Construction, identification and expression of recombinant Schistosoma japonicum vaccine mediated by Enterococcus faecalis / 中华地方病学杂志

Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.

Treatment Following Erythema Multiforme in the Oral Mucosa After qHPV Vaccine with Low-Level Laser Therapy: Case Report

ABSTRACT: Erythema multiforme (EM) is a mucocutaneous condition of uncertain etiology, although the hypersensitivity reaction to a wide variety of agents may be related to the onset of the lesions. In about half of the affected patients it is possible to identify a previous infection. This article aims to report a case of EM in the oralmucosa after qHPV vaccine (Gardasil®), to highlight the diagnostic process and the proposed treatment. Female patient, 16 years old, after 10 days of receiving the first dose of the qHPV vaccine. On physical examination, she presented multiple ulcers and hemorrhagic crusts to the touch, based on the clinical picture and the history of the disease, a diagnostic hypothesis was EM. Low-level laser therapy (LLLT) was chosen as an alternative treatment, since the exercises applied were not successful. The patient was followed up, reported decreased pain and burn and, after one year of treatment, there was no recurrence of the lesions. Laser treatment showed an effective treatment alternative, in addition to the low cost and ease of application.

RESUMEN: El eritema multiforme (EM) es una afección mucocutánea de etiología incierta, aunque la reacción de hipersensibilidad a una amplia variedad de agentes puede estar relacionada con la aparición de las lesiones. En aproximadamente la mitad de los pacientes afectados es posible identificar una infección previa. Este artículo tiene como objetivo informar un caso de EM en la mucosa oral después de la vacuna qHPV (Gardasil®), para resaltar el proceso de diagnóstico y el tratamiento propuesto. Paciente de 16 años, después de 10 días de recibir la primera dosis de la vacuna qHPV. En el examen físico, presentó múltiples úlceras y costras hemorrágicas al tacto, según el cuadro clínico y la historia de la enfermedad, una hipótesis diagnóstica fue EM. La terapia con láser de baja potencia (TLBP) se eligió como un tratamiento alternativo, ya que los ejercicios aplicados no tuvieron éxito. La paciente fue seguida, informó disminución del dolor y las quemaduras y, después de un año de tratamiento, no hubo recurrencia de las lesiones. El tratamiento con láser mostró una alternativa de tratamiento efectivo, además del bajo costo y la facilidad de aplicación.

Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

Immunogenicity of a Recombinant Gonadotropin-releasing Hormone Associated to the B Subunit of Escherichia coli Heat-labile Enterotoxin Expressed in Pichia pastoris and Escherichia coli Platforms

Abstract Gonadotropin-releasing hormone (GnRH) is one of the main targets for the development of immunocontraceptives vaccines. The aim of this study was to clone and express the recombinant GnRH fused to the B subunit of Escherichia coli heat-labile enterotoxin (LTB) molecule in Pichia pastoris and Escherichia coli platforms and evaluate their immunogenicity in mice. P. pastoris (pGnRH/LTB) and E. coli (eGnRH/LTB) platforms were able to express GnRH/LTB expected band with ~ 21 kDa. Both constructions were immunogenic in mice. Similar IgG kinetics was observed for both construction when it was used as ELISA antigen respectively, showing significant (p<0.05) IgG levels 5-fold higher than a commercial vaccine and 14-fold higher than the controls. The histological effects of pGnRH/LTB as well as eGnRH/LTB proteins demonstrated a significant effect on the gonads, characterized by atrophy of seminiferous tubules, absence of spermatogenesis and reduction of Leydig cells. Both constructions were able to induce antibodies that block the hormone effect, suggesting the potential of GnRH/LTB, independently of the P. pastoris or E. coli platform used, as a vaccine candidate for immunocontraception.

Vacuna del papilomavirus en el Perú / Papillomavirus vaccine in Peru

RESUMEN El cáncer de cuello uterino es un problema de salud pública. La vacuna contra el virus del papiloma humano (VPH) protege contra la infección por el VPH. Ha mostrado ser efectiva para prevenir lesiones premalignas y cáncer de cérvix, así como lesiones de la vulva, vagina, canal anal, pene y orofaringe. Forma parte del calendario nacional de vacunación, es costo efectiva en la introducción de la estrategia nacional de vacunación y es la herramienta ideal ante sistemas de salud donde la prevención secundaria no ha dado resultado a lo largo del tiempo. La implementación del programa de vacunación en Perú se inició en el 2011. Actualmente, la indicación de la vacunación es con la vacuna tetravalente a niñas del 5° grado de primaria de los colegios públicos y privados, en 2 dosis a los 0 y 6 meses. En el 2019, la cobertura fue de 87% (234 535 niñas) para la primera dosis y 78% (211 339) para la segunda dosis.

ABSTRACT Cervical cancer is a public health concern. The human papillomavirus (HPV) vaccine protects against infection with HPV. The vaccine has been shown to be effective in preventing premalignant lesions and cervical cancer, as well as lesions of the vulva, vagina, anal canal, penis, and oropharynx. It has also proven to be cost effective and supports the idea of introducing a national vaccination strategy. The HPV vaccine could be the ideal tool for health systems where secondary prevention has not been successful over time. The implementation of the vaccination program in Peru began in 2011. Currently, in Peru, the indication for vaccination is with the quadrivalent vaccines for 5th grade girls from public and private schools. It is administered in 2 doses, 0-6 months. In 2019, coverage in Peru was 87% (234 535 girls) for the first dose and 78% (211 339 girls) for the second dose.

HPV vacina: conhecer e aceitar para assegurar a eficácia / HPV vaccine: knowledge and acceptance to ensure effectiveness

The new Vaccine technologies against transmissible and non-transmissible diseases, such as cancer, have had an impact on international public health. The human papillomavirus (HPV) vaccine is used on a large scale in immunization programs in more than 58 countries, with resultant efficacy and safety for precursor lesions of cervical cancer, in addition to anogenital lesions. After the introduction of quadrivalent HPV vaccine (6,11,16 and 18) in Brazil in 2014, monitoring the vaccination coverage and the development of HPV prevalence incidence of cervical abnormalities and precancerous lesions must be observed, as well as morbidity and mortality trends from in situ and invasive cancer. Encouraging information, counseling and continuing education is recommended as a strategy to broaden vaccine acceptance in order to sediment its implementation and ensure effectiveness in reducing new cases of cervical cancer in the future.

As novas tecnologias em vacina contra doenças transmissíveis e não transmissíveis como o câncer, tiveram impacto na saúde pública internacional, especificamente a vacina para o papiloma vírus humano (HPV) utilizada em larga escala nos programas de imunização em mais de 58 países, com resultados de eficácia e segurança para lesões precursoras do câncer de colo do útero além de lesões anogenitais. Após a introdução em território Nacional da vacina quadrivalente para o HPV (6,11,16 e 18) desde 2014, ressalta-se a importância do monitoramento da cobertura vacinal e o desenvolvimento de estudos de prevalência de HPV em logo prazo, de incidência de anormalidades cervicais e lesões pré-cancerosas bem como de tendência de morbimortalidade por câncer in situ e invasivo. O incentivo às informações, aconselhamento e educação continuada é recomendado como uma estratégia para ampliar a aceitação da vacina a fim de sedimentar sua implantação e assegurar a eficácia na redução dos novos casos de câncer de colo do útero para o futuro.

Avaliação da imunogenicidade de diferentes formas alélicas da proteí­na recombinante PvAMA-1expressa em Pichia pastoris: impacto da diversidade antigênica / Evaluation of the immunogenicity of different allelic forms of PvAMA-1 protein expressed in Pichia pastoris: impact of antigenic diversity

A malária é um problema de saúde pública no Brasil e no mundo. Em 2016, o número de casos estimado pela Organização Mundial de Saúde foi de 216 milhões. Plasmodium falciparum é a espécie mais prevalente e responsável pelo maior número de mortes no mundo, sobretudo no continente africano. Por outro lado, o Plasmodium vivax é conhecido por sua ampla distribuição geográfica, sendo a espécie que predomina nas Américas, incluindo o Brasil. Nos últimos 20 anos, nosso grupo tem gerado e caracterizado diversas proteínas recombinantes baseadas em antígenos imunodominantes de P. vivax que podem servir como base para o desenvolvimento de uma vacina contra malária. Entre os antígenos de merozoítas, uma das principais proteínas em estudo pelo nosso grupo é o Antígeno 1 de Membrana Apical de P. vivax (PvAMA-1), caracterizado previamente como altamente imunogênico em infecções naturais e em camundongos imunizados, na presença de diferentes adjuvantes. O objetivo do presente estudo foi investigar o efeito da diversidade antigênica dessa proteína no reconhecimento por anticorpos específicos e na indução de imunidade contra o parasita. Para isso, foram geradas seis novas proteínas representando diferentes alelos descritos na natureza: PvAMA-1-Belem, PvAMA-1-Sal-I, PvAMA-1-Chesson-I, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX e PvAMA-1-PNG_62_MU. As proteínas recombinantes foram expressas em leveduras Pichia pastoris e purificadas em duas etapas cromatográficas. Em seguida, as imunizações em camundongos C57BL/6 foram realizadas com as proteínas administradas de forma isolada, ou em combinação, na presença do adjuvante agonista de TLR3 (Poly I:C). Por ELISA, observamos que todas as formulações foram capazes de induzir anticorpos IgG contra as proteínas homólogas e heterólogas, o que sugere que a diversidade antigênica entre as formas alélicas não compromete o reconhecimento. Os dados gerados no presente trabalho sugerem que uma formulação contendo mistura de diferentes alelos representando a proteína AMA-1 pode ser explorada para o desenvolvimento de uma vacina de ampla cobertura contra o P. vivax

Malaria is a public health problem in Brazil and throughout the world. In 2016, the World Health Organization estimated there were 216 million cases of malaria. Plasmodium falciparum is the most prevalent species and is responsible for the largest number of deaths, especially in the African continent. However, Plasmodium vivax is known for its wide geographic distribution, being the species that prevails in the Americas, including Brazil. In the last 20 years, our group has generated and characterized several recombinant proteins based on immunodominant antigens of P. vivax that can serve as a basis for the development of a malaria vaccine. Among the merozoite antigens, one of the main proteins studied by our group is P. vivax apical membrane antigen-1 (PvAMA-1), previously characterized as highly immunogenic in natural infections and immunized mice, in the presence of different adjuvants. The objective of this study was to investigate the effect of antigenic diversity of this protein in the recognition of specific antibodies and the induction of immunity against the parasite. For this, six new proteins were generated representing different alleles described in nature: PvAMA-1-Belem, PvAMA-1-Sal-i, PvAMA-1-Chesson-i, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX, and PvAMA-1-PNG_62_MU. Recombinant proteins were expressed in Pichia pastoris yeast and purified by two chromatographic stages. Then, C57BL/6 mice were immunized with these proteins administered in isolation or in combination, in the presence of the TLR3 agonist adjuvant, Poly I:C. Using an enzyme-linked immunosorbent assay, we observed that all formulations induced IgG antibodies against homologous and heterologous proteins. This indicates that antigenic diversity between allele forms does not compromise recognition. This finding suggests that a formulation containing a mixture of different alleles representing the PvAMA-1 protein can be exploited for developing of a wide coverage vaccine against P. vivax

Conocimientos sobre el virus del papiloma humano y su vacuna en padres de familia de Rivera, Huila en el 2015 / Knowledge of human papillomavirus and its vaccine in parents of Rivera, Huila in 2015

RESUMEN Introducción: la vacuna contra el virus del papiloma humano es la medida preventiva más reciente y controversial para el cáncer de cuello uterino. En 2014 Colombia tuvo una serie de supuestas reacciones adversas ante la vacuna que provocó la caída de su cobertura. Objetivo: describir los conocimientos frente al virus del papiloma humano y su vacuna en los padres de Rivera, Huila. Materiales y método: estudio transversal con muestra por conveniencia de 124 padres de niños menores de 18 años, a través de una encuesta sociodemográfica donde fue medido el conocimiento acerca del virus, su vacuna y el cáncer de cérvix. Análisis cuantitativo por medio de la técnica de frecuencias simples. Resultados: el nivel de conocimiento fue bajo (44,35%); el 43,54% identifican al virus del papiloma humano como agente etiológico del cáncer de cuello uterino; sólo el 7,4% considera útil la vacuna como método preventivo y la vacuna fue aceptada por el 87% de los padres. Conclusiones: El conocimiento del virus del papiloma humano y el cáncer de cuello uterino es bajo y se ve afectado por el desconocimiento de las diferentes estrategias preventivas del cáncer de cuello uterino, así como por la falta de información sobre la acción de la vacuna contra el virus. Sin embargo, la aceptabilidad de la vacuna es alta. MÉD.UIS. 2017;30(1):13-9.

ABSTRACT Introduction: Human Papillomavirus Vaccine is the most recent and controversial step for cervical cancer prevention. During 2014, in Colombia, a series of suspected adverse reactions to the vaccine caused a decline in its coverage. Objective: the aim of this study is to describe the knowledge about Human Papillomavirus and its vaccine from parents of Rivera, Huila. Materials and method: cross-sectional study with a convenient sample of 124 parents whose children were under 18 years old; through a socio-demographic questionnaire, knowledge about issues related to the virus, related to cervical cancer and its vaccine was measured. A quantitative analysis was performed by simple frequency analysis. Results: the level of knowlodge was low (44,35%); 43,54% of the parents identify Human Papillomavirus as an etiological cervical cancer agent, just 7,4% of the parents consider the vaccine as an useful preventive method and the vaccine was accepted by 87% of the parents. Conclusions: the knowledge level of the parents of Rivera, Huila about Human Papillomavirus and cervical cancer is low and it is affected by the ignorance of the different cervical cancer preventive strategies, as well as the lack of information of the action of Human Papillomavirus vaccine. However, the vaccine acceptability is high. MÉD.UIS. 2017;30(1):13-9.

