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Assessing the Immunogenicity of GTU®-based HIV-1 Multigene DNA Vaccines in Murine Models

Väitöskirjatyössä arvioitiin alun perin Tampereen yliopistossa ja myöhemmin FIT Biotech Oy:ssä kehitetyn HIV-1 rokotteen tehoa ja kykyä synnyttää HIV-spesifinen suojaava immuunivaste hiirimalleissa. Terapeuttisen HIV-1 rokotteen tarkoituksena on herättää immuunipuolustus tuhoamaan elimistön HI-viruksen infektoimia soluja. Rokotteen osoitettiin tehokkaasti aktivoivan antigeenispesifisiä tappajasoluja ja herättävän suojaavan immuunivasteen kokeellisissa hiirimalleissa. Kehitetty geneettinen rokote, GTU®-MultiHIV, muodostaa multiantigeenin eli yhdistelmäproteiinin, joka koostuu HIV-1 Rev, Nef, Tat ja Gag p17/p24 -proteiineista ja T-solujen epitooppialueista. MultiHIV-proteiini tuotetaan elimistössä GTU® -vektorista, jonka on osoitettu muodostavan antigeeniä pidempikestoisesti ja tehokkaammin kuin perinteisten rokotevektoreiden. GTU®-MultiHIV immunisoinnin osoitettiin synnyttävän rokotespesifisen solu- ja vasta-ainevälitteisen immuunivasteen jokaista vektorin tuottamaa antigeeniä kohtaan. Immuunivasteen herättämisen osoitettiin olevan immunisaatioreitistä ja käytetystä annostuksesta riippuvainen, ihonsisäinen immunisointi todettiin lihaksensisäistä immunisaatiota tehokkaammaksi. Soluvälitteistä immuunivastetta arvioitiin immunisoitujen hiirien lymfosyyttien antigeenispesifisellä gamma interferonin erityksellä. HIV-spesifisen immuunivasteen suojatehoa arvioitiin kahdessa kokeellisessa hiirimallissa, joista ensimmäisessä mallissa arvioitiin immunisoinnin kykyä estää MultiHIV-antigeeniä kantavien kasvainsolujen lisääntymistä. Toisessa mallissa saatiin viitteitä rokotteen suojatehosta eri HIV-1 kantoja vastaan käyttämällä hiirien pseudovirusaltistukseen HIV-1 kantoja, jotka poikkesivat rokotteen HIV-1 antigeenien kannoista. Lopuksi tutkittiin dendriittisolujen oleellista merkitystä immuunivasteen syntymisessä GTU®-MultiHIV DNA immunisoinnin jälkeen uutta lähestymistapaa käyttäen.HIV-1 vaccine development has proven an extremely challenging task, largely related to the highly variable nature of the virus which generates constantly new variants able to escape the immune system surveillance and lack of correlates of protection. DNA vaccines have the potential to encode multiple viral antigens, thereby eliciting immune responses that could lead to improved containment of the HIV-1 virus in a relatively safe way. Furthermore, the endogenous synthesis of the plasmid encoded antigen mimics the viral replication and enables the antigen presentation in a natural way for immune system cells. The first aim of this study was to evaluate the immunogenicity of the HIV-1 multigene DNA plasmid vaccine, encoding for Rev, Nef, Tat, p17, p24 and selected T cell epitopes of HIV-1 pol and env in mice. GTU® vector encoding the multigene is an advanced expression vector resulting in higher expression level and longer maintenance of the plasmid in dividing cells compared to conventional DNA plasmids. In the first part of the work, we demonstrated that GTU®-MultiHIV DNA induces cellular and humoral immune responses in mice directed to all components of the HIV-1 multigene. Delivery route and DNA dose used were shown to be major determinants for the efficiency of the immunization. Biolistic gene gun delivery induced strong immune responses with very low DNA doses, whereas intradermal and intramuscular administrations were dependent on high DNA doses. The induced cellular immune responses as measured by IFN-gamma secretion were shown to correlate with cytotoxic T cell activity in vitro and in vivo. To evaluate the protective efficacy of the HIV-1 DNA vaccine induced immune responses, we developed a novel tumor challenge model. We showed that HIV-1 specific cellular immune responses were able to significantly delay the growth of the HIV-1 antigen expressing tumor, thereby demonstrating the cytotoxic activity of the induced T lymphocytes, which is an important characteristic of HIV-1 vaccine. Furthermore, the HIV-1 specific T cells activated by immunization were shown to efficiently clear the HIV-1/MuLV infected cells used for the challenge in another experimental challenge model. Indication of cross-clade protection was demonstrated by evaluating the protection induced by immunization with a multiclade specific plasmid cocktail, containing antigens derived from HIV-1 strains A C and F-H and subsequently using different HIV-1 subtypes for the challenge. Finally, we briefly addressed the significant role of dendritic cells in eliciting immune responses by GTU®-MultiHIV DNA immunization

