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Introducción: el programa de vacunación es una intervención de salud pública cuyo propósito es controlar o eliminar enfermedades inmunoprevenibles. El objetivo de este trabajo fue estimar la evolución del presupuesto en vacunas entre 2007 y 2022, y el impacto de potenciales mejoras en el calendario de vacunaciones de Paraguay. Método: se estimó la evolución del presupuesto en vacunas según los cambios entre 2007 y 2022, y el impacto de esquemas alternativos versus el actual, que incluyen la vacuna contra la influenza cuádruple, séxtuple en lactantes, el agregado de la vacuna contra papiloma en varones y meningococo ACYW en adolescentes. La perspectiva del análisis fue la del Ministerio de Salud Pública y el horizonte temporal de un año. Los resultados de las alternativas se expresan como impacto presupuestal versus el año 2022. Resultados: entre 2007 y 2022 la cantidad de biológicos del Programa Ampliado de Inmunizaciones pasó de 11 a 18 e incorporó indicaciones de algunas vacunas. Se estima que el presupuesto se incrementó de 3,8 a 29,9 millones de dólares entre los extremos de la serie. Las alternativas implicarían un incremento de 13%, 35%, 5% y 10%, individualmente. El incremento en conjunto alcanza el 62%. Conclusiones: el aumento del gasto en vacunas fue de ocho veces entre 2007 y 2022. Se estima el impacto presupuestal en diferentes escenarios que se interpretan como mejoras comparadas con el actual de 2022, siendo el incremento más exigente de un 35%. La evidencia generada puede colaborar en el proceso de toma de decisiones acerca de esta política pública en Paraguay.

Introduction: the vaccination program is a public health intervention aimed at controlling or eliminating vaccine-preventable diseases. The objective of the study was to estimate the evolution of the vaccine budget between 2007 and 2022 and the impact of potential improvements in Paraguay's vaccination schedule. Method: the evolution of the vaccine budget was estimated considering the changes introduced between 2007 and 2022 and the impact of alternative schedules versus the current one. These alternatives include the addition of the quadrivalent influenza vaccine, the hexavalent vaccine for infants, the inclusion of the HPV vaccine for boys, and the ACYW meningococcal ACYW vaccine for adolescents. The analysis was conducted from the perspective of the Ministry of Health, with a time horizon of one year. The results of the alternatives are expressed as budget impact compared to the year 2022. Results: between 2007 and 2022, the number of biological products in the EPI increased from 11 to 18, and additional indications for some vaccines were incorporated. The budget is estimated to have increased from 3.8 to 29.9 million USD over the series. The alternatives would result in individual increases of 13%, 35%, 5%, and 10%. The combined increase reaches 62%. Conclusions: the increase in vaccine expenditure was eightfold between 2007 and 2022. The budget impact was estimated in different scenarios, interpreted as improvements compared to the current 2022 scenario, with the most demanding increase being 35%. The generated evidence can assist in the decision-making process regarding this public policy in Paraguay.

Introdução: o programa de vacinação é uma intervenção de saúde pública cuja finalidade é controlar ou eliminar doenças imunopreveníveis. O objetivo deste estudo foi estimar a evolução do orçamento de vacinas entre 2007 e 2022 e o impacto de possíveis melhorias no cronograma de vacinação do Paraguai. Métodos: estimamos a evolução do orçamento de vacinas de acordo com as mudanças ocorridas entre 2007 e 2022 e o impacto de esquemas alternativos em relação ao atual, que incluem a vacina quádrupla contra a gripe, a sextupla em bebês, a adição da vacina contra o papilomavírus em homens e a vacina meningocócica ACYW em adolescentes. A perspectiva da análise foi a do Ministerio de Salud e o intervalo de tempo foi de um ano. Os resultados das alternativas são expressos como impacto orçamentário em relação a 2022. Resultados: Entre 2007 e 2022, o número de produtos biológicos do PAI aumentou de 11 para 18 e incorporou indicações para algumas vacinas. Estima-se que o orçamento tenha aumentado de US$ 3,8 milhões para US$ 29,9 milhões entre os extremos da série. As alternativas implicariam em um aumento de 13%, 35%, 5% e 10% individualmente. O aumento geral chega a 62%. Conclusões: o aumento nos gastos com vacinas foi de oito vezes entre 2007-2022. O impacto orçamentário é estimado em diferentes cenários que são interpretados como melhorias em comparação com o cenário de 2022, sendo que o aumento mais exigente é de 35%. As evidências geradas podem contribuir para o processo de tomada de decisão relacionado a essa política pública no Paraguai.