Dynamic investigation of cytokines in mice immunizedwith Echinococcus granulosus / 西安交通大学学报(医学版)

Objective To investigate the dynamic changes of IL-2, IFN-γ, TNF-α, IL-4, IL-5 and IL-10 in mice immunized with Eg14-3-3 and then challenged by Echinococcus granulosus protoscoleces.Methods ICR mice were subcutaneously immunized every two weeks for altogether three times with rEg14-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally four weeks after the third immunization.The PBS control group mice were operated in the same way.We collected mouse serum from tail vessel at different time points after immunization and challenge.The serum levels of IL-2, IFN-γ, TNF-α, IL-4, IL-5 and IL-10 were examined with enzyme-linked immunosorbent assay.Results The levels of the six cytokines were higher after immunization and challenge infection in rEg14-3-3 group than in PBS control group.The immunized mice generated a great deal of Th1 cells, namely, IL-2, IFN-γ and TNF-α, after immunization and showed a peak at week 10 (acute infection phase), then the level decreased rapidly at week 30 (chronic infection phase), and maintained a high level for a long time.In contrast, Th2 cells like IL-4, IL-5 and IL-10 kept a low level until week 18, peaked at week 30, and then decreased gradually but maintained a relatively high level for a long time.For PBS control group, IL-2, IFN-γ and TNF-α did not increase obviously before infection, but increased rapidly after challenge infection and peaked at week 18;then the level decreased gradually and maintained a high level for a long time.On the other hand, IL-4, IL-5 and IL-10 kept a low level, but increased gradually after challenge infection.IL-4 peaked at week 18 while IL-5 and IL-10 peaked at week 30, and then they all decreased slowly but maintained a relatively high level for a long time.Conclusion Eg14-3-3 can induce significant antibody response by Th2 cells and cell-mediated immunity response by Th1 cells.Both Th1 and Th2 cells participate in the anti-echinococcusis protective immunity.

Construction and identification of recombinant rBCG-Rv2029c vaccine of Mycobacterium tuberculosis / 中国人兽共患病学报

We constructed a recombinant vaccine of Mycobacterium tuberculosis rBCG-Rv2029c,and then identified it.Rv2029c antigen encoding gene was amplified by PCR.The enzyme digestion products were ligated into rpMV261-2029c recombinant plasmid,after double digestion of Rv2029c and pmv261 vector,and then we introduced the plasmid into BCG to construct rBCG-Rv2029c recombinant vaccine by electroporation method.Finally,we analyzed the expression of the recombinant protein by SDS-PAGE and Western blotting.A total of 1 020 bp Rv2029c gene successfully amplified by PCR was inserted into the plasmid pmv261,then the fusion gene was successfully transduced into BCG.After identified by double enzyme digestion,confirmed by gene alignment and by thermally induced with Western blotting,the recombinant protein had a free primary.The recombinant live vaccine of M.tuberculosis rBCG-Rv2029c is successfully constructed,which lay a foundation for the study of the immune mechanism of recombinant vaccine.

Acute toxicity investigation of recombinant trivalent human papillomavirus vaccine / 药物评价研究

objective To evaluate the acute toxicity of recombinant trivalent human papillomavirus (HPV) vaccines.Methods Wistar rats were randomly divided into negative control and HPV groups with 20 rats in each group.Rats in HPV groups were sc administered with a single-dose of 1.5 mL of HPV vaccines (three times of human dose/each rat),while rats in negative control group were given equal volume of saline.The clinical state of animals was observed,and the body mass changes were detected.Rats were dissected after 15 d of treatment and examined by gross pathology.Results Following a single injection with high-dose HPV vaccines,there were not obvious abnormalities in clinical symptom.Compared with negative control group,the body weight of rats from HPV vaccines-treated group had no significant difference.No obvious macro-pathological change was found in all animals.Conclusion Single sc injection with recombinant trivalent HPV vaccines is well tolerated and no obvious toxicological change is found in Wistar rats.These results will facilitate further preclinical safety studies of HPV vaccines.

Acute toxicity investigation of recombinant trivalent human papillomavirus vaccine / 药物评价研究

objective To evaluate the acute toxicity of recombinant trivalent human papillomavirus (HPV) vaccines.Methods Wistar rats were randomly divided into negative control and HPV groups with 20 rats in each group.Rats in HPV groups were sc administered with a single-dose of 1.5 mL of HPV vaccines (three times of human dose/each rat),while rats in negative control group were given equal volume of saline.The clinical state of animals was observed,and the body mass changes were detected.Rats were dissected after 15 d of treatment and examined by gross pathology.Results Following a single injection with high-dose HPV vaccines,there were not obvious abnormalities in clinical symptom.Compared with negative control group,the body weight of rats from HPV vaccines-treated group had no significant difference.No obvious macro-pathological change was found in all animals.Conclusion Single sc injection with recombinant trivalent HPV vaccines is well tolerated and no obvious toxicological change is found in Wistar rats.These results will facilitate further preclinical safety studies of HPV vaccines.