Parenterally Administered Norovirus GII.4 Virus-Like Particle Vaccine Formulated with Aluminum Hydroxide or Monophosphoryl Lipid A Adjuvants Induces Systemic but Not Mucosal Immune Responses in Mice

Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH)3) or monophosphoryl lipid A (MPLA) would induce mucosal antibodies in mice. Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)3 and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses

Development of T cell immunity to norovirus and rotavirus in children under five years of age

Most of the research effort to understand protective immunity against norovirus (NoV) has focused on humoral immunity, whereas immunity against another major pediatric enteric virus, rotavirus (RV), has been studied more thoroughly. The aim of this study was to investigate development of cell-mediated immunity to NoV in early childhood. Immune responses to NoV GI.3 and GII. 4 virus-like particles and RV VP6 were determined in longitudinal blood samples of 10 healthy children from three months to four years of age. Serum IgG antibodies were measured using enzyme-linked immunosorbent assay and production of interferon-gamma by peripheral blood T cells was analyzed by enzyme-linked immunospot assay. NoV-specific T cells were detected in eight of 10 children by the age of four, with some individual variation. T cell responses to NoV GII.4 were higher than those to GI.3, but these responses were generally lower than responses to RV VP6. In contrast to NoV-specific antibodies, T cell responses were transient in nature. No correlation between cell-mediated and antibody responses was observed. NoV exposure induces vigorous T cell responses in children under five years of age, similar to RV. A role of T cells in protection from NoV infection in early childhood warrants further investigation.Peer reviewe

Development and maturation of norovirus antibodies in childhood

The burden of norovirus (NoV) gastroenteritis is substantial in young children. Maternal antibodies are thought to protect a child from NoV infection in early infancy but subsequent development of NoV-specific protective immunity in children is still largely unexplored. We have determined NoV-specific antibody seroconversion to GII.4 virus-like particles as an indicator of NoV infection in two children prospectively followed from birth to eight years of age. Blocking activity and affinity maturation of maternal and serum IgG antibodies were evaluated. Our results show that multiple infections occur in children up to eight years of age. The titer, blocking activity and avidity of maternal antibodies determined susceptibility of an infant to NoV infection. NoV GII.4-specific antibodies with high blocking potential and avidity were developed at two to three years of age and were retained throughout the follow-up. Subsequent NoV infections may have contributed to the duration of protective NoV-specific immune responses that lasted for several years. This study adds to current understanding of the duration of passive protection by maternal antibodies and the duration and quality of acquired immunity following primary and subsequent NoV infections in infants and young children, who are the main target group for NoV vaccine development. (C) 2016 The Authors. Published by Elsevier Masson SAS on behalf of Institut Pasteur.Peer reviewe

Second-generation antipsychotics and pregnancy complications

Purpose To study if second-generation antipsychotic (S-GA) use during pregnancy is associated with an increased risk of pregnancy and neonatal complications. Methods A population-based birth cohort study using national register data extracted from the "Drugs and Pregnancy" database in Finland, years 1996-2016. The sampling frame included 1,181,090 pregnant women and their singleton births. Women were categorized into three groups: exposed to S-GAs during pregnancy (n = 4225), exposed to first-generation antipsychotics (F-GAs) during pregnancy (n = 1576), and unexposed (no purchases of S-GAs or F-GAs during pregnancy, n = 21,125). Pregnancy outcomes in S-GA users were compared with those in the two comparison groups using multiple logistic regression models. Results Comparing S-GA users with unexposed ones, the risk was increased for gestational diabetes (adjusted odds ratio, OR 1.43; 95% CI 1.25-1.65), cesarean section (OR 1.35; 95% CI 1.18-1.53), being born large for gestational age (LGA) (OR 1.57; 95% CI 1.14-2.16), and preterm birth (OR 1.29; 95% CI 1.03-1.62). The risk for these outcomes increased further with continuous S-GA use. Infants in the S-GA group were also more likely to suffer from neonatal complications. Comparing S-GA users with the F-GA group, the risk of cesarean section and LGA was higher (OR 1.25, 95% CI 1.03-1.51; and OR 1.89, 95% CI 1.20-2.99, respectively). Neonatal complications did not differ between the S-GA and F-GA groups. Conclusions Prenatal exposure to S-GAs is associated with an increased risk of pregnancy complications related to impaired glucose metabolism. Neonatal problems are common and occur similarly in S-GA and F-GA users.Peer reviewe

