4.3 (95% CI: 3.4–5.3) between randomization groups after intervention and 1.5 (95% CI: 1.3–1.9) after routine MMR. Reactogenicity was independent of randomization (HR, 1.0; 95% CI: 0.9–1.1). Severe adverse events occurred in 25 infants (HR, 1.8; 95% CI: 0.8–4.0); none deemed vaccine related. Interpretation: Early MMR elicited low SPRs but did not negatively impact short-term responses to a subsequent MMR. MMR at 5–7 months was safe and not associated with higher rates of reactogenicity than placebo. Funding: Innovation Fund Denmark.

The adaptive immune system involves many complex communication pathways that are essential for providing an adequate immune response to foreign pathogens. So far, multiple platforms and tools have been developed to study the adaptive immune system, and the discovery of powerful genetic tools like CRISPR/Cas9 and a myriad of vectors that allow for introduction of complexes like CRISPR/Cas9 into the cells. The development of viral-based vectors for the delivery of genetic material into immune cells, such as lentivirus-based vectors have also allowed for greater study of the immune system with the ability to target specific cells by pseudotyping the lentivirus with other viral glycoproteins. This master thesis project will cover what is currently known about lentiviral and CRISPR/Cas9 technologies, as well as exploring options for using lentiviral vectors to potentially carry the CRISPR/Cas9 system into specific immune cells, with the primary goal of creating a CRISPR/Cas9-mediated knock out in B-cells using a lentiviral vector with specific tropism for B- cells, as well as using transiently expressed CRISPR/Cas9 in a platform that normally does not support transient expression of inserts. Finally, this thesis will discuss approaches to potentially improve the lentiviral vector’s ability to infect B-cells, which has notoriously been elusive when it comes to generating lentiviral vectors with specific tropism.

The bacterium Coxiella burnetii can cause the disease Q-fever in a wide range of animal hosts. Ruminants, including sheep, are thought to play a pivotal role in the transmission of C. burnetii to humans; however, the only existing livestock vaccine, namely, Coxevac® (Ceva Animal Health Ltd., Libourne, France), a killed bacterin vaccine based on phase I C. burnetii strain Nine-Mile, is only approved for use in goats and cattle. In this study, a pregnant ewe challenge model was used to determine the protective effects of Coxevac® and an experimental bacterin vaccine based on phase II C. burnetii against C. burnetii challenge. Prior to mating, ewes (n = 20 per group) were vaccinated subcutaneously with either Coxevac®, the phase II vaccine, or were unvaccinated. A subset of pregnant ewes (n = 6) from each group was then challenged 151 days later (~100 days of gestation) with 106 infectious mouse doses of C. burnetii, Nine-Mile strain RSA493. Both vaccines provided protection against C. burnetii challenge as measured by reductions in bacterial shedding in faeces, milk and vaginal mucus, and reduced abnormal pregnancies, compared to unvaccinated controls. This work highlights that the phase I vaccine Coxevac® can protect ewes against C. burnetii infection. Furthermore, the phase II vaccine provided comparable levels of protection and may offer a safer and cost-effective alternative to the currently licensed vaccine.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of infection. Vaccination is a key control measure and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin preparations. Subunit vaccines based on recombinant proteins offer significant advantages, as they can be designed to reduce reactogenicity and can be co-designed with defined antigen serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 ORFs in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different C. burnetii exposure statuses. However, C. burnetii exposure did result in more uniform seroreactivity across species. Through the implementation of three different vaccine candidate methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic, out of which 65 have been previously described. This is the first study to investigate seroreactivity against the entire C. burnetii genome presented as overlapping linear peptides and provides the basis for se

The apicomplexan parasite Theileria parva is the causative agent of East Coast fever, usually a fatal disease for cattle, which is prevalent in large areas of eastern, central, and southern Africa. Protective immunity against T. parva is mediated by CD8+ T cells, with CD4+ T-cells thought to be important in facilitating the full maturation and development of the CD8+ T-cell response. T. parva has a large proteome, with >4000 protein-coding genes, making T-cell antigen identification using conventional screening approaches laborious and expensive. To date, only a limited number of T-cell antigens have been described. Novel approaches for identifying candidate antigens for T. parva are required to replace and/or complement those currently employed. In this study, we report on the use of immunopeptidomics to study the repertoire of T. parva peptides presented by both BoLA-I and BoLA-DR molecules on infected cells. The study reports on peptides identified from the analysis of 13 BoLA-I and 6 BoLA-DR datasets covering a range of different BoLA genotypes. This represents the most comprehensive immunopeptidomic dataset available for any eukaryotic pathogen to date. Examination of the immunopeptidome data suggested the presence of a large number of coprecipitated and non-MHC-binding peptides. As part of the work, a pipeline to curate the datasets to remove these peptides was developed and used to generate a final list of 74 BoLA-I and 15 BoLA-DR-presented peptides. Together, the data demonstrated the utility of immunopeptidomics as a method to identify novel T-cell antigens for T. parva and the importance of careful curation and the application of high-quality immunoinformatics to parse the data generated.

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue-resident memory (TRM) CD8+ T-cells is of great interest, but limited data exist on TRM-like cells within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8+ T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and a transcriptionally TRM-like profile distinct from blood CD8+ T-cells. In PLWH, CD8+ TRM-like cells are expanded and adopt a more cytolytic, activated, and exhausted phenotype not reversed by antiretroviral therapy (ART). This phenotype was enhanced in HIV-specific CD8+ T-cells from tonsils compared to matched blood suggesting a higher antigen burden in tonsils. Single-cell transcriptional and clonotype resolution showed that these HIV-specific CD8+ T-cells in the tonsils express heterogeneous signatures of T-cell activation, clonal expansion, and exhaustion ex-vivo. Interestingly, this signature was absent in a natural HIV controller, who expressed lower PD-1 and CXCR5 levels and reduced transcriptional evidence of T-cell activation, exhaustion, and cytolytic activity. These data provide important insights into lymphoid tissue-derived HIV-specific CD8+ TRM-like phenotypes in settings of HIV remission and highlight their potential for immunotherapy and targeting of the HIV reservoirs.