Newcastle disease virus vectored vaccines as bivalent or antigen delivery vaccines

Recent advances in reverse genetics techniques make it possible to manipulate the genome of RNA viruses such as Newcastle disease virus (NDV). Several NDV vaccine strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens. The safety, immunogenicity, and protective efficacy of these NDV-vectored vaccines have been evaluated in pre-clinical and clinical studies. The vaccines are safe in mammals, humans, and poultry. Bivalent NDV-vectored vaccines against pathogens of economic importance to the poultry industry have been developed. These bivalent vaccines confer solid protective immunity against NDV and other foreign antigens. In most cases, NDV-vectored vaccines induce strong local and systemic immune responses against the target foreign antigen. This review summarizes the development of NDV-vectored vaccines and their potential use as a base for designing other effective vaccines for veterinary and human use.

Impacto precoce na redução de anormalidades cervicais em campos dos goytacazes, rj, brasil, depois da introdução de vacina quadrivalente de hpv em meninas de 11 a 15 anos: é hora de pensar em vacinas hpv para rapazes? / Early impact in reducing cervical abnormalities in campos dos goytacazes, rj, brazil, after introduction of the quadrivalent hpv vaccine for girls 11­15: is it time to think about hpv vaccine in boys?

O papilomavírus humano (HPV) é uma grande preocupação na saúde pública e privada. A prevenção dessa condição é a combinação do uso do exame de Papanicolaou, de preservativos e de vacinas contra o HPV. Campos dos Goytacazes é o primeiro município brasileiro a implementar em setembro de 2010 a vacina quadrivalente contra o HPV (Gardasil®) para meninas de 11 a 15 anos de idade em uma estratégia híbrida de vacinação (escolas e centros de saúde). Em 2014, a vacinação começou para os meninos na mesma época em que o Ministério da Saúde introduziu a vacina para as meninas. Objetivo: O objetivo do estudo foi analisar as tendências na redução de anormalidades cervicais de baixo grau cinco anos depois da introdução da vacina quadrivalente de HPV na cidade (resultado primário). Além disso, esta investigação avaliou o risco relativo de cada grupo analisado, de maneira a explicar o efeito protetor da vacina (resultado secundário). Métodos: A análise ecológica avaliou o impacto da vacinação contra o HPV como um fator protetor contra o baixo risco de anormalidades pelo HPV. Os resultados do teste de Papanicolaou, obtidos por meio do Sistema de Informação do Câncer do Colo do Útero (Siscolo) do Ministério da Saúde, foram categorizados em anormalidades de baixo grau (LGA) e anormalidades de alto grau (HGA). Este estudo preliminar foi centrado nas taxas de LGA, as quais foram estimadas para o período de um mês e estratificadas por quatro grupos etários (<20; 20-24; 25- 30; >30 anos) de 2007 a 2014. A comparação quantitativa das tendências temporais de LGA antes e depois da vacinação foi feita com análise de regressão de Quase-Poisson. O efeito protetor da vacina ao longo do tempo foi avaliado por cálculo do risco relativo em cada grupo de idade. Resultados: O estudo mostrou diminuição significativa de mais de 60% em LGA em mulheres de <20 anos de idade e de pelo menos cerca de 50% para os outros grupos. A vacina contra o HPV foi um fator de proteção, por causa do resultado do risco relativo de <0,0001 em todas as idades. Conclusões: Embora os estudos mostrem que as lesões pré-neoplásicas do HPV possam ser reversíveis espontaneamente, é inegável que a vacina contribuiu grandemente para as taxas elevadas de redução, associadas com a alta cobertura vacinal. Esses resultados são os primeiros no Brasil e podem dirigir no futuro a necessidade de discutir a vacinação dos meninos no contexto dos mesmos resultados obtidos na Austrália

Human papillomavirus (HPV) is a huge concern in public and private health. The prevention of this condition is the combination of the use of Papanicolaou smear test, condoms and HPV vaccines. Campos dos Goytacazes, RJ, is the first Brazilian municipality to implement in September 2010 the quadrivalent HPV vaccine (Gardasil®) for girls in the age group of 11­15 years, in a hybrid strategy of vaccination (schools and health centers). In 2014, the vaccination was started also for boys, at the same time that the Ministry of Health introduced it for girls. Objective: The aim of the study was to analyze the trends in reduction of low-grade cervical abnormalities five years after the introduction of the quadrivalent HPV vaccine in this municipality (primary outcome). Furthermore, this study evaluated the relative risk (RR) of each of the groups studied, in order to explain the protective effect of the vaccine (secondary outcome). Methods: The ecological analysis evaluated the impact of HPV vaccination as a protective factor against low risk of HPV abnormalities. Results of the Pap smear test obtained from the Brazilian Ministry of Health's Sistema de Informação do Câncer do Colo do Útero (SISCOLO) were categorized in low-grade abnormalities (LGA) and high-grade abnormalities (HGA). This preliminary study focused in LGA rates, which were estimated for a 1-month period and then stratified by four age groups (<20; 20­24; 25­30; >30 years) from 2007 to 2014. A quantitative comparison of LGA temporal trends before and after vaccination was done with Quasi-Poisson regression analysis. The protective effect of the vaccine over time was evaluated by calculating the RR in each age group. Results: The study showed significant decrease of more than 60% in LGA in women <20 years old, and less, almost 50% for the other groups. HPV vaccine was a protective factor, because the RR result was <0.0001 in all age groups. Conclusions: Although the studies show that the pre-HPV neoplastic lesions may be reversible spontaneously, it is undeniable that the vaccine contributes greatly to the high reduction rates, associated with high vaccination coverage. These results are the first in Brazil and in future may address the necessity to discuss the vaccination for boys in the context of the same results obtained in Australia.