Simultaneous Immunization with Multivalent Norovirus VLPs Induces Better Protective Immune Responses to Norovirus Than Sequential Immunization

Human noroviruses (NoVs) are a genetically diverse, constantly evolving group of viruses. Here, we studied the effect of NoV pre-existing immunity on the success of NoV vaccinations with genetically close and distant genotypes. A sequential immunization as an alternative approach to multivalent NoV virus-like particles (VLPs) vaccine was investigated. Mice were immunized with NoV GI.3, GII.4-1999, GII.17, and GII.4 Sydney as monovalent VLPs or as a single tetravalent mixture combined with rotavirus VP6-protein. Sequentially immunized mice were primed with a trivalent vaccine candidate (GI.3 + GII.4-1999 + VP6) and boosted, first with GII.17 and then with GII.4 Sydney VLPs. NoV serum antibodies were analyzed. Similar NoV genotype-specific immune responses were induced with the monovalent and multivalent mixture immunizations, and no immunological interference was observed. Multivalent immunization with simultaneous mix was found to be superior to sequential immunization, as sequential boost induced strong blocking antibody response against the distant genotype (GII.17), but not against GII.4 Sydney, closely related to GII.4-1999, contained in the priming vaccine. Genetically close antigens may interfere with the immune response generation and thereby immune responses may be differently formed depending on the degree of NoV VLP genotype identity

Hope in hospitalized patients with terminal cancer

Hope is of great importance for patients diagnosed with cancer, especially those that are terminally ill. The diagnosis often puts an end to the realization of personal, social, and professional goals. The aim of this study was to characterize the hope of hospitalized patients diagnosed with cancer in the terminal phase of the disease. The research tool used in the study was Block’s hope test (NCN-36; NCN- Nadzieja Chorych Nowotworowych—Hope of Cancer Patients), designed for patients with life-threatening diseases. The results showed that the patients were characterized by a moderate level of global hope. The highest levels of hope were noted in the spiritual-religious area and the lowest levels of hope concerned curing the disease. Patients exhibited varied levels of hope and varied internal structures of hope. They presented four different types of hope: optimistic, moderate, religious, and weak. Optimistic hope was found most frequently in patients diagnosed with a terminal phase of cancer, while weak hope was represented by the smallest group of these patients

Norovirus-Specific Memory T Cell Responses in Adult Human Donors

Norovirus (NoV) is a leading cause of acute gastroenteritis in people of all ages worldwide. NoV-specific serum antibodies which block the binding of NoV virus-like particles (VLPs) to the cell receptors have been thoroughly investigated. In contrast, only a few publications are available on the NoV capsid VP1 protein-specific T cell responses in humans naturally infected with the virus. Freshly isolated peripheral blood mononuclear cells of eight healthy adult human donors previously exposed to NoV were stimulated with purified VLPs derived from NoV GII.4-1999, GII.4-2012 (Sydney), and GI.3, and IFN-γ production was measured by an ELISPOT assay. In addition, 76 overlapping synthetic peptides spanning the entire 539-amino acid sequence of GII.4 VP1 were pooled into two-dimensional matrices and used to identify putative T cell epitopes. Seven of the eight subjects produced IFN-γ in response to the peptides and five subjects produced IFN-γ in response to the VLPs of the same origin. In general, stronger T cell responses were induced with the peptides in each donor compared to the VLPs. A CD8+ T cell epitope in the shell domain of the VP1 (134SPSQVTMFPHIIVDVRQL151) was identified in two subjects, both having human leukocyte antigen (HLA)-A∗02:01 allele. To our knowledge, this is the first report using synthetic peptides to study NoV-specific T cell responses in human subjects and identify T cell epitopes