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BACKGROUND: Argentina currently uses a pentavalent vaccine containing diphtheria, tetanus, pertussis (whole cell), Haemophilus influenza type b and hepatitis B antigens, administered concomitantly with the inactivated polio vaccine (IPV) (DTwP-Hib-HB plus IPV) in its childhood vaccination schedule. However, hexavalent vaccines containing acellular pertussis antigens (DTaP-Hib-HB-IPV) and providing protection against the same diseases are also licensed, but are only available with a private prescription or for high-risk pre-term infants in the public health program. We analyzed the cost of switching from the current schedule to the alternative schedule with the hexavalent vaccine in Argentina, assuming similar levels of effectiveness. METHODS: The study population was infants ≤ 1 year of age born in Argentina from 2015 to 2019. The analysis considered adverse events, programmatic, logistic, and vaccine costs of both schemes from the societal perspective. The societal costs were disaggregated to summarize costs incurred in the public sector and with vaccination pre-term infants in the public sector. Costs were expressed in 2021 US Dollars (US$). RESULTS: Although the cost of vaccines with the alternative scheme would be US$39.8 million (M) more than with the current scheme, these additional costs are in large part offset by fewer adverse event-associated costs and lower programmatic costs such that the overall cost of the alternative scheme would only be an additional US$3.6 M from the societal perspective. The additional cost associated with switching to the alternative scheme in the public sector and with the vaccination of pre-term infants in the public sector would be US$2.1 M and US$84,023, respectively. CONCLUSIONS: The switch to an alternative scheme with the hexavalent vaccine in Argentina would result in marginally higher vaccine costs, which are mostly offset by the lower costs associated with improved logistics, fewer separate vaccines, and a reduction in adverse events.

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The Epstein-Barr nuclear antigen 1 (EBNA1) is essential for DNA replication and episome segregation of the viral genome, and participates in other gene regulatory processes of the Epstein-Barr virus in benign and malignant diseases related to this virus. Despite the participation of other regions of the protein in evading immune response, its DNA binding, dimeric beta-barrel domain (residues 452-641) is necessary and sufficient for the main functions. This domain has an unusual topology only shared by another viral origin binding protein (OBP), the E2 DNA binding domain of papillomaviruses. Both the amino acid and DNA target sequences are completely different for these two proteins, indicating a link between fold conservation and function. In this work we investigated the folding and stability of the DNA binding domain of EBNA1 OBP and found it is extremely resistant to chemical, temperature, and pH denaturation. The thiocyanate salt of guanidine is required for obtaining a complete transition to a monomeric unfolded state. The unfolding reaction is extremely slow and shows a marked uncoupling between tertiary and secondary structure, indicating the presence of intermediate species. The Gdm.SCN unfolded protein refolds to fully soluble and spherical oligomeric species of 1.2 MDa molecular weight, with identical fluorescence centre of spectral mass but different intensity and different secondary structure. The refolded spherical oligomers are substantially less stable than the native recombinant dimer. In keeping with the substantial structural rearrangement in the oligomers, the spherical oligomers do not bind DNA, indicating that the DNA binding site is either disrupted or participates in the oligomerization interface. The puzzling extreme stability of a dimeric DNA binding domain from a protein from a human infecting virus in addition to a remarkable kinetically driven folding where all molecules do not return to the most stable original species suggests a co-translational and directional folding of EBNA1 in vivo, possibly assisted by folding accessory proteins. Finally, the oligomers bind Congo red and thioflavin-T, both characteristic of repetitive beta-sheet elements of structure found in amyloids and their soluble precursors. The stable nature of the "kinetically trapped" oligomers suggest their value as models for understanding amyloid intermediates, their toxic nature, and the progress to amyloid fibers in misfolding diseases. The possible role of the EBNA1 spherical oligomers in the virus biology is discussed.

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Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.

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