Expression of Hemagglutinin-Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L kanamycin and 30 mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

The mc2-CMX vaccine induces an enhanced immune response against Mycobacterium tuberculosis compared to Bacillus Calmette-Guérin but with similar lung inflammatory effects

Although the attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine has been used since 1921, tuberculosis (TB) control still proceeds at a slow pace. The main reason is the variable efficacy of BCG protection against TB among adults, which ranges from 0-80%. Subsequently, the mc2-CMX vaccine was developed with promising results. Nonetheless, this recombinant vaccine needs to be compared to the standard BCG vaccine. The objective of this study was to evaluate the immune response induced by mc2-CMX and compare it to the response generated by BCG. BALB/c mice were immunised with both vaccines and challenged withMycobacterium tuberculosis (Mtb). The immune and inflammatory responses were evaluated by ELISA, flow cytometry, and histopathology. Mice vaccinated with mc2-CMX and challenged with Mtb induced an increase in the IgG1 and IgG2 levels against CMX as well as recalled specific CD4+ T-cells that produced T-helper 1 cytokines in the lungs and spleen compared with BCG vaccinated and challenged mice. Both vaccines reduced the lung inflammatory pathology induced by the Mtb infection. The mc2-CMX vaccine induces a humoral and cellular response that is superior to BCG and is efficiently recalled after challenge with Mtb, although both vaccines induced similar inflammatory reductions.

Seroconversión frente a primovacunación reforzada contra hepatitis B en niños con cáncer / Seroconversion in response to a reinforced primary hepatitis B vaccination in children with cancer

Introducción: La respuesta inmune a los antígenos de las vacunas está disminuida en los niños con cáncer. El objetivo de este estudio fue evaluar la seroconversión frente a vacuna ADN recombinante contra hepatitis B al momento del inicio de la quimioterapia y/o remisión en niños con cáncer. Pacientes y método: Estudio prospectivo, bicéntrico, controlado, no aleatorizado de niños con diagnóstico reciente de cáncer pareados con niños sanos. Los casos fueron vacunados a tiempo 0, 1 y 6 meses, a dosis de 20 y 40 μg si eran < ó > 10 años, respectivamente, con vacuna ADN recombinante contra hepatitis B, en el momento del diagnóstico en el caso de los tumores sólidos y luego de la remisión en el caso de los tumores hematológicos. El grupo control recibió el mismo esquema, con dosis de 10 o 20 μg respectivamente. Se midieron anticuerpos séricos anti-HBs a los 2, 8 y 12 meses posvacunación. Seroconversión se definió como títulos anti-HBs > 10 mUI/ml al octavo mes. Resultados: Un total de 78 niños con cáncer y 25 controles fueron evaluados con títulos anti-HBs al octavo mes. La tasa de seroconversión fue de 26,9%, en niños con cáncer, sin diferencia por edad, género ni tipo de tumor (p = 0,13; 0,29; y 0,44, respectivamente), y de 100% en el grupo control (p < 0,0001, comparado con los niños con cáncer). En el seguimiento a los 12 meses solo el 31,9% de los niños con cáncer presentaba títulos anti-HBs > 10 mUI/ml. Conclusiones: La vacunación contra hepatitis B con vacuna ADN recombinante, con esquema reforzado de 3 dosis, en el momento del inicio de la quimioterapia y/o remisión provee una respuesta inmune insuficiente en la mayoría de los niños con cáncer. En esta población debieran evaluarse vacunas de tercera generación, con adyuvantes más inmunogénicos, esquemas reforzados a los 0, 1, 2 y 6 meses, medición de títulos de anticuerpos al octavo y duodécimo mes, eventual uso de refuerzos y reevaluación de inmunogenicidad si correspondiese.

Introduction: Immune response against vaccine antigens may be impaired in children with cancer. The aim of this study was to evaluate the seroconversion response against hepatitis B vaccination (HBV) at the time of chemotherapy onset and/or remission in children with cancer. Patients and method: Prospective, two-centre, controlled, non-randomised study conducted on children recently diagnosed with cancer, paired with healthy subjects. Cases received HBV at time 0, 1 and 6 months with DNA recombinant HBV at a dose of 20 and 40 μg if < or > than 10 years of age, respectively, at the time of diagnosis for solids tumours and after the remission in case of haematological tumours. Controls received the same schedule, but at of 10 and 20 μg doses, respectively. HBs antibodies were measured in serum samples obtained at 2, 8 and 12 months post-vaccination. Protective titres were defined as > 10 mIU/ml at 8th month of follow up. Results: A total of 78 children with cancer and 25 healthy controls were analysed at month 8th of follow up. Seroconversion rates in the cancer group reached 26.9%, with no differences by age, gender or type of tumour (P = .13, .29, and .44, respectively). Control group seroconversion was 100% at the 8th month, with P < .0001 compared with the cancer group. At month 12 of follow up, just 31.9% of children with cancer achieved anti-HBs antibodies > 10 mIU/ml. Conclusions: Vaccination against hepatitis B with three doses of DNA recombinant vaccine at an increased concentration, administrated at the time of onset of chemotherapy and/or remission provided an insufficient immune response in a majority of children with cancer. More immunogenic vaccines should be evaluated in this special population, such as a third generation, with more immunogenic adjuvants, enhanced schedules at 0, 1, 2, 6 month, evaluation of antibody titres at month 8 and 12 h to evaluate the need for further booster doses.

Resposta de linfonodos em bovinos inoculados a campo com a vacina recombinante rSBm7462 anti Rhipicephalus (Boophilus) microplus) / Lymph nodes response of cattle inoculated at field with a recombinant vaccine rSBm7462 against Rhipicephalus (Boophilus) microplus

A dependência exclusiva de compostos químicos para o controle de Rhipicephalus (Boophilus) microplus tornou-se uma das maiores preocupações científicas e econômicas dos últimos anos, e como consequência, estão sendo realizadas pesquisas para o desenvolvimento de vacinas. O objetivo deste trabalho foi avaliar a resposta de linfonodos de bovinos imunizados a campo com o peptídeo rSBm7462 anti R. (B.) microplus. Foram utilizados 14 bovinos mestiços (Bos taurus x Bos indicus), com idades entre 4-10 meses, mantidos em duas propriedades rurais do norte do estado de Minas Gerais. Os animais receberam três imunizações do peptídeo rSBm7462, aplicados por via subcutânea, com intervalo de 30 dias. Após 15 dias de cada imunização, os linfonodos pré-escapulares foram coletados e fixados por 18 horas em formol. Posteriormente, foram incluídos em Paraplast e as amostras foram coradas pela técnica hematoxilina-eosina (HE) para a observação de eventos celulares. Para a identificação do antígeno nos linfonodos dos animais imunizados, foi realizada a técnica de imuno-histoquímica (IHQ) com o método peroxidase-anti-peroxidase (PAP). A resposta de linfonodos dos bovinos inoculados foi avaliada pelas análises de formação de centros germinais (CG), hiperplasia de cordões medulares (CM) e a presença do antígeno rSBm7462 em células PAP+, demonstrando que o peptídeo recombinante rSBm7462 induz uma resposta imune adaptativa T-dependente, caracterizada nos tecidos linfóides secundários pela formação de estruturas que conferem afinidade e memória imunológica.

Exclusive chemicals dependence for the control of Rhipicephalus (Boophilus) microplus has become one of the largest scientific and economical concerns in recent years, and as a result, research to vaccine development are being undertaken. The objective of this study was evaluating the lymph nodes response of cattle immunized at field with the rSBm7462 anti-R. (B.) microplus peptide. Fourteen crossbred cattle (Bos taurus x Bos indicus), aged 4-10 months, were used. The animals were maintained on two farms in the north of Minas Gerais state and received three immunizations with the peptide rSBm7462 applied subcutaneously at 30-day intervals. Pre-scapular lymph nodes were collected surgically 15 days after each immunization and fixed in formalin for 18 hours, then, they were embedded in Paraplast subsequently and the samples were stained with Hematoxylin-Eosin (HE) technique for cellular events observation. On the other hand, in order to antigens identifying in immunized animals lymph nodes, the immunohistochemistry (IHC) with peroxidase-anti peroxidase (PAP) method was performed. Lymph node response of cattle inoculated was evaluated by analysis of germinal centers (GC) formation, medullary cords hyperplasia (MC) and antigen rSBm7462 presence in PAP+ cells. This study shows that the recombinant peptide rSBm7462 induces a T-dependent adaptive immune response characterized on secondary lymphoid tissues by structure formation for affinity and immunological memory.

Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E / Imunogenicidade de uma cepa inativada do herpesvírus bovino tipo 5 defectiva na timidina quinase e glicoproteína E

The immunogenicity of an inactivated, experimental vaccine based on a bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E (BoHV-5 gE/TKΔ) was evaluated in cattle and the results were compared with a vaccine containing the parental BoHV-5 strain (SV507/99). To formulate the vaccines, each virus (wildtype SV507/99 and BoHV-5 gE/TK∆) was multiplied in cell culture and inactivated with binary ethyleneimine (BEI). Each vaccine dose contained approximately of 10(7.5) TCID50 of inactivated virus mixed with an oil-based adjuvant (46:54). Forty calves, 6 to 9-months-old, were allocated into two groups of 20 animals each and vaccinated twice (days 0 and 22pv) by the subcutaneous route with either vaccine. Serum samples collected at day 0 and at different intervals after vaccination were tested for virus neutralizing (VN) antibodies against the parental virus and against heterologous BoHV-5 and BoHV-1 isolates. The VN assays demonstrated seroconversion to the respective homologous viruses in all vaccinated animals after the second vaccine dose (mean titers of 17.5 for the wildtype vaccine; 24.1 for the recombinant virus). All animals remained reagents up to day 116 pv, yet showing a gradual reduction in VN titers. Animals from both vaccine groups reacted in similar VN titers to different BoHV-1 and BoHV-5 isolates, yet the magnitude of serological response of both groups was higher against BoHV-5 field isolates. Calves vaccinated with the recombinant virus did not develop antibodies to gE as verified by negative results in a gE-specific ELISA, what would allow serological differentiation from naturally infected animals. Taken together, these results indicate that inactivated antigens of BoHV-5 gE/TK recombinant virus induced an adequate serological response against BoHV-5 and BoHV-1 and thus can be used as an alternative, differential vaccine candidate.

A imunogenicidade de vacina experimental inativada, produzida com uma cepa do herpesvírus bovino tipo 5 defectiva nos genes da timidina quinase e glicoproteína E (BoHV-5 gE/TKΔ) foi avaliada em bovinos e o resultado foi comparado com a resposta induzida pela cepa parental do BoHV-5 (SV507/99). Para a formulação da vacina, cultivos de células infectados com cada um dos vírus (SV507/99 ou BoHV-5 gE/TKΔ) foram inativados com etilenamina binária. Cada dose de vacina continha aproximadamente 107,5 TCID50 de um dos vírus inativados emulsionado em adjuvante oleoso (46:54). Quarenta bezerros de raças cruzadas, com idade entre seis a nove meses, foram alocados em dois grupos de 20 animais cada e vacinados duas vezes (dia 0 e 22 pv) pela via subcutânea com uma das vacinas. Amostras de soro foram coletadas no dia 0 e a vários intervalos após vacinação para a pesquisa de anticorpos neutralizantes frente ao vírus homólogo ou frente a isolados de BoHV-5 e BoHV-1. Os testes de soroneutralização (SN) demonstraram que todos os animais soroconverteram após a segunda dose da vacina (títulos médios de 17,5 para o grupo SV507/99; 24,1 para o grupo BoHV-5 gE/TKΔ). Todos os animais mantiveram níveis de anticorpos neutralizantes até o dia 116 pv, no entanto foi observada uma redução gradual no títulos. A sorologia cruzada com amostras heterólogas do BoHV-5 e BoHV-1 indicou que ambos os grupos vacinais reagiram em níveis similares frente ao mesmo vírus, no entanto a magnitude da resposta sorológica foi maior frente a amostras de BoHV-5. Os animais vacinados com a cepa recombinante não desenvolveram anticorpos contra a gE detectáveis por um ELISA específico, o que permitiria a sua diferenciação sorológica de animais infectados naturalmente. Esses resultados demonstram que a vacina contendo antígenos inativados do vírus recombinante BoHV-5 gE/TKΔ induziu resposta sorológica em níveis satisfatórios, constituindo-se, assim, em alternativa a cepa ...

Immune strategies using single-component LipL32 and multi-component recombinant LipL32-41-OmpL1 vaccines against leptospira

Genes encoding lipoproteins LipL32, LipL41 and the outer-membrane protein OmpL1 of leptospira were recombined and cloned into a pVAX1 plasmid. BALB/c mice were immunized with LipL32 and recombined LipL32-41-OmpL1 using DNA-DNA, DNA-protein and protein-protein strategies, respectively. Prime immunization was on day 1, boost immunizations were on day 11 and day 21. Sera were collected from each mouse on day 35 for antibody, cytokine detection and microscopic agglutination test while spleen cells were collected for splenocyte proliferation assay. All experimental groups (N = 10 mice per group) showed statistically significant increases in antigen-specific antibodies, in cytokines IL-4 and IL-10, as well as in the microscopic agglutination test and splenocyte proliferation compared with the pVAX1 control group. The groups receiving the recombined LipL32-41-OmpL1 vaccine induced anti-LipL41 and anti-OmpL1 antibodies and yielded better splenocyte proliferation values than the groups receiving LipL32. DNA prime and protein boost immune strategies stimulated more antibodies than a DNA-DNA immune strategy and yielded greater cytokine and splenocyte proliferation than a protein-protein immune strategy. It is clear from these results that recombination of protective antigen genes lipL32, lipL41, and ompL1 and a DNA-protein immune strategy resulted in better immune responses against leptospira than single-component, LipL32, or single DNA or protein immunization.