Showing papers by "Carlos A. Guzmán published in 2023"

T cell repertoire breadth is associated with the number of acute respiratory infections in the LoewenKIDS birth cohort

Abstract: We set out to gain insight into peripheral blood B and T cell repertoires from 120 infants of the LoewenKIDS birth cohort to investigate potential determinants of early life respiratory infections. Low antigen-dependent somatic hypermutation of B cell repertoires, as well as low T and B cell repertoire clonality, high diversity, and high richness especially in public T cell clonotypes reflected the immunological naivety at 12 months of age when high thymic and bone marrow output are associated with relatively few prior antigen encounters. Infants with inadequately low T cell repertoire diversity or high clonality showed higher numbers of acute respiratory infections over the first 4 years of life. No correlation of T or B cell repertoire metrics with other parameters such as sex, birth mode, older siblings, pets, the onset of daycare, or duration of breast feeding was noted. Together, this study supports that-regardless of T cell functionality-the breadth of the T cell repertoire is associated with the number of acute respiratory infections in the first 4 years of life. Moreover, this study provides a valuable resource of millions of T and B cell receptor sequences from infants with available metadata for researchers in the field.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Abstract: Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

Pulmonary Application of Novel Antigen-Loaded Chitosan Nano-Particles Co-Administered with the Mucosal Adjuvant C-Di-AMP Resulted in Enhanced Immune Stimulation and Dose Sparing Capacity

Abstract: The most successful medical intervention for preventing infectious diseases is still vaccination. This effective strategy has resulted in decreased mortality and extended life expectancy. However, there is still a critical need for novel vaccination strategies and vaccines. Antigen cargo delivery by nanoparticle-based carriers could promote superior protection against constantly emerging viruses and subsequent diseases. This should be sustained by the induction of vigorous cellular and humoral immunity, capable of acting both at the systemic and mucosal levels. Induction of antigen-specific responses at the portal of entry of pathogens is considered an important scientific challenge. Chitosan, which is widely regarded as a biodegradable, biocompatible and non-toxic material for functionalized nanocarriers, as well as having adjuvant activity, enables antigen administration via less-invasive mucosal routes such as sublingual or pulmonic application route. In this proof of principle study, we evaluate the efficacy of chitosan nanocarriers loaded with the model antigen Ovalbumin (OVA) co-administrated with the STING agonist bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) given by pulmonary route. Here, BALB/c mice were immunized with four doses of the formulation that stimulates enhanced antigen-specific IgG titers in sera. In addition, this vaccine formulation also promotes a strong Th1/Th17 response characterized by high secretion of IFN-γ, IL-2 and IL-17, as well as induction of CD8+ T cells. Furthermore, the novel formulation exhibited strong dose-sparing capacity, enabling a 90% reduction of the antigen concentration. Altogether, our results suggest that chitosan nanocarriers, in combination with the mucosal adjuvant c-di-AMP, are a promising technology platform for the development of innovative mucosal vaccines against respiratory pathogens (e.g., Influenza or RSV) or for therapeutic vaccines.

Protective Efficacy of a Mucosal Influenza Vaccine Formulation Based on the Recombinant Nucleoprotein Co-Administered with a TLR2/6 Agonist BPPcysMPEG

Abstract: Current influenza vaccines target highly variable surface glycoproteins; thus, mismatches between vaccine strains and circulating strains often diminish vaccine protection. For this reason, there is still a critical need to develop effective influenza vaccines able to protect also against the drift and shift of different variants of influenza viruses. It has been demonstrated that influenza nucleoprotein (NP) is a strong candidate for a universal vaccine, which contributes to providing cross-protection in animal models. In this study, we developed an adjuvanted mucosal vaccine using the recombinant NP (rNP) and the TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl-poly-ethylene-glycol (BPPcysMPEG). The vaccine efficacy was compared with that observed following parenteral vaccination of mice with the same formulation. Mice vaccinated with 2 doses of rNP alone or co-administered with BPPcysMPEG by the intranasal (i.n.) route showed enhanced antigen-specific humoral and cellular responses. Moreover, NP-specific humoral immune responses, characterized by significant NP-specific IgG and IgG subclass titers in sera and NP-specific IgA titers in mucosal territories, were remarkably increased in mice vaccinated with the adjuvanted formulation as compared with those of the non-adjuvanted vaccination group. The addition of BPPcysMPEG also improved NP-specific cellular responses in vaccinated mice, characterized by robust lymphoproliferation and mixed Th1/Th2/Th17 immune profiles. Finally, it is notable that the immune responses elicited by the novel formulation administered by the i.n. route were able to confer protection against the influenza H1N1 A/Puerto Rico/8/1934 virus.

Influenza vaccine is able to prevent neuroinflammation triggered by H7N7 IAV infection

Abstract: Influenza A virus (IAV) subtypes are a major cause of illness and mortality worldwide and pose a threat to human health. Although IAV infection is considered a self-limiting respiratory syndrome, an expanded spectrum of cerebral manifestations has been reported following IAV infection. Neurotropic IAVs, such as the H7N7 subtype, are capable of invading the central nervous system (CNS) and replicating in brain cells, resulting in microglia-induced neuroinflammation. Microglial cells, the brain’s resident immune cells, are instrumental in the inflammatory response to viral infection. While activation of microglia is important to initially contain the virus, excessive activation of these cells leads to neuronal damage. Previous studies have shown that acute and even long-term IAV-induced neuroinflammation leads to CNS damage. Therefore, the search for possible preventive or therapeutic strategies is of great importance. In this study, we investigated the potential effect of vaccination against acute neuroinflammation induced by H7N7 infection and subsequent neuronal damage in the hippocampus, a particularly vulnerable brain region, comparing young and aged mice. Immunosenescence is one of the striking pathophysiological changes during mammalian aging that leads to “inflammaging” and critically limits the protection by vaccines in the elderly. The results suggest that formalin-inactivated H7N7 vaccine has a preventive effect against the inflammatory responses in the periphery and also in the CNS after H7N7 infection. Cytokine and chemokine levels, increased microglial density, and cell volume after H7N7 infection were all attenuated by vaccination. Further structural analysis of microglial cells also revealed a change in branching complexity after H7N7 infection, most likely reflecting the neuroprotective effect of the vaccination. In addition, synapse loss was prevented in vaccinated mice. Remarkably, engulfment of post-synaptic compartments by microglia can be proposed as the underlying mechanism for spine loss triggered by H7N7 infection, which was partially modulated by vaccination. Although young mice showed better protection against neuroinflammation and the resulting deleterious neuronal effects upon vaccination, a beneficial role of the vaccine was also observed in the brains of older mice. Therefore, vaccination can be proposed as an important strategy to prevent neurological sequelae of H7N7 infection.

Systems immunology reveals the molecular mechanisms of heterogeneous influenza vaccine response in the elderly

Abstract: Vaccination-induced protection against influenza is greatly diminished and increasingly heterogeneous with age. We investigated longitudinally (up to five timepoints) a cohort of 234 elderly influenza vaccinees across two independent seasons including up to six modalities (multi-omics and immunological parameters). System-level analyses revealed responders exhibited time-dependent changes attributed to a productive vaccine response across all omics layers whereas non-responders did not follow such dynamics, suggestive of systemic dysregulation. Through multi-omics integration, we identified key metabolites and proteins and their likely role in immune response to vaccination. High pre-vaccination IL-15 concentrations negatively associated with antibody production, further supported by experimental validation in mice revealing an IL-15-driven NK-cell axis with a suppressing role on antibody production. Finally, we propose certain long-chain fatty acids as modulators of persistent inflammation in non-responders. Our findings highlight the potential for stratification of vaccinees and open avenues for possible pharmacological interventions to enhance vaccine responses.