individualized-reconstitution-human-milk-microbiota-feasible-approach

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

Mother&#39;s own milk (MOM) is the most complete nutritional resource for newborns. In cases where mothers are unable to produce sufficient milk or cannot ...

Federal University of Santa Catarina

The circumsporozoite protein (CSP), the most abundant surface antigen of sporozoites, has been extensively studied in different expression platforms as a vaccine candidate. Clinical trials have shown the necessity of broad and highly avid humoral immune responses together with high numbers of CSP-specific TCD4+ and TCD8+ cells, especially those producing IFN-γ, to induce protection. To this aim, we designed two distinct recombinant immunogens based on previously-described antigenic fragments of Plasmodium vivax CSP (PvCSP) to be used as vaccine candidates. The first one is a virus-like particle (VLP) comprising the repeat region of PvCSP (B and TCD4+ epitopes) within the loop of the hepatitis B virus core antigen (HBcAgPvCSP). The second one is a PvCSP multi-epitope polypeptide, rPvCSP-ME, designed based on antigenic regions of PvCSP recognized by lymphocytes of individuals from endemic areas. Mice immunized with 2 doses of these proteins, administered individually or combined and formulated in Montanide ISA 720 adjuvant, were able to induce strong effector and memory humoral responses with IgG titers ranging from 10(4) to 10(5) and avidity indexes toward full-length PvCSP reaching up to 66%, even 3 months after the last immunization. Furthermore, balanced Th1/Th2 responses were generated, as determined by titers of IgG subclasses and further confirmed by ELISPOT analyses, which detected that these vaccination protocols were able to elicit long-term IFN-γ and IL-2-secreting memory T-cells. Overall, these results show that our vaccine candidates generate, in mice, immune responses against regions within PvCSP that have been associated with protection against malaria in humans.

Long-lasting humoral and cellular immune responses elicited by immunization with recombinant chimeras of the Plasmodium vivax circumsporozoite protein.

Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties.

Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

Integrins mediate the lymphocyte migration into an infected tissue, and these cells are essential for controlling the multiplication of many intracellular parasites such as , the causative agent of Chagas disease. Here, we explore LFA-1 and VLA-4 roles in the migration of specific CD8 T cells generated by heterologous prime-boost immunization during experimental infection with . To this end, vaccinated mice were treated with monoclonal anti-LFA-1 and/or anti-VLA-4 to block these molecules. After anti-LFA-1, but not anti-VLA-4 treatment, all vaccinated mice displayed increased blood and tissue parasitemia, and quickly succumbed to infection. In addition, there was an accumulation of specific CD8 T cells in the spleen and lymph nodes and a decrease in the number of those cells, especially in the heart, suggesting that LFA-1 is important for the output of specific CD8 T cells from secondary lymphoid organs into infected organs such as the heart. The treatment did not alter CD8 T cell effector functions such as the production of pro-inflammatory cytokines and granzyme B, and maintained the proliferative capacity after treatment. However, the specific CD8 T cell direct cytotoxicity was impaired after LFA-1 blockade. Also, these cells expressed higher levels of Fas/CD95 on the surface, suggesting that they are susceptible to programmed cell death by the extrinsic pathway. We conclude that LFA-1 plays an important role in the migration of specific CD8 T cells and in the direct cytotoxicity of these cells.

LFA-1 Mediates Cytotoxicity and Tissue Migration of Specific CD8 T Cells after Heterologous Prime-Boost Vaccination against  Infection.

Vaccine development against Plasmodium vivax malaria lags behind that for Plasmodium falciparum. To narrow this gap, we administered recombinant antigens based on P. vivax circumsporozoite protein (CSP) to mice. We expressed in Pichia pastoris two chimeric proteins by merging the three central repeat regions of different CSP alleles (VK210, VK247, and P. vivax-like). The first construct (yPvCSP-All) contained the fused repeat regions flanked by N- and C-terminal regions. The second construct (yPvCSP-All) contained the fused repeat regions and the C-terminal domain, plus RI region. Mice were vaccinated with three doses of yPvCSP in adjuvants Poly (I:C) or Montanide ISA720. We also used replication-defective adenovirus vectors expressing CSP of human serotype 5 (AdHu5) and chimpanzee serotype 68 (AdC68) for priming mice which were subsequently boosted twice with yPvCSP proteins in Poly (I:C) adjuvant. Regardless of the regime used, immunized mice generated high IgG titres specific to all CSP alleles. After challenge with P. berghei ANKA transgenic parasites expressing Pb/PvVK210 or Pb/PvVK247 sporozoites, significant time delays for parasitemia were observed in all vaccinated mice. These vaccine formulations should be clinically tried for their potential as protective universal vaccine against P. vivax malaria.

Prime-boost vaccination with recombinant protein and adenovirus-vector expressing Plasmodium vivax circumsporozoite protein (CSP) partially protects mice against Pb/Pv sporozoite challenge.

Oscar Bruna-Romero

Laboratory of Applied Immunology,
LIA,
Department of Microbiology,
Immunology,
and Parasitology,
Center for Dysbiosis Control,
Laboratory of Applied Immunology, LIA, Department of Microbiology, Immunology, and Parasitology, Center for Dysbiosis Control

Federal University of Santa Catarina

